Antigenic features of foot-and-mouth disease virus serotype Asia1 as revealed by monoclonal antibodies and neutralization-escape mutants

Neutralizable antigenic sites/epitopes of serotype Asia1 foot-and-mouth disease virus (strain IND63/72) were identified using monoclonal antibodies (mabs) and their neutralization-escape mutants. Relative affinity/reactivity of the mabs for viral (both native and trypsin-cleaved) and subviral antigens in enzyme-linked immunosorbent assay (ELISA) showed dominance of trypsin-sensitive and conformation-dependent neutralizable antigenic sites. Characterization of neutralization escape mutants identified at least four independent trypsin-sensitive neutralizable antigenic sites on Asia1 FMD virus. One site was identified by mabs B3, 1A, 24, 2A, 40 and 63, second site by mabs 34 and 81, third site by mab 72 and fourth site by mab 89. The reaction profile of the mabs with selected field isolates in ELISA identified four different neutralization epitopes within the site B3/1A/24/2A/40/63.     

Characterization of anti-idiotypic antibodies generated against foot-and-mouth disease virus neutralizing monoclonal antibodies

A series of seven neutralizing monoclonal antibodies (nMAbs) against type A12 foot-and-mouth disease virus (FMDV) was used to induce polyclonal anti-idiotypic antibodies (anti-ids) in rabbits. The anti-ids were semi-purified through isotype affinity columns and assayed by solid-phase radioimmunoassay for cross-reactivity. nMAbs which map to the same epitope on the virion appear to contain a common idiotype, and the corresponding anti-ids competitively inhibited the virus-nMAb reaction. Using a modified ELISA assay, it was possible to demonstrate binding of purified anti-ids to FMDV susceptible tissue culture cells. Such antibodies however, did not interfere with the binding of virus to cells, and the binding of anti-ids to FMDV receptor-negative cells could also be demonstrated. Mice were inoculated with purified anti-ids, and two elicited anti-viral antibodies, although these antibodies were non-neutralizing. Thus anti-ids to anti-FMDV nMAbs failed to react with cellular receptors for the virus, but were able to induce anti-viral antibody and thus should be examined as an alternative vaccine strategy for this virus.     

抗MDV-1 VP22羧基端单克隆抗体的制备与免疫学特性鉴定

目的：制备抗血清Ⅰ型马立克氏病病毒（MDV-1）VP22的单克隆抗体（mAb），并鉴定其免疫学特性。方法：在原核系统中表达VP22羧基端区域（94～243aa），获得融合表达产物GST-VP22C。将该表达产物切胶免疫小鼠，利用杂交瘤技术制备抗MDV-1VP22C的mAb，并通过ELISA、间接免疫荧光（IFA）、Western blot鉴定其特性。结果：获得了2株可稳定分泌抗MDV-1VP22C的mAb的杂交瘤细胞，命名为3F7、4FA。IFA鉴定表明，两株mAb均能与感染MDV-1的成纤维细胞反应，其中，3F7mAb染色呈现MDV空斑，而4FAmAb呈现整个单层的细胞核荧光。ELISA和Westernblot分析表明，3F7能与杆状病毒表达的VP22反应，4FA不具备该特性。对3F7mAb进一步鉴定，确定了该mAb针对的具体位置在94～193aa处，是蛋白转导域的预测位置。结论：成功地制备了抗MDV-1VP22C的mAb，为深入研究VP22蛋白的转导功能提供了有用的试剂。     

应用噬菌体展示技术筛选、鉴定抗汉滩病毒单克隆抗体的模拟表位

目的 :应用噬菌体 12肽文库筛选抗汉滩病毒单抗 (mAb)F3、B11的模拟配体肽 ,并对其免疫学特性进行初步分析。方法 :以纯化的mAb为筛选配基 ,进行噬菌体肽库的生物亲和淘选。用ELISA法鉴定筛选克隆的结合活性 ,对阳性克隆进行序列测定和分析。用动物免疫试验初步分析噬菌体颗粒展示的抗原肽的免疫特性。结果 :通过 3～ 4轮生物淘选 ,ELISA显示筛选到的多数噬菌体克隆均可与mAb特异性结合 ;与mAbF3结合的阳性克隆的氨基酸序列高度一致 ,均为 MHGP TKNQMWHT ,与HTNV/SEOVM蛋白G2区第 75 0～ 75 9位氨基酸具有较高的同源性 ;而mAbB11特异性的结合肽在氨基酸水平上表现出一定的多态性 ,其序列的基序尚未能确定 ;动物免疫试验表明 ,噬菌体肽抗原具有良好的免疫反应性和免疫原性 ,是天然病毒抗原较好的免疫原模拟物。结论 :获得了具有良好结合活性的模拟表位肽 ,为基于表位的HFRSV多肽疫苗及DNA疫苗的研制奠定了基础。     

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.     

Isolation of therapeutic human monoclonal antibodies for varicella-zoster virus and the effect of light chains on the neutralizing activity

Therapeutic antibodies against varicella-zoster virus (VZV) were isolated from a combinatorial library of human antibodies using a phage-display system. Purified gH:gL was used to screen the library, and approximately 300 clones were isolated. Eight kinds of Fab-cp3-fused molecules (clones 10, 24, 36, 60, 94, 120, 192, and 431) neutralized viral infectivity. After conversion of Fab-cp3 antibodies to the Fab-protein A form, the concentrations of antibodies showing 50% inhibition of plaque formation ranged from 0.12 to 400 nM. Clones 10, 24, 94, 120 and 431 neutralized wild strains without showing strain specificity and were further converted to human IgG(1). Two clones (24 and 94) were confirmed to react with gH:gL and VZV-infected cells. IgG of clone 94 prevented spreading of infected cells. Thus these antibodies exhibited the typical phenotype of anti-gH antibody. Next the contribution of light (L) chains to neutralizing activity was analyzed by comparing the effect of L chain of clones 10, 120, and 192 with the identical heavy chain on their neutralizing activity. The L chain in the Fab form of clone 94 was replaced by L chains of clones 10, 24, 36, and 60 and the neutralizing activity of these replaced antibodies was weaker than that of the prototype clone 94. When the kappa-L chain of clone 94 was replaced by the lambda-L chain of clone 24, this antibody possessed neutralizing activity despite the kappa-lambda class change. Thus, human antibody library against VZV-gH has been established and characterized the role of the L chain in VZV-neutralizing activity to engineering further an antibody with stronger neutralizing activity.     

Permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the V3 loop of HIV-1

Neutralizing monoclonal antibodies (mAb) specific for the third variable (V3) domain of gp120, the HIV-1 surface envelope protein, are mainly isolate specific. We have studied the composition and the permissivity of the minimal epitopes interacting with two of these, the 110-A and 19.26.4 mAb, which are strictly LAI isolate specific. Screening a hexapeptide phage library displayed on the surface of filamentous phage with the 110-A mAb has allowed selection of 49 phage sequences, permitting the definition of a consensus sequence. Based on this sequence, substituted synthetic peptides were prepared and used in binding assays. Our results show that both mAb interact with the same narrow region (316–320) of the V3 domain. The minimal epitope of the 110-A mAb was identified as a five amino acid sequence, Hy x R G p, where Hy represents any non-aromatic hydrophobic amino acid. By contrast, the minimal epitope of the 19.26.4 mAb was identified as x Q Pos G P, where Pos is any positively charged amino acid. Core residues of the epitope, critical for the binding to the mAb (written in uppercase letters), were set apart from permissive amino acid positions that tolerate substitutions (written in lowercase letters). Interestingly, the identified core residues Q_2/317 (19.26.4 mAb) and R_3/318 (110-A mAb) do not tolerate substitution and correspond to the QR insertion in the V3 domain, characteristic of the LAI isolate as compared to other isolates. This result may explain the strict isolate specificity of most anti-V3 LAI mAb. The two epitopes have totally different patterns of permissivity; thus, the effect of substitutions will differ depending on the mAb involved in the interaction. This suggests that the diversity of the antibody response is high enough to delay the emergence of HIV-1 variants resistant to neutralization by V3-specific antibodies.     

Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C

Abstract

Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150?kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity.     

Preparation and characterization of three monoclonal antibodies against HIV-1 p24 capsid protein

HIV-1 p24 detection provides a means to aid the early diagnosis of HIV-1 infection, track the progression of disease and assess the efficacy of antiretroviral therapy. In the present study, three monoclonal antibodies （mAbs） p3JB9, p5F1 and p6F4 against HIV-1 p24 were generated. All mAbs could detect p24 of HIV-1ⅢB, HIV-1Ada-M, HIV-174v mAbs p5F1 and p6F4 could detect HIV-1KM018, while p3JB9 could not. Three mAbs did not react with HIV-2ROD, HIV-2CBL-20 and SIVagmTYO-1. The recognized epitope of p5F1 was located on the Gag amino acid region DCKTILKALGPAATLEEMMTAC. The p5F1 was used to establish a modified sandwich ELISA with rabbit anti-p24 serum and showed good specificity and high sensitivity, which has been used to measure HIV-1 p24 antigen levels in research. Cellular ＆ Molecular Immunology.     

H1亚型流感病毒HA蛋白单克隆抗体的制备及部分特性鉴定

目的:制备针对H1亚型流感病毒HA蛋白的单克隆抗体(mAb),并分析其反应特性。方法:分别以2009年甲型H1N1、季节性A1流感病毒裂解疫苗为免疫原,常规法免疫、融合、克隆化,获得各抗原特异性mAb。应用ELISA、HI试验和Western blot等技术研究mAb的反应性和特异性。结果:获得稳定分泌抗H1亚型流感病毒HA蛋白的杂交瘤细胞97株。其中株特异性mAb39株,29株具有HI活性;亚型特异性mAb7株,5株具有HI活性;2009年流行株与季节性A1、A3流行株共同抗原的mAb16株,9株具有HI活性;针对流感病毒共同抗原mAb35株,22株具有HI活性。结论:两种疫苗均具有较好的免疫原性和免疫保护活性,这些mAb的获得为流感病毒株特异、亚型特异性诊断试剂盒及流感病毒通用诊断试剂盒的制备提供了实验资料,为进一步研究H1N1流感病毒HA的抗原表位奠定了基础。     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Antigenic variation of the Epstein–Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

The Epstein–Barr virus (EBV) nuclear antigen EBNA1 plays an essential role in the replication of EBV episomes in latently infected cells and is the only viral protein that is consistently expressed in all programs of latent EBV gene expression. In this study, four monoclonal antibodies (MoAbs) directed to a region (amino acid residues 442–530) of EBNA1 were generated. Competitive enzyme-linked immunosorbent assay (ELISA) experiments using biotinylated MoAbs showed that they recognized distinct epitopes. Reactivity of these MoAbs with various laboratory EBV strains and field EBV isolates was shown to be heterogeneous in that EBNA1 from certain strains (isolates) was recognized and that from others was not. All four MoAbs showed such heterogeneous reactivity, and moreover, each MoAb showed a distinct spectrum of reactivity with these EBV strains (isolates). These results demonstrate an extensive structural variation in this region of EBNA1 as predicted by previous sequencing studies. These MoAbs will be useful as probes to dissect this structural heterogeneity of EBNA1.     

New polymorphic HLA-DR epitopes recognized by three monoclonal antibodies produced against DR103 transfected L cells

Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non-MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103-transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA-DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B-lymphoblastoid cell lines (B-LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA-DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B-LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA-DR products and react in western blots with DR alpha/beta-dimer but not with free alpha- or beta-chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.     

抗肠道病毒EV71型外壳蛋白VP1单克隆抗体的制备和鉴定

目的 制备具有中和活性的抗肠道病毒EV71型外壳蛋白VP1的单克隆抗体.方法 人工合成SP55和SP70(分别包含VP1的第163-177,208-222位氨基酸)两段VP1的多肽,分别免疫BALB/c小鼠,常规杂交瘤技术进行细胞融合,用间接酶联免疫吸附试验(ELISA)筛选阳性杂交瘤细胞并测定效价.用分泌的单抗和EV71病毒在RD细胞上进行中和试验以检验其中和活性.结果 得到2株能稳定分泌抗肠道病毒EV71型VP1蛋白单克隆抗体的杂交瘤细胞株,2株单抗的中和效价分别为1:8和1:16.结论 成功制备出2株具有中和活性的抗肠道病毒EV71型VP1蛋白单克隆抗体,为其下一步应用打下基础.     

用噬菌体肽库筛选IBDV单克隆抗体的模拟表位

目的:筛选法氏囊病毒(IBDV)单克隆抗体所针对的模拟抗原表位。方法:以纯化的3株IBDV单克隆抗体为靶分子用噬菌体12肽库筛选相应抗原模拟表位;应用间接和竞争ELISA鉴定所筛选的阳性样品;最后对与抗体高亲和结合的噬菌体进行测序分析。结果:经过四轮淘洗,随机挑取30个克隆经间接ELISA鉴定,发现其中22个克隆结合活性较高。进一步应用竞争ELISA,获得14个噬菌体克隆,其抑制率高达50%以上。对这些噬菌体进行DNA测序,并分析比对了IBDV相应序列,确定了3个抗原模拟表位。结论:通过噬菌体随机肽库成功筛选出3个IBDV模拟表位,为进一步研究IBDV抗原性质奠定了基础。     

Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes

The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein–Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases.     

Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis A virus infection in vitro and in vivo

Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.     

O型口蹄疫病毒VP1蛋白在大肠埃希菌中的可溶性表达、纯化及纳米样结构观察

目的本实验旨在高效表达可溶性的O型口蹄疫病毒VP1蛋白,并形成纳米样颗粒。方法根据O型口蹄疫病毒核酸序列,得到FMDV O/MYA/7/98株的VP1蛋白基因,并进行截短和优化,共73个氨基酸;同时,从肠道沙门菌(Salmonella enterica)中分离得到135个氨基酸铁蛋白(Fn)基因片段,将O型口蹄疫病毒VP1蛋白与铁蛋白串联,设计并合成了口蹄疫病毒VP1蛋白-铁蛋白基因片段,命名为Se Fnt16798。构建了Se Fnt16798融合Grifin、GST、MBP、Sumo、Thioredoxin、γ-crystallin、Ars C、Ppi B、Ce HSP17等9种不同可溶性标签的表达重组载体。分别转化至大肠埃希菌BL21(DE3)中,异丙基硫代半乳糖苷(IPTG)诱导,SDS-PAGE电泳对融合蛋白的可溶性表达进行检测,筛选高效可溶性表达的Se Fnt16798融合蛋白。重组蛋白通过Ni-NTA Agarose亲和纯化,进行电子显微镜检测。结果实验成功构建9个Se Fnt16798表达载体;9个标签中,MBP与Se Fnt16798蛋白相融合的可溶性表达效果最好,并获得了高纯度的MBP-Se Fnt16798重组蛋白质;电子显微镜结果显示,MBP-Se Fnt16798形成了纳米样颗粒。结论本实验建立了稳定获得Se Fnt16798重组蛋白质的方法。