The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 10⁶ and 1.3 × 10⁷ transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.     

开封农村地区婴幼儿中肠道病毒71型和柯萨奇病毒A组16型中和抗体水平及流行趋势

目的了解开封农村地区婴幼儿中肠道病毒71型(EV71)和柯萨奇病毒A组16型(CA16)中和抗体水平及流行趋势。方法应用微量细胞病变法,对2004年开封农村地区采集的349名7～30月龄婴幼儿血清进行EV71和CA16中和抗体检测。结果7～30月龄婴幼儿EV71和CA16中和抗体阳性率分别为36.7%(128/349)和36.5%(123/337),但二者流行趋势不同。EV71抗体阳性率由10～12月龄组的18.2%上升至25～30月龄组的81.3%,上升约4.5倍,抗体几何平均滴度(GMTs)也缓慢上升;CA16抗体阳性率由13～15月龄组的23.0%上升至22～24月龄组的47.6%,上升约2.1倍,GMTs在125～338之间波动;不同月龄婴幼儿中存在EV71和CA16混合感染的情况,19～24和25～30月龄组的EV71和CA16混合感染率显著高于7～12和13～18月龄组。结论开封农村地区婴幼儿中EV71和CA16中和抗体阳性率高,但二者上升的趋势不同,18月龄后的婴幼儿中EV71和CA16混合感染的比例显著升高。     

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.     

By-Passing Large Screening Experiments Using Sequencing as a Tool to Identify scFv Fragments Targeting Atherosclerotic Lesions in a Novel In Vivo Phage Display Selection

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. To interrogate the molecular components involved in this process, single-chain variable fragments (scFvs) from a semi-synthetic human antibody library were selected on the lesions induced in a rabbit model of atherosclerosis after two rounds of in vivo phage display. Homing Phage-scFvs were isolated from (1) the injured endothelium, (2) the underlying lesional tissue and (3) the cells within the intima. Clones selected on the basis of their redundancy or the presence of key amino acids, as determined by comparing the distribution between the native and the selected libraries, were produced in soluble form, and seven scFvs were shown to specifically target the endothelial cell surface and inflamed intima-related regions of rabbit tissue sections by immunohistology approaches. The staining patterns differed depending on the scFv compartment of origin. This study demonstrates that large-scale scFv binding assays can be replaced by a sequence-based selection of best clones, paving the way for easier use of antibody libraries in in vivo biopanning experiments. Future investigations will be aimed at characterizing the scFv/target couples by mass spectrometry to set the stage for more accurate diagnostic of atherosclerosis and development of therapeutic strategies.     

柯萨奇病毒A组16型VP1蛋白的原核表达及其免疫原性

目的原核表达柯萨奇病毒A组16型(Coxsackievirus group A type 16,CA16)VP1蛋白,并检测其免疫原性。方法通过RT-PCR法从CA16病毒青岛株中扩增VP1基因,克隆至原核表达载体pET43.1a(+)中,构建重组表达质粒pET43.1a-VP1,转化感受态E.coli Rossatte(DE3),IPTG诱导表达。表达的重组CA16 VP1蛋白通过Ni柱亲和层析纯化后,采用不同剂量(5、10、20、40μg)免疫BALB/c小鼠,ELISA法检测血清中特异性IgG、IgG1、IgG2a、IgG2b、IgG3抗体效价,微量细胞病变抑制法检测血清中和抗体效价。结果重组表达质粒pET43.1a-VP1经双酶切及测序证实构建正确;表达的重组CA16 VP1蛋白相对分子质量约为34 000,主要以包涵体形式存在,表达量占菌体总蛋白的15%;纯化的重组CA16 VP1蛋白纯度可达95%以上,可与猴CA16抗血清反应;不同剂量的重组CA16 VP1蛋白免疫BALB/c小鼠,可诱导产生CA16特异性抗体,血清中总IgG、IgG1、IgG2a、IgG2b、IgG3抗体效价均明显高于对照组,且抗体效价与免疫剂量存在一定的量效关系;各剂量CA16 VP1组免疫小鼠血清中和抗体效价均小于1∶8。结论已成功在大肠杆菌中表达了重组CA16 VP1蛋白,纯化的重组蛋白可诱导小鼠特异性体液免疫应答,为进一步研究CA16的结构、功能及相关疫苗的研制奠定了基础。     

33株柯萨奇病毒A组16型分离株的全基因序列分析

目的对2008～2010年北京和青岛地区流行的33株柯萨奇病毒A组16型(Coxsackie virus A16,CA16)分离株的全基因序列进行分析。方法收集2008～2010年北京和青岛地区CA16感染患者咽拭子标本,采用Vero细胞对病毒进行分离,并经噬斑纯化。提取病毒RNA,采用RT-PCR法分段扩增CA16全长基因,经序列测定和拼接后,利用DNAStar和MEGA5.10软件分析全基因序列。结果经病毒分离和噬斑纯化,共获得33株CA16分离株;33个分离株之间的核苷酸序列同源性大于90.9%,与CA16国际标准株G10各区段的核苷酸序列同源性为72.7%～89.0%,氨基酸序列同源性为79.3%～100.0%;与近年中国分离的CA16 SZ/HK08-7株同源性较高,全基因组同源性大于91.5%,各区段核苷酸序列同源性均大于83.7%;在基于VP1序列的种系进化树中,BJWG16、BJWG17、BJWG20等23个分离株属于C1亚型,其余10个分离株属于C3亚型。结论 2008～2010年北京和青岛地区流行的33株CA16分离株均为C基因型。本研究对我国CA16分子流行病学、毒力位点的研究以及疫苗株的选择具有重要意义。     

Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10(-2) μg/mL and contaminating mycelium at a quantity of 10(-2) mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.