Human JC virus-like particles as a gene delivery vector

INTRODUCTION: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy. AREAS COVERED: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed. EXPERT OPINION: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.     

Human JC virus-like particles as a gene delivery vector

Introduction: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy.

Areas covered: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed.

Expert opinion: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.     

Virus-like particles as a modular system for novel vaccines

Induction of protective immune responses with recombinant antigens is a major challenge for the vaccine industry. Here we present a molecular assembly system that renders antigens of choice highly repetitive. Using this method, efficient antibody responses may be induced in the absence of adjuvants resulting in protection from viral infection and allergic reactions.     

耐核糖核酸酶病毒样颗粒的构建和表达

目的：为耐核糖核酸酶（RNase)的RNA标准品和质控品的表达制备提供1个通用载体平台。方法：将MS2噬菌体基因组中成熟酶蛋白和包膜蛋白基因编码序列的1．7kb cDNA目的片段，用Hind Ⅲ和EcoR I酶切后，用于相同内切酶酶切的表达载体质粒PET28b中，并在T4 DNA连接酶的存在下连接，构建一新的表达载体pI NCCL,再转化BL21－DE3 E.Coli进行原核表达。结果：成功构建出新的表达载体pI NCCL,经原核表达为耐RNase的病毒样颗粒。结论：本研究得到的pI NCCL表达载体及原核表达系统，可作为1个耐RNase的RNA标准品与质控品的构建和制备表达通用载体平台，以促进有关标准品和质控品的研究。     

Antibody-based diagnostic assays with recombinant virus-like particles

Virus-like particles were produced with 4 genogroup I Norwalk-like viruses(NLVs) and 7 genogroup II NLVs by a baculovirus expression system, and used to detect the antibody to NLVs by enzyme-linked immunosorbent assays(ELISA). Very little cross reactivity was observed between the genogroup I NLVs and the genogroup II NLVs when the antibody ELISA was done with hyperimmune sera. Infections by several genotypes of the NLVs were thought to occur in oyster-associated acute gastroenteritis, while an infection by a single genotype of NLVs was found in the illness occurred in a hospital. A high prevalence of antibody to 11 NLVs was observed in samples collected from healthy adults in Japan.     

The use of virus-like particles for gene transfer

A major challenge in the field of gene therapy is the development of new carrier/delivery systems that lack the disadvantages of current transfer systems. In the past, some time has been spent developing such modified or alternative vectors. A new candidate is represented by virus-like particles (VLPs). It has been shown that recombinant expression of the major structural proteins of many viruses leads to the formation of VLPs. Such VLPs exhibit morphology similar to the empty capsids of the virus from which they are derived. VLPs are non-infectious, have a similar tropism to the natural virus, and show comparable cellular uptake and intracellular trafficking. Since its discovery, VLP technology has gained importance in biomedical research. Although most investigations into VLP technology have dealt with vaccine development, some research groups have demonstrated that VLPs could also represent a useful gene therapy delivery system. This review will focus on studies performed with VLPs from members of the Papillomaviridae and Polyomaviridae families.     

Comparison of human papillomavirus type 16 L1 chimeric virus-like particles versus L1/L2 chimeric virus-like particles in tumor prevention

Chimeric human papillomavirus (HPV) virus-like particles (cVLPs) with the HPV16 E7 antigen fused to either the major capsid protein, L1, or the minor capsid protein, L2, have been used independently to protect against the formation of HPV-induced tumors in animal models. However, the advantages and disadvantages of both types of particles with respect to production and vaccine efficacy have never been analyzed. Therefore, in this study, we compared cVLPs with the HPV16 E7 antigen fused to L1 versus cVLPs with E7 fused to L2 with respect to their ability to protect mice from tumor challenge. The first 57 amino acids of E7 were used to overcome the size limitation and limited VLP production imposed by inserting polypeptides into L1 cVLPs. C57BL/6 mice were immunized with the above cVLPs at various doses. Tumor challenge was then performed with HPV16 E7-positive TC-1 cells. HPV16 L1-E7((1-57)) was superior to HPV16 L1/L2-E7((1-57)) in eliciting tumor protection at equivalent doses, although both types of particles were able to protect mice. Both cVLPs induced a specific cytotoxic T lymphocyte (CTL) response to the H2-D(b)-restricted E7 peptide (E7(49-57)) as determined by an ELISPOT assay and tetramer staining; however, immunization with the L1-E7((1-57)) cVLPs resulted in twofold higher CTL precursor frequencies. Our results demonstrate that cVLPs with the antigen fused to L1 are a more efficient vaccine with respect to tumor prevention than cVLPs with the antigen fused to L2. At the same time, however, L1 cVLPs are limited by the size of the antigen that can be incorporated and in the amount of cVLP that can be obtained from cultures when compared to L1/L2 cVLPs. This balances out their superior ability to induce protective immunity.     

Production and biomedical applications of virus-like particles derived from polyomaviruses

Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargos and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.     

Insight into N-terminal localization and dynamics of engineered virus-like particles

Virus-like particles composed of the cowpea chlorotic mottle virus (CCMV) capsid protein (CP) have been extensively studied as carrier systems in nanoscience. One well-established method to improve their stability under physiological conditions is to fuse a stimulus-responsive elastin-like polypeptide (ELP) to the N-terminus of the CPs. Even though the N-terminus should in principle be localized in the inner cavity of the protein cage, studies on the native CCMV revealed its accessibility on the particle surface. We verified that such phenomenon also applies to ELP-CCMVs, by exploiting the covalent functionalization of the CP N-terminal domain via a sortase A-mediated reaction. Western-blot analysis and Förster resonance energy transfer (FRET) experiments furthermore revealed this to be caused by both the external display of the N-termini and the interchange of CPs among preformed capsids. Our findings demonstrate the tunability of ELP-CCMV stability and dynamics and their potential effect on the exploitation of such protein cages as a drug delivery system.

The N-terminal localization and dynamic intermixing of engineered cowpea chlorotic mottle virus-like particles were studied independently from each other.     

含风疹病毒部分核酸序列的耐核糖核酸酶病毒样颗粒的构建和表达

目的构建原核表达系统，表达内含风疹病毒部分核酸序列的由噬菌体包膜蛋白构成的病毒样颗粒，并包被重组RNA，使其具耐核糖核酸酶(RNase)的特性。方法用逆转录聚合酶链反应(RT-PCR)扩增风疹减毒活疫苗中风疹病毒糖蛋白E1的部分保守区域，进行TA克隆后，用限制性内切酶HindⅢ酶切，获得所需要的目的片段，并与用HindⅢ酶切的表达载体pNCCL1相连接，构建一新的表达载体pNCCL1-Ru。再转化E．Coli BL21-DE3，用IPTG诱导表达噬菌体MS2包膜蛋白后，再自我组装成病毒样颗粒，并将风疹病毒部分序列包裹到病毒样颗粒内。结果成功构建了新的表达载体pNCCL1-Ru，其原核表达产物病毒样颗粒中所包裹的风疹病毒RNA序列与风疹BRDⅡ株同源性高达98％，所包裹的RNA具有耐RNase消化的特性以及良好的稳定性。结论我们构建的表达载体pNCCL1-Ru及原核表达系统，可作为构建和制备耐RNase的风疹病毒核酸标准品和质控品的平台，可为实验室进行风疹病毒核酸的检测提供稳定的无传染性的标准品和质控品。     

A precipitation reaction found in patients with hepatitis C as a marker for the purification of virus-like particles

Even after the molecular cloning of the hepatitis C virus (HCV), an HCV-specific precipitation reaction has not yet been identified. We attempted to develop a precipitation system exclusively for anti-HCV-positive sera as a first step in finding an HCV-specific antigen and HCV-associated particles. In some patients being in a final stage of different liver diseases, we found sera (179/132,761; designated 'a-CK') which specifically precipitated with anti-HCV- and HCV-RNA-positive sera (designated 'CK'). When CK-positive sera were searched for in patients with various liver diseases using standard a-CK-positive plasma, CK was detected in 420 (57.9%) of 726 anti-HCV-positive sera and in none of the 1,630 anti-HCV-negative ones. The nature of CK and a-CK has not been fully clarified yet; CK demonstrated inter-betagamma mobility, whereas a-CK showed beta-globulin mobility; CK was not detected in cryoprecipitate, but HCV RNA was present in precipitates of CK-positive plasma incubated with one that was a-CK positive. Transmission electron microscopy revealed two size ranges of particles in the precipitate of CK- and a-CK-positive plasmas, 23-38 nm and 48-65 nm. We have found a novel precipitation system which is exclusive to anti-HCV-positive sera and which specifically precipitates an HCV-RNA-containing serum fraction and particles. This system can be useful for the purification and characterization of the circulating particles. Furthermore, it may be a new approach to the nature of HCV-RNA-carrying material.     

耐核糖核酸酶内含长片段嵌合体RNA的病毒样颗粒的构建和表达

目的 通过改变原噬菌体ms2包膜蛋白RNA包装位点(19碱基的茎环结构)的数量及亲和力,构建新的原核表达系统,探讨表达内含长片段(达到理论上的1 900 bp)RNA的耐RNase病毒样颗粒的可能性.方法 首先设计含Hind Ⅲ和Not Ⅰ酶切位点的引物,扩增ms2包膜蛋白的编码成熟酶蛋白和衣壳蛋白的1 700 bp序列,并将原来的19mer的包装位点序列改变为C-5变异体(19 bp stem-loop结构中-5位的尿嘧啶改变为胞嘧啶),Hind Ⅲ和Not Ⅰ酶切后,与用同样酶切的表达载体pET-28(b)相连接,得到重组载体pET-ms2-pac.应用重叠PCR扩增3种病毒的5段嵌合体序列(包括3段SARS-CoV基因、一段HCV基因和一段HSN1基因),并在SARS-CoV3和HCV序列之间插入一个19mer的变异体包装位点序列,在设计引物时,使嵌合体两端含有Not Ⅰ酶切位点,与Not Ⅰ酶切的重组载体pET-ms2-pac相连接,构建得到具有2个变异包装位点的表达载体pET-ms2-3V.同时构建3种对照重组表达载体,分别测定N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V 4种重组表达质粒表达产物的260 nm吸光度(A260)值,根据公式A160=0.125 mg/ml计算4种表达产物的表达效率.结果 成功构建了4种原核表达载体:pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P和N-P3V-pET-C.pET-ms2-3V和P-3V-pET-P经原核表达后得到含全长为1 891的5段嵌合体RNA的病毒样颗粒;N-P3V-pET-P、N-P3V-pET-C其原核表达产物病毒样颗粒中仅包装了1 200 bp的目的 嵌合体RNA.N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V的表达效率分别为0.23、0.35、0.35和0.51 mg/ml.N-P3V-pET-C比N-P3V-pET-P表达效率高52%,而pET-ms2-3V比P-3V-pET-P表达效率高38%.所包装的RNA具有耐RNase和DNase消化的特性以及良好的不同温度条件下的稳定性.结论 通过改变噬菌体ms2 RNA包装位点(19碱基的茎环结构)的数量,可构建能表达内含达到理论上的约1 900 bp外源RNA的耐RNase病毒样颗粒的原核表达载体平台.通过应用包装位点的变异体C-变异体,可以增加病毒样颗粒的表达效率.     

Isolation and characterization of a neprilysin-like protein from Venturia canescens virus-like particles

Maternal protein secretions from endoparasitoid wasps are evolutionary adaptations to regulate host physiology as part of an extended wasp phenotype. Virus-like particles (VLPs) produced in the calyx region of Venturia canescens wasps are involved in immune evasion of the developing parasitoid inside the host. In contrast to polydnaviruses (PDVs), VcVLPs are devoid of any nucleic acids. To understand the role of these particles in the regulation of host physiology and phylogenetic relationship between VLPs and PDVs, it is essential to identify particle proteins. In this paper, we describe the isolation and molecular cloning of a neprilysin-like gene (VcNEP) coding for a 94 kDa VcVLP protein and discuss its possible role in host regulation.     

Gene transfer with synthetic virus-like particles via the integrin-mediated endocytosis pathway.

The interaction between cationic DNA-containing particles and cell surface anionic proteoglycans is an efficient means of entering cultured cells. Therapeutic in vivo gene delivery levels, however, require binding to less ubiquitous molecules. In an effort to follow adenovirus, thiol-derivatized polyethylenimine (PEI) was conjugated to the integrin-binding peptide CYGGRGDTP via a disulfide bridge. The most extensively conjugated derivative (5.5% of the PEI amine functions) showed physical properties of interest for systemic gene delivery. In the presence of excess PEI-RGD, plasmid DNA was condensed into a rather homogeneous population of 30-100 nm toroidal particles as revealed by electron microscopy images in 150 mM salt. Their surface charge was close to neutrality as a consequence of the shielding effect of the prominent zwitterionic peptide residues. Transfection efficiency of integrin-expressing epithelial (HeLa) and fibroblast (MRC5) cells was increased by 10- to 100-fold as compared with PEI, even in serum. This large enhancement factor was lost when aspartic acid was replaced by glutamic acid in the targeted peptide sequence (RGD/RGE), confirming the involvement of integrins in transfection. PEI-RGD/DNA complexes thus share with adenovirus constitutive properties such as size and a centrally protected DNA core, and 'early' properties, i.e. cell entry mediated by integrins and acid-triggered endosome escape.     

Chimeric Trop2 virus-like particles: a potential immunotherapeutic approach against pancreatic cancer

Trop2 is a recently discovered cell surface glycoprotein overexpressed in pancreatic cancer which could potentially be used as an immunotherapeutic target. Enveloped virus-like particles (VLPs) are highly immunogenic and versatile immune stimulatory agents which can be modified to incorporate exogenous proteins on their membrane envelope to use as cancer vaccines. In this study, we investigated the effects of murine Trop2 (mTrop2) VLP immunization in a pancreatic cancer syngeneic murine model. VLPs incorporating mTrop2 were used to immunize C57BL/6 tumor-bearing mice. Immunization with mTrop2 VLPs led to a significant reduction in tumor growth accompanied by a broad activation and tumor infiltration of CD4(+) and CD8(+) T cells as well as natural killer and natural killer T cells. VLP immunization generated mTrop2-specific cytotoxic T lymphocytes and antibodies with no measurable induction of autoimmunity. Importantly, VLP immunization decreased the population of regulatory T cells and myeloid-derived suppressor cells inside the tumor tissue resulting in decreased levels of immunosuppressive cytokines like interleukin-10 and transforming growth factor-β while promoting the activation of immature macrophages and dendritic cells. Furthermore, combination of VLP immunization with gemcitabine treatment showed an improved effect significantly increasing the survival of tumor bearing mice. Our results demonstrate that mTrop2 VLP immunization can activate broad antitumor immune responses and affect key players in the tumor microenvironment overcoming a major barrier, which has limited the efficacy of cancer vaccines. This study presents a novel immunotherapeutic approach which could potentially be used as an alternative treatment option in combination therapies to treat pancreatic cancer patients.     

T载体克隆在病毒样颗粒表达载体构建中的应用

目的提高带某些限制酶切位点聚合酶链反应(PCR)扩增产物克隆入表达载体的效率。方法将带Hind Ⅲ酶切位点的PCR扩增产物与T载体连接，转化BL21-DE3 E．Coli，提取质粒后，用Hind Ⅲ酶切，电泳分离并提取纯化的目的片段。然后，与经过相同的限制性酶酶切的表达载体pNCCL1连接。结果经T载体克隆酶切构建的内含严重急性呼吸综合征冠状病毒(SARS—CoV)核酸的病毒样颗粒表达载体，方法简单快速，每次均可获得成功。而采用直接酶切扩增产物的构建方法，未获得成功构建的表达载体。结论T载体克隆可用于对直接酶切效果不好(如Hind Ⅲ)的PCR扩增片段的表达载体的连接构建，方法简便高效。     

Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae

OBJECTIVES: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. METHODS: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. RESULTS: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. CONCLUSION: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.     

Peptide mimotopes as candidate vaccines

The development of combinatorial peptide libraries, where random peptide sequences are displayed either on the surface of a phage or on a solid support, provides researchers with a powerful tool for analysis and study of the specificity of immune responses. The strength of this technology lies in the large amount of molecular diversity displayed that can be easily obtained and rapidly tested. As a result of screening peptide libraries, novel peptide sequences can be identified, which mimic native protective epitopes (mimotopes), and have the potential for use as vaccine candidates.