Newly attenuated Mycobacterium bovis mutants as vaccines against bovine tuberculosis, particularly for possums

Cattle vaccinated with a single dose of subunit vaccine containing the capsid and 3C proteinase coding regions of foot-and-mouth disease virus (FMDV) Asia I/HNK/CHA/05 strain were protected when challenged 28 days later with a homologous virus. Here, the 50% bovine protective dose (PD(50)) test was performed to assess the potency of the subunit vaccine. When challenged with two Chinese isolates, the subunit vaccine could achieve 6.5 PD(50) (challenged with Asia I/HNK/CHA/05 strain) and 5.2 PD(50) (challenged with Asia I/JSL/05 strain) per dose.     

Genome analysis and development of infectious cDNA clone of a virulence-attenuated strain of foot-and-mouth disease virus type Asia 1 from China

The RNA genome sequence of the rabbit passage-attenuated strain of foot-and-mouth disease virus (FMDV) Asia 1, ZB/CHA/58(att), was determined to be 8165 nt in length excluding the poly(C) tract in the 5′ UTR and the poly(A) tail at the 3′ end. ZB/CHA/58(att) was most similar to the vaccine strain Asia 1/YNBS/58 in genome sequence and there were no deletions or insertions within the deduced polyprotein between ZB/CHA/58(att) and YNBS/58, but there were a total of 25 substitutions at the amino acid level and an extra 19-nt stretch in the 5′ UTR was found in ZB/CHA/58(att). An infectious full-length cDNA clone of ZB/CHA/58(att) was developed. Infectious virus could be recovered in BHK-21 cells transfected with the synthetic viral RNA transcribed in vitro. The plaque morphology, growth kinetics and antigenic profile of the infectious clone-derived virus (termed tZB) were indistinguishable from those induced by the parental virus. Furthermore, the virulence properties of ZB/CHA/58(att) and tZB were found to be highly similar in the mouse model. The availability of genome sequence information and infectious cDNA clone of the FMDV ZB/CHA/58(att) lays a new ground for further investigation of FMDV virulence determinants and development of new potent vaccine to FMD.     

Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.     

A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response, and an immunostimulatory peptide

The epitopes of the capsid of foot-and-mouth disease virus (FMDV) play important roles in the construction of highly immunogenic subunit vaccines. However few epitopes have been found for FMDV serotype Asia1. In this study we screened for epitopes of the VP1 and VP2 proteins of FMDV serotype Asia1 isolate, YNBS/58. Fragments consisting of amino acids 133-163 of VP1 and amino acids 1-33 of VP2 contained epitopes, and both induced lymphoproliferation in guinea pigs. Only the VP1 fragment induced neutralizing antibodies but the VP2 peptide dramatically increased the neutralizing antibody response induced by the VP1 peptide.     

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.     

Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C)

Epitope-based vaccines are always questioned for their cross-protection against the antigenically variable foot-and-mouth disease virus (FMDV). In this study, we proved the cross-protection effect of a multi-epitope vaccine incorporated with poly(I:C) against three topotypes of O type FMDV. A total of 45 naïve pigs were vaccinated with different doses of multi-epitope protein vaccine incorporated with poly(I:C). At 28 days post-vaccination, 45 vaccinated and 6 unvaccinated control pigs (two pigs for each group) were challenged with three topotypes of virulent O type FMDV, namely, O/Mya/98 (Southeast Asia topotype), O/HN/CHA/93 (Cathay topotype) and O/Tibet/CHA/99 (PanAsia topotype) strains. All unvaccinated pigs developed generalised FMD clinical signs. Results showed that all pigs (n = 15) conferred complete protection against the O/Mya/98 and O/HN/CHA/93 FMDV strains, 11 of which were protected against the O/Tibet/CHA/99 FMDV strain. The 50% protective dose values of the vaccine against the O/Mya/98, O/HN/CHA/93 and O/Tibet/CHA/99 FMDV strains were 15.59, 15.59 and 7.05, respectively. Contact challenge experiment showed that transmission occurred from the donors to the unvaccinated but not to vaccinated pigs. These results showed that vaccination with multi-epitope protein vaccine incorporated with poly(I:C) can efficiently prevent FMD in pigs.     

鸭坦布苏病毒E蛋白具有独特交叉反应性中和表位的鉴定

利用抗鸭坦布苏病毒(DTMUV)中和单抗1G2,结合E蛋白多肽扫描技术,鉴定了最小抗原表位²²⁷GSSAGTWQN²³⁵。Western blot显示,残基²³¹G或²³³W的突变后,表位完全失去了与1G2抗体识别的功能,表明²³¹G或²³³W是抗体1G2识别的关键氨基酸位点。通过免疫荧光分析(IFA)显示,MAb 1G2与JEV、WNV和ZIKV的E蛋白发生交叉反应,表明该表位是黄病毒的交叉反应性表位。蛋白质和病毒模型显示,表位定位于成熟病毒粒子可接近的表面,在E蛋白结构域Ⅱ的hi环中。本研究首次证明,中和抗体1G2靶向交叉反应表位定位在E蛋白结构域Ⅱ的hi环,该表位的鉴定有助于提高对黄病毒血清诊断中存在交叉反应问题的认识和理解。     

Induction of protective immune response against both PPRV and FMDV by a novel recombinant PPRV expressing FMDV VP1

Peste des petits ruminants (PPR) and foot-and-mouth disease (FMD) are both highly contagious diseases of small domestic and wild ruminants caused by the PPR virus (PPRV) and the FMD virus (FMDV). In this study, a recombinant PPRV expressing the FMDV VP1 gene (rPPRV/VP1) was generated and FMDV VP1 expression did not impair replication of the recombinant virus in vitro and immunogenicity in inducing neutralizing antibody against PPR in goats. Vaccination with one dose of rPPRV/VP1 induced FMDV neutralizing antibody in goats and protected them from challenge with virulent FMDV. Our results suggest that the recombinant PPRV expressing the FMDV VP1 protein is a potential dual live vectored vaccine against PPRV and FMDV.     

Kinetics of immune response to foot-and-mouth disease virus (type Asia 1) in experimental cattle

Humoral and mucosal (secretory antibody)immune response to FMDV type Asia 1 in cattle was analyzed after vaccination and infection using virus neutralizing test (VNT). Vaccination (1/16th the usual dose) failed to protect cattle from generalized clinical disease following experimental FMDV Asia 1 infection. Our results showed that infection induced higher and prolonged serum antibody titres indicating antigen mass is important for optimal immune response. Experimental FMDV infection induced significant secretory antibody (mucosal) response in cattle. Though, there was no difference in the serum antibody response between the cattle that developed generalized infection (unprotected) and those with only localized infection (protected), secretory antibody response differed, wherein the unprotected cattle had higher secretory response than protected cattle. Thus, FMDV Asia 1 infection stimulates a similar serum antibody response and a unique secretory antibody response among the infected cattle. An erratum to this article can be found at     

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.     

A common neutralizing epitope on envelope glycoprotein E2 of different pestiviruses: implications for improvement of vaccines and diagnostics for classical swine fever (CSF)?

The Pestivirus genus within the family of Flaviviridae consists of at least three species; classical swine fever virus (CSFV) found in swine and wild boar, bovine viral diarrhoea virus type 1 and type 2 (BVDV-I and BVDV-II) mainly isolated from cattle and border disease virus (BDV) preferably replicating in ovine species. Many features demonstrate differences between CSFV and other pestiviruses, BVDV-I, BVDV-II and BDV, here defined as nonCSFV, whereas other features show similarities between all different species of pestiviruses. Focussing on the major envelope glycoprotein E2, the immunodominant protein of pestiviruses, CSFV seems to be a more distinct species within the Pestivirus genus. Here we confirm on one hand the more separated grouping of CSFV by isolation of monoclonal antibodies (MAbs) raised against E2 of BVDV-I and BVDV-II. None of these MAbs recognize E2 of CSFV strains. On the other hand, only one MAb, MAb 912, was isolated against E2 of BDV. MAb 912 binds to E2 of CSFV strains and partly neutralizes CSFV. The epitope of MAb 912 is mapped in antigenic domain B of CSFV-E2. This common epitope of CSFV strains and nonCSFV strains could have implications for development of DIVA vaccines and serological diagnostics for CSF.     

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。     

O型口蹄疫病毒结构蛋白VP1的原核表达与抗原性分析

研究分析了O型口蹄疫病毒(FMDV)结构蛋白VP1与当前猪FMDV疫苗血清的免疫反应性.将VP1基因克隆至原核表达载体pET32c,并在大肠埃希菌BL21中得到了表达,Western blot分析表明该重组蛋白与豚鼠O型FMDV标准阳性血清具有良好免疫反应性.目的蛋白经纯化后用ELISA分析其与猪疫苗血清的免疫反应性,结果显示该重组VP1蛋白(rVP1)只能与部分O型FMDV疫苗血清反应.推测当前使用的不同O型FMDV疫苗毒株在VP1重要中和抗原位点G-H环(134 aa～158 aa)与C末端(200 aa～213 aa)存在较大差异.     

Specific antibody responses and generation of antigenic variants in chickens immunized against a virulent avian influenza virus.

To examine the specificity of the antibody response to the influenza hemagglutinin and the generation of antigenic variants, chickens were immunized against the highly virulent H5 virus A/Ty/Ont/7732/66 (H5N9) and then challenged with a lethal dose of the virus. The antibody responses of these chickens to the hemagglutinin (HA) were examined with an enzyme-linked immunosorbent assay (ELISA) in which their sera were titrated for the ability to block the binding of monoclonal antibodies (MAbs) to five distinct neutralizing epitopes on the viral HA. Based on the ELISA results, a majority (5/6) of the chickens produced antibodies to three of the five neutralizing epitopes on the viral HA. After challenge, two of six immunized chickens shed virus and died; antigenic comparisons of isolates from these two chickens indicated the presence of an antigenic variant; i.e., there was a change in one neutralizing epitope on the HA of virus shed by one chicken. None of the chickens had produced antibodies to this particular epitope on the viral HA. Inoculation of chickens with this variant resulted in 100% mortality, demonstrating that a change in this particular epitope did not alter the virulence of the virus. These studies indicate that chickens immunized against highly virulent influenza viruses may excrete virulent variants following challenge with live virus.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs,swine and cattle

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD₅₀) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法.豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验.豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平.统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极...     

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.     

口蹄疫O、Asia1分型彩色胶乳试纸条诊断方法的建立

为建立一种快速、简便、直观的口蹄疫O、Asia 1分型彩色胶乳免疫层析的方法,作者选择2种不同颜色的单分散胶乳,将纯化的Asia 1/China/05流行毒株兔抗体制备蓝色免疫胶乳;将纯化的O/China/99流行毒株兔抗体制备红色免疫胶乳.将2种免疫胶乳分别喷涂于不同玻璃纤维上,晾干叠加制成彩色胶乳反应垫.再分别将纯化的Asia 1/YN/BSh/58/B和AV99(L)标准毒株的豚鼠抗体固定于同一硝酸纤维素膜的不同区域上作为2条检测带.最后组装成口蹄疫O、Asia 1分型彩色胶乳试纸条.通过对阳性参考样品的检测,结果显示试纸条有很好的灵敏度,可检测到病毒含量为3.90×104LD50;在检测与口蹄疫临床症状相似病原,如猪水泡病病毒(SVDV)、水泡性口炎(VS)、水泡性疹(VES)抗原时无交叉反应,特异性好;不同批次间、同一批次内的试纸条,在检测阴、阳性参考样品时,结果完全一致,说明试纸条重复性好;试纸条对检测已知血清型的74份田间样品的符合性试验中,O型、Asia 1型样品的阳性符合率分别为95.24%、96.30%,阴性样品符合率为100%.本试验研发的口蹄疫O、Asia 1分型彩色胶乳试纸条具有很好的灵敏度、特异性、重复性和符合性,而且可以通过不同颜色进行定型检测,适合国内流行的口蹄疫病原型别的分型检测.