Effects of disinfectants against norovirus virus‐like particles predict norovirus inactivation

Human noroviruses (NoVs) are a major cause of epidemic and sporadic acute gastroenteritis worldwide. Public and personal hygiene is one of the most important countermeasures for preventing spread of NoV infection. However, no a practicable cell culture system for NoV had been developed, initial tests of the virucidal effectiveness of anti‐NoV disinfectants and sanitizers have been performed using surrogate viruses. In this study, NoV virus‐like particles (VLPs) were used as a new surrogate for NoVs and a method for evaluating NoV inactivation using them developed. This method is based on morphological changes in VLPs after treatment with sodium hypochlorite. VLP specimens were found to become deformed and degraded in a concentration‐dependent manner. Based on these results, the effects of sodium hypochlorite on VLPs were classified into four phases according to morphological changes and number of particles. Using the criteria thus established, the efficacy of ethanol, carbonates and alkali solutions against VLPs was evaluated. Deformation and aggregation of VLPs were observed after treatment with these disinfectants under specific conditions. To determine the degradation mechanism(s), VLPs were examined by SDS‐PAGE and immunoblotting after treatment with sodium hypochlorite and ethanol. The band corresponding to the major capsid protein, VP1, was not detected after treatment with sodium hypochlorite at concentrations greater than 500 ppm, but remained after treatment with ethanol. These results suggest that VLPs have excellent potential as a surrogate marker for NoVs and can be used in initial virucidal effectiveness tests to determine the mechanism(s) of chemical agents on NoVs.     

Monoclonal antibody to fibulin-1 generated by genetic immunization

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.     

Incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2

While virus-like particles (VLPs) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. In this study, we successfully established a cell line stably expressing the precursor membrane/envelope (PrM/E) proteins of dengue virus type 2 (DENV2), which can package a DENV2 replicon with deletion of PrM/E genes and produce single-round infectious VLPs. Moreover, it can package a similar replicon of different serotype, dengue virus type 4, and produce infectious chimeric VLPs. To our knowledge, this study reports for the first time replicon-containing VLPs of dengue virus. Moreover, this convenient system has potential as a valuable tool to study encapsidation of dengue virus and to develop novel chimeric VLPs containing dengue virus replicon as vaccine in the future.     

消减免疫法制备克伦特罗单克隆抗体

目的：制备克伦特罗（CL）单克隆抗体。方法：重氮化法制备克伦特罗完全抗原,消减免疫法免疫BALB／c小鼠,常规杂交瘤技术制备抗克伦特罗单克隆抗体。结果：消减免疫法使小鼠获得了对BSA的耐受,单抗筛选过程中获得了高达8．2％的阳性率。最后得到了针对CL的特异性抗体。结论：用消减免疫法制备针对小分子污染物的单克隆抗体,可减少在制备单克隆抗体时筛选的工作量,可增加获得预期抗体的机会。     

Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles

Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.     

Versatile RHDV virus-like particles: incorporation of antigens by genetic modification and chemical conjugation

Virus‐like particles have proved to be excellent molecular scaffolds, yet the individual characteristics and immune responses generated against each VLP requires the development of a wide range of capsids for use as vaccines, molecular delivery vessels, and nanoscale templates. Here we describe the development of Rabbit haemorrhagic disease virus (RHDV)‐like particles as a rapidly versatile molecular workbench, overcoming limitations imposed by established genetic antigen incorporation procedures with chimeric VLP. Production of the RHDV capsid protein in a baculovirus system led to the self‐assembly of VLP which were recovered at over 99% purity and manipulated both genetically and chemically. Fusion of small peptide sequences to RHDV VLP was well tolerated, forming chimeric capsids that enhanced the presentation of foreign peptide to hybridoma T helper cells 700‐fold. Rapid and simple conjugation techniques employing the hetero‐bifunctional chemical linker sulfo‐SMCC enabled both small peptides and whole proteins to be conjugated to the surface of RHDV VLP, overcoming limitations imposed on VLP formation and yield experienced with chimeric VLP. Administration of VLP/ovalbumin conjugate provoked high titre ovalbumin‐specific antibody in mice, demonstrating the immune stimulatory properties of the capsid were conferred to conjugated foreign antigen. VLP facilitated delivery of conjugated antigen to dendritic cells, eliciting proliferative responses in naïve TCR transgenic T helper cells that were at least 10‐fold greater than ovalbumin antigen delivered alone. Biotechnol. Bioeng. 2007;98: 968–977. © 2007 Wiley Periodicals, Inc.     

Effect of MOI ratio on the composition and yield of chimeric infectious bursal disease virus-like particles by baculovirus co-infection: deterministic predictions and experimental results

Virus-like particles (VLPs) are empty particles consisting of virus capsid proteins that closely resemble native virus but are devoid of the native viral nucleic acids and therefore have attracted significant attention as noninfectious vaccines. A recombinant baculovirus, vIBD-7, which encodes the structural proteins (VP2, VP3, and VP4) of infectious bursal disease virus (IBDV), produces native IBD VLPs in infected Spodoptera frugiperda insect cells. Another baculovirus, vEDLH-22, encodes VP2 that is fused with a histidine affinity-tag (VP2H) at the C-terminus. By co-infection with these two baculoviruses, hybrid VLPs with histidine tags were formed and purified by immobilized metal affinity chromatography (Hu et al., 1999). Also, we demonstrated that varying the MOI ratio of these infecting viruses altered the extent of VP2H incorporated into the particles. A dynamic mathematical model that described baculovirus infection and VLP synthesis (Hu and Bentley, 2000) was adapted here for co-infection and validated by immunofluorescence labeling. It was shown to predict the VLP composition as a dynamic function of MOI. A constraint in the VP2H content incorporated into the particles was predicted and shown by experiments. Also, the MOI ratio of both infecting viruses was shown to be the major factor influencing the composition of the hybrid particles and an important factor in determining the overall yield. ELISA results confirmed that VP2H was exhibited to a varied extent on the outer surface of the particles. This model provides insight on the use of virus co-infection in virus-mediated recombinant protein expression systems and aids in the optimization of chimeric VLP synthesis.     

不同粒径口蹄疫病毒样颗粒-ZIF-8复合物诱导小鼠的体液免疫效果分析

为进一步提高口蹄疫(foot-and-mouthdisease,FMD)病毒样颗粒(virus-likeparticles,VLPs)疫苗的免疫效果,本研究采用仿生矿化方法,将Zn2+和2-甲基咪唑按照不同浓度配比制备了不同粒径的FMDV VLPs-沸石咪唑骨架-8 (zeolitic imidazolate framework-8, ZIF-8)复合物,以探究尺寸效应对免疫效果的影响。结果显示,成功制备出3种不同粒径的FMDV VLPs-ZIF-8,粒径分别约为70、100、1 000 nm。细胞毒性和组织病理学试验表明,3种复合物均具有良好的生物安全性。小鼠免疫试验表明,3种复合物均能明显提高中和抗体和特异性抗体水平,并且随着复合物体积的减小,其免疫效果也随之增强。本研究表明,ZIF-8包封FMDV VLPs可显著增强其免疫效果,且具有尺寸依赖性。     

Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl- or sucrose-stressed cultures, the specific antibody production rate wasincreased up to 2.6-fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl-stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. (c) 1994 John Wiley & Sons, Inc.     

埃博拉病毒GP和VP40蛋白在哺乳动物细胞中的共表达及病毒样颗粒装配

目的：制备基因重组埃博拉病毒样颗粒,为疫苗研究及埃博拉病毒特异抗原、抗体检测提供基础。方法：根据埃博拉病毒扎伊尔株的GP和VP40蛋白氨基酸序列,以哺乳动物细胞基因表达密码子偏好性进行基因优化设计;化学合成GP和VP40基因片段并分别构建于表达质粒pcDNA3.1或同时构建到具有双表达单元的质粒pBudCE4.1;重组质粒经lipofectamine2000转染293FT细胞;以Western blot检测重组蛋白GP和VP40的表达;通过电镜观察病毒样颗粒。结果：构建的重组质粒经酶切鉴定及测序分析证实构建成功;Western blot结果显示,共转染分别表达GP和VP40的两个质粒或转染共表达两个蛋白的质粒都发现GP特异反应条带产生,且大小与预期相符,此外,转染共表达质粒产生的GP蛋白表达明显强于两个质粒共转染,并同时可检测到VP40的表达;电镜观察到典型的丝状的埃博拉病毒样颗粒。结论：在293FT细胞中基因优化的埃博拉病毒GP和VP40可有效表达并装配为病毒样颗粒,为进一步研究奠定了基础。     

Immobilization of gene vectors on polyurethane surfaces using a monoclonal antibody for localized gene delivery

BACKGROUND: Conventional strategies of gene therapy using viral vectors result in suboptimal localization and potentially dangerous distal spread of vector. We hypothesized that localized delivery of adenoviral gene vectors could be achieved from a polyurethane (PU) film through a mechanism involving anti-viral antibody tethering. METHODS: PU films were formulated with a collagen coating. Anti-adenoviral monoclonal antibodies were covalently bound to the collagen surface. These antibodies enabled tethering of replication-defective adenoviruses [Ad-GFP (encoding green fluorescent protein)] through highly specific antigen-antibody affinity. The binding stability and in vitro delivery of virus bound on PU films were investigated. Cell culture studies with rat arterial smooth muscle cells (A10) assessed transduction on or near the PU matrix. In vivo experiments with collagen-coated PU films investigated atrial epicardial implant and subdermal implant models in Yorkshire swine. RESULTS: We report for the first time successful PU film-based gene delivery using antibody-tethered adenovirus encoding the green fluorescent protein (GFP), demonstrating efficient and highly localized gene delivery to arterial smooth muscle cells in cell culture and pig implant. In comparison, direct injections of viral vectors into subcutaneous sites gave sparse, needle-track-oriented GFP expression patterns. CONCLUSION: We conclude that PU film is a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using PU film-based anti-viral antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies, and for further device-based gene delivery therapeutic strategies.     

Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.     

Modification of monoclonal antibody carbohydrates by oxidation,conjugation, or deoxymannojirimycin does not interfere with antibody effector functions

Site-specific attachment of metal chelators or cytotoxic agents to the carbohydrate region of monoclonal antibodies results in clinically useful immunoconjugates [Doerr et al. (1991) Ann Surg 214: 118, Wynant et al. (1991) Prostate 18: 229]. Since the capacity of monoclonal antibodies (mAb) to mediate tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) may accentuate the therapeutic effectiveness of immunoconjugates, we determined whether site-specific modification of mAb carbohydrates interfered with these functions. The chemical modifications examined consisted of periodate oxidation and subsequent conjugation to either a peptide linker/chelator (GYK-DTPA) or a cytotoxic drug (doxorubicin adipic dihydrazide). mAb-associated carbohydrates were also modified metabolically by incubating hybridoma cells in the presence of a glucosidase inhibitor deoxymannojirimycin to produce high-mannose antibody. All four forms (unaltered, oxidized, conjugated and high-mannose) of murine mAb OVB-3 mediated tumor cell lysis via CDC. Similarly, equivalent ADCC was observed with native and conjugated forms of mAb OVB-3 and EGFR.1. ADCC was achieved with different murine effector cells such as naive (NS), poly (I^*C)- and lipopolysaccharide-stimulated (SS) spleen cells, orCorynebacterium-parvum-elicited peritoneal cells (PEC). All murine effector cell types mediated tumor cell lysis but differed in potency such that PEC>SS>NS. Excellent ADCC activity was also demonstrable by human peripheral blood mononuclear cells with OVB-3-GYK-DTPA and high-mannose OVB-3 mAb. ADCC activity was detectable in vivo: both native and conjugated OVB-3 inhibited growth of OVCAR-3 xenografts in nude mice primed withC. parvum. In conclusion, modification of mAb carbohydrates did not compromise their in vivo or in vitro biological functions. Therefore, combination therapy using immunomodulators to enhance the effector functions of site-specific immunoconjugates could be seriously contemplated.     

Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

重组单克隆抗体药物的工程细胞培养工艺研究

单克隆抗体(monoclonal antibody, mAb)是一类重要的基因工程生物技术衍生药物,其药物适应证包括自身免疫性疾病、移植后并发症、心血管疾病、感染性疾病和各种类型的癌症。此类适应证药物通常使用时间长、使用剂量大,因此需具备大批量生产的能力。由于mAb药物的生产成本以及产品质量控制的复杂性,要求有效且经济地提供批间一致性较好的高质量药物。中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞是生物技术产业中用于生产治疗性糖蛋白最常用的真核表达宿主,也是生产治疗性抗体药物最常使用的细胞。在细胞培养过程中,工程细胞株、培养基和工艺条件是mAb药物生产中影响其质量的三个重要因素。现分别从培养基、培养参数和培养模式等对生产mAb药物的细胞培养技术作一概述。     

Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production

The quality‐by‐design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design‐of‐experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus‐like particles from CHO‐cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

Here we characterize virus-like particles (VLPs) by three very distinct, orthogonal, and quantitative techniques: electrospray differential mobility analysis (ES-DMA), asymmetric flow field-flow fractionation with multi-angle light scattering detection (AFFFF-MALS) and transmission electron microscopy (TEM). VLPs are biomolecular particles assembled from viral proteins with applications ranging from synthetic vaccines to vectors for delivery of gene and drug therapies. VLPs may have polydispersed, multimodal size distributions, where the size distribution can be altered by subtle changes in the production process. These three techniques detect subtle size differences in VLPs derived from the non-enveloped murine polyomavirus (MPV) following: (i) functionalization of the surface of VLPs with an influenza viral peptide fragment; (ii) packaging of foreign protein internally within the VLPs; and (iii) packaging of genomic DNA internally within the VLPs. These results demonstrate that ES-DMA and AFFFF-MALS are able to quantitatively determine VLP size distributions with greater rapidity and statistical significance than TEM, providing useful technologies for product development and process analytics.