Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

Study on the porcinophilic foot-and-mouth disease virus I. production and characterization of monoclonal antibodies against VP1

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

Foot-and-mouth disease virus C3 Resende subtype analysed by means of competition RIA using neutralizing monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) was analysed using 30 monoclonal antibodies (MAbs) obtained from Balb/c mice immunized with FMDV C3 Resende (C3R) subtype 7 and 14 days before fusion No. 15 and 16 respectively. Fourteen MAbs were neutralizing and by means of competition radioimmuno assay it was possible to classify them into four groups. The first group consisted of MAbs specific for three sequential and three conformational epitopes. The second group consisted of MAbs specific for two conformational and for one sequential epitope. The third and the fourth groups consisted only of one MAb each, being specific for conformationally and one sequentially dependent epitope, respectively.     

Identification of a conformational epitope on the VP1 G-H Loop of type Asia1 foot-and-mouth disease virus defined by a protective monoclonal antibody

Although neutralizing antigenic sites of foot-and-mouth disease virus (FMDV) can be defined by selection of monoclonal antibody (MAb) escape mutants, no conformational neutralizing epitope on the major antigenic site located on the G-H loop of type Asia1 FMDV has been precisely mapped. In this study, we generated a potent neutralizing MAb 3E11, which recognized a conformation-dependent epitope and neutralized FMDV Asia1/YS/CHA/05 in vitro. Importantly, a dose of 5.5 NT(50) of the MAb 3E11 completely protected suckling mice from a dose of 10 LD(50) of homologous virus challenge in vivo. Through a 12-mer random peptide phage display, synthetic peptide analysis and constructing a series of FMDV Asia1/YS/CHA/05 mutants using reverse genetic system, we finely mapped the neutralizing epitope as the 12-amino acid peptide (141)SXRGXLXXLXRR(152). These results provide additional insights into the virus-MAb interaction at the amino acid level and may help in the development of an epitope-based Asia1 FMDV vaccine.     

口蹄疫病毒亚洲1型TaqMan实时定量RT—PCR快速定型检测方法研究

采用TaqMan方法,根据口蹄疫病毒亚洲1型VP1的基因编码区基因(1D)序列,设计合成多对引物和多条探针,通过对引物、探针的筛选,反应条件的选择和优化,建立了口蹄疫病毒亚洲1型荧光RT-PCR检测技术,试验体系采用一对引物和两条探针配对,有效降低了由于变异导致的漏检率。经一系列的试验表明建立的荧光PCR检测技术快速、敏感、特异,检测时限3个小时以内,与口蹄疫病毒A型、O型和其它病毒不发生交叉反应,适用于样品中口蹄疫病毒亚洲1型的直接检测。口蹄疫病毒亚洲1型荧光PCR快速检测技术的建立,为我国养殖场口蹄疫亚洲1型疫情的快速诊断进而采取相应防制措施、减少疫情散播,以及动物产品中口蹄疫病毒亚洲1型的快速检测提供了一种可靠方法。     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A_93–143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD₅₀ (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.     

Asia I型口蹄疫疫苗效力检验替代方法的初步研究

使用三株针对AsiaⅠ型口蹄疫146S抗原的杂交瘤细胞株(1#G3C2、2#G6B9、10#C2G1)大量生产腹水单克隆抗体,粗提纯化并按一定的比例进行混合。初步建立混合腹水单克隆抗体介导的间接夹心ELISA方法,使用该方法测定AsiaⅠ型病毒灭活抗原、O型病毒灭活抗原、正常BHK21细胞培养液,结果表明该方法特异性好,所测定OD值和AsiaⅠ型抗原稀释倍数呈明显线性关系。用该方法对11批AsiaⅠ型、O型单价或双价成品疫苗破乳抗原进行测定,结果表明不同批次不同企业生产的疫苗完整病毒颗粒抗原含量有差异,进一步完善有望用于AsiaⅠ型口蹄疫成品疫苗中有效颗粒抗原含量的测定,从而发展成一种疫苗效力检验替代方法。     

ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立

猪口蹄疫免疫防御是控制猪口蹄疫疫情的重要手段,免疫水平指标通常用免疫抗体水平判定。对于其细胞免疫水平检测尚缺乏成熟可靠的技术,本试验利用市售ELISpot试剂盒建立猪口蹄疫特异γ干扰素检测方法,刺激物分别采用疫苗全病毒颗粒（O/Mya98/XJ/2010）、口蹄疫病毒T细胞表位多肽池,以植物血凝素（PHA）为阳性对照,对细胞浓度、刺激物浓度、孵育时间等进行优化。优化条件为：外周血PBMC新鲜提取或冻存细胞成活率90%以上,最佳细胞数为2×10⁵个/孔,最佳反应时间是16 h,病毒粒子146 S含量为100 ng/mL,多肽最佳浓度10 μg/mL,PHA的最佳浓度是20 μg/mL。ELISpot技术检测口蹄疫病毒感染猪γ干扰素方法的建立,为口蹄疫免疫力评价及口蹄疫疫苗免疫效果评估及进一步研究其与疫苗保护力（PD₅₀）的相关性奠定基础。     

非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定

旨在制备非洲猪瘟病毒(ASFV)p54蛋白的特异性单克隆抗体。本研究利用大肠杆菌表达系统表达p54蛋白,免疫BALB/c小鼠,取其脾细胞与SP2/0细胞进行细胞融合。利用纯化的p54蛋白作为包被抗原,采用间接ELISA方法筛选获得阳性杂交瘤细胞。经4次亚克隆后,取杂交瘤细胞上清进行单克隆抗体亚型鉴定,利用体内诱生法制备单克隆抗体并进行纯化。间接ELISA方法检测单克隆抗体的效价,利用交叉反应性试验、间接免疫荧光试验和蛋白印迹对所获单克隆抗体的特异性进行鉴定。根据预测的p54蛋白二级结构,采用逐步截短法分析鉴定单克隆抗体识别的抗原表位区域,并在p54的三级结构中进行标注。结果显示：成功筛选了6株分泌p54单克隆抗体的杂交瘤细胞,分别命名为28G12-1、31G7-1、31G7-2、35F10-1、35F10-2、38D3-1。其中28G12-1、31G7-1、31G7-2重链为IgG2a型,35F10-1、35F10-2、38D3-1重链为IgG1型;轻链均为κ链。单克隆抗体的最低效价为1∶25 600,与猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪流行性腹泻病毒、猪细小病毒、猪急性腹泻综...     

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies.

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.     

Production and characterization of monoclonal antibodies against an avian group A rotavirus.

Fifteen monoclonal antibodies (MAbs) against an avian group A rotavirus were cloned and characterized. Eight of the 15 MAbs had neutralizing activity (N-MAbs). Five of the N-MAbs (1G1, 5B8, 4E2, 3G1, 2E3) were VP4-specific by radioimmunoprecipitation assay (RIPA), and two N-MAbs (2D11, 6E8) were possibly VP7-specific (faint bands by RIPA). One N-MAb (4H12) of undefined protein specificity cross-reacted with serotype 3 simian rotaviruses. The other seven N-MAbs did not cross-react with any of the eight distinct serotypes of human and mammalian rotaviruses tested. Of the seven non-neutralizing MAbs, three were VP6-specific (3H10, 4B12, 5F6), two were VP8-specific (6C9, 1D1), one was VP4-specific (4E9), and one was of undefined protein specificity (1B11). Four non-neutralizing MAbs recognized only avian group A rotavirus in cell-culture immunofluorescence tests (6C9, 1D1, 4E9 and 5F6), whereas two MAbs (3H10 and 4B12) cross-reacted with all human and animal rotaviruses tested. The MAb 1B11 did not recognize any human rotavirus serotypes but cross-reacted with all nonhuman animal rotavirus serotypes. The MAbs produced in this study should be useful for the detection and further characterization of avian group A rotaviruses.     

北京地区犬瘟热病毒分离株H基因的分型

根据发表的犬瘟热病毒(CDV)参考毒株Onderstepoort的序列设计了一对引物,以感染犬瘟热病毒的犬的粪便总RNA为模板,RT-PCR扩增出17株CDV的H全基因1877bp的片段,将这个片段连接到pGEM-Teasy载体上,经酶切鉴定得到阳性克隆,将阳性质粒进行序列测定,并与国内外毒株进行同源性比较和系统发育分析,结果显示17株均为Asia-1型。北京地区流行的毒株主要是America-1型和Asia-1型,其中2007年主要流行America-1(疫苗型),而2008年主要是Asia-1型。     

悬浮培养在口蹄疫疫苗中的应用

通过生物反应器中进行BHK21细胞悬浮培养并逐级放大,分别接种口蹄疫OJMS/2000株与Asia 1/JSL株,纯化灭活后制备50批疫苗,结果均符合《口蹄疫O型、亚洲I型二价疫苗（OJMS株＋JSL株）制造及检验规程》（以下称规程）所规定的各项标准,病毒146S抗原含量比转瓶培养提高10倍以上、疫苗的不良反应得到进一步改善。     

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.     

O型、A型及Asia-1型口蹄疫病毒多重RT-PCR检测方法的建立

根据口蹄疫病毒VP1基因序列,利用Primer Premier 5.0软件设计4条特异性引物,通过对退火温度等反应条件进行优化,建立能够同时扩增出O型、A型、Asia-1型口蹄疫病毒的多重RT-PCR检测方法。结果表明,建立的方法最低可以检测出约含1 pg/μL的病毒样品;该方法对猪瘟病毒、猪细小病毒、猪伪狂犬病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒等其他相关病毒的检测结果均为阴性,特异性良好。建立的方法能够对口蹄疫O型、A型、Asia-1型病毒准确定型,可广泛用于口蹄疫病毒3个血清型的快速检测和分子流行病学调查。     

口蹄疫疫苗的悬浮培养工艺研究

通过生物反应器中进行BHK21细胞悬浮培养并逐级放大,分别接种口蹄疫OJMS/2000株与Asia 1/JSL株,纯化灭活后制备50批疫苗,结果均符合《口蹄疫O型、亚洲Ⅰ型二价疫苗(OJMS株+ JSL株)制造及检验规程》（以下称规程）所规定的各项标准,病毒146S抗原含量比转瓶培养提高10倍以上、疫苗的不良反应得到进一步改善.     

Production and Characterization of Monoclonal Antibodies to Peste des Petits Ruminants (PPR) Virus

Peste des petits ruminants (PPR) is an acute, febrile viral disease of small ruminants, caused by a virus of the genus Morbillivirus. PPR and rinderpest viruses are antigenically related and need to be differentiated serologically. In the present study, 23 mouse monoclonal antibodies were produced by polyethyleneglycol (PEG)-mediated fusion of sensitized lymphocytes and myeloma cells. Among these, two belong to the IgM class and the remaining 21 to various subclasses of IgG. The MAbs from the IgG class designated 4B6 and 4B11 neutralized PPR virus in vitro. In radioimmunoprecipitation assay, 10 MAbs recognized nucleoprotein, 4 recognized the matrix protein and one each haemagglutinin and phosphoprotein. The remaining 7 MAbs failed to precipitate any defined viral protein. The reactivity pattern of the monoclonal antibodies in indirect ELISA indicated a close antigenic relationship within three Indian PPR (lineage 4) virus isolates and also within two rinderpest vaccine strains. All PPR virus isolates could be distinguished from rinderpest vaccine viruses on the basis of the reactivity pattern of all MAbs and anti-N protein MAbs. A set of six monoclonal antibodies specific to PPR virus could also be identified from the panel. From the panel of MAbs available, two MAbs were selected for diagnostic applications, one each for the detection of antigens and antibodies to PPR virus.     

口蹄疫灭活疫苗抗原稳定性研究进展

口蹄疫是一种感染牛、羊和猪等偶蹄动物、具有高度传染性的动物疫病。疫苗免疫是控制该病的关键措施之一,口蹄疫灭活疫苗在口蹄疫流行地区广泛使用。灭活疫苗中,完整口蹄疫病毒粒子(146S粒子)是至关重要的免疫抗原,它的数量和稳定性决定了疫苗的免疫效果。不同血清型,甚至同型不同毒株的口蹄疫病毒粒子稳定性不同,146S粒子在一定温度、酸、碱条件下容易分解为五聚体(12S粒子),导致疫苗免疫效力大幅下降。近年来口蹄疫病毒结构及其稳定性的分子基础研究取得了一定进展,为研究口蹄疫病毒稳定性提高疫苗质量,开发新型口蹄疫空衣壳疫苗提供了重要的理论基础。本文简要介绍了口蹄疫病毒结构基础、稳定性研究方法和研究进展。为同行了解口蹄疫病毒结构与免疫的关系,评价新型疫苗提供一些参考信息。     

抗猪口蹄疫病毒单克隆抗体的抗原识别位点分析

用ELISA叠加试验，对5株具有ELISA反应特性的抗猪口蹄疫病毒(FMDV)单克隆抗体(McAb—A6、F9、G17、G52、S25)的抗原识别位点进行检测。抗体反应增值结果表明，5株单抗分别针对4个不同抗原住点，McAb—G17、G52、S25识剐的住点各不相同，其中McAb—G7、G52识别的位点有部分重叠，McAb—A6、F9识别同一位点，位于G17、G52位点的重叠区内。