Studies on the antigenic structure of Japanese encephalitis virus using monoclonal antibodies

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama-RFVL strain in hemagglutination-inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus-specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species-specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species-specific HI antibody, NARMA 6 is a Nakayama strain-specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain-specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme-linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species-specific HI site and the flavivirus genus-specific HI site are topologically distinct.     

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.     

Antigenic analysis of flaviviruses with monoclonal antibodies against Negishi virus

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.     

Inactivation and stability of viral diagnostic reagents treated by gamma radiation

The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 (Atomic Energy of Canada, Ltd., Ottawa, Canada) was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with greater than 2.4 Mrad radiation at temperatures above 4 degrees C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is reliable and effective replacement for BPL in preparing diagnostic reagents.     

Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.     

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the p _epitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012–2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6–93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years.     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备和鉴定

目的:获得分泌抗H9亚型禽流感病毒(AIV)血凝素单克隆抗体的杂交瘤细胞。方法:以H9N2亚型AIV为免疫原,免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14,在PEG4000的作用下进行细胞融合,通过血凝抑制(HI)试验筛选分泌抗H9亚型AIV血凝素单克隆抗体的杂交瘤细胞。结果:经过连续3～4次克隆化,获得能稳定分泌抗H9亚型AIV血凝素的单克隆抗体细胞系6株,分别命名为1B2、1C10、1G2、2B7、2E3和5E11。6株细胞培养上清HI效价为24～28,腹水HI效价为210～213。除1G2为IgM外,其余5株均为IgG1。Western blotting结果显示,1B2、1C10、2B7和2E3能与AIVH9蛋白在Mr为75000处反应,表明其是针对AIVH9亚型血凝素蛋白的单抗。特异性试验表明该6株单抗均只与H9亚型AIV发生特异性HI反应,而不与其他14个HA亚型的AIV及新城疫病毒、传染性支气管炎病毒发生交叉反应,显示出良好的特异性。结论:制备了针对H9亚型禽流感病毒血凝素的单克隆抗体,为禽流感的快速诊断和病毒的抗原性分析等奠定了基础。     

抗禽流感病毒多表位DNA疫苗的构建及其免疫效力研究

多表位DNA疫苗是建立在常规DNA疫苗基础上的一种新型疫苗。它是用表位作免疫原,这样就比较容易在一个表达载体上克隆病原体的多个抗原基因中具有免疫活性的部分。本试验以H5N1亚型禽流感病毒的HA和NP基因及其表位为基础构建了4个重组质粒：1 pIRES/HA（表达全长的HA基因）;2 pIRES/tHA（只表达HA基因的主要抗原表位区）;3 pIRES/tHANpep（融合表达HA基因的抗原表位区和NP基因的3个CTL表位）;4 pIRES/tHANpep-IFN-γ（用鸡的IFN-γ基因取代质粒pIRES/tHANpep中的neo基因）。分别用这4个重组质粒和空载体质粒pIRES1neo肌注免疫30日龄SPF鸡。免疫3次,间隔为2周,每次每只鸡的剂量为200μg。第3次免疫后两周以高致病性禽流感病毒H5N1强毒攻击,免疫及攻毒前后均采血检测HI抗体效价和外周血CD4⁺、CD8⁺T细胞的变化。结果发现,攻毒前各质粒免疫组均检测不到HI抗体,攻毒后1周存活鸡HI抗体效价迅速升高到64～256。流式细胞仪检测显示外周血CD4⁺、CD8⁺T细胞在疫苗免疫后都有不同程度的升高。空载体质粒对照组鸡（10只）在攻毒后3～8 d内全部死亡,其他各重组质粒免疫组鸡都获得了部分保护,保护率分别是：pIRES/HA组为545%（6/11）,pIRES/tHA组为30%（3/10）,pIRES/tHANPep组为36.3%（4/11）, pIRES/tHANPepIFNγ组为50%（5/10）。这些结果表明我们构建的多表位DNA疫苗能够诱导机体产生特异性免疫应答,并在同型禽流感强毒攻击时对鸡只提供了一定的保护。     

Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.     

A Single Amino Acid in the Stalk Region of the H1N1pdm Influenza Virus HA Protein Affects Viral Fusion,Stability and Infectivity

The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production.     

Elucidation of the topography and determination of the protective epitopes on the E glycoprotein of Saint Louis encephalitis virus by passive transfer with monoclonal antibodies

We have identified and characterized eight antigenic epitopes on the 53,000 dalton envelope (E) glycoprotein of Saint Louis encephalitis (SLE) virus by using monoclonal antibodies. One of these epitopes (E-1c) encoded for the type-specific biologic functions of hemagglutination (HA) and neutralization (N). Injection of 50 ng of anti-E-1c antibody protected the majority of mice from peripheral challenge with 100 i.p. LD50 of SLE virus. Similar levels of protection with antibodies specific for other epitopes usually required greater than or equal to 1000-fold additional antibody. Attempts to block N or protection at the E-1c antigenic domain by using antibody to several other SLE epitopes that strongly competed for the E-1c site were unsuccessful. Enhancement of protection was observed with mixtures of the more cross-reactive antibodies. The E-1c antibody was also effective in abrogating SLE virus replication until neural invasion occurred. On the basis of these findings, the topologic arrangement and function of the eight SLE E glycoprotein epitopes on the virion spike is proposed.     

整合HA蛋白的HIV假病毒展示禽流感病毒感染宿主细胞机制

通过将高致病性禽流感病毒HA蛋白整合到HIV颗粒,包装成表达HA蛋白的假病毒粒子（命名为HIV/H5-HA）,并对所包装的假病毒的生物学功能进行了研究.通过RT PCR获得了H5N1亚型禽流感病毒完整的血凝素基因(HA)并克隆到真核表达载体pcDNA3.1(+)上,通过与假病毒构建体系的2种质粒pCMV△8.2和pHR′-CMVLacZ共转染293T细胞,包装成假病毒颗粒.利用LacZ染色和HA假病毒颗粒感染MDCK等6种细胞株并对标记基因LacZ进行检测.结果表明,HIV/H5-HA与天然的禽流感病毒相似,具有广泛的细胞嗜性; Western 印迹和FACS检测结果,和HA假病毒颗粒的电镜照片确认了HA基因在假病毒颗粒表面得到了表达;HIV/H5-HA能够凝集鸡红细胞,并且pH值依赖性测定表明,HA假病毒需要低pH值才能实现正确的入侵宿主细胞.本研究结果显示：禽流感病毒H5N1亚型的HA基因得到了有效的包装,并且所包装的假病毒颗粒能够表达具有高度生物活性的HA蛋白.同时,假病毒模型的建立为进一步研究禽流感病毒与宿主之间的免疫应答提供了一种新的途径.     

Covalent inactivation of factor VIII antibodies from hemophilia A patients by an electrophilic FVIII Analog

The antigen-binding sites of antibodies (Abs) can express enzyme-like nucleophiles that react covalently with electrophilic compounds. We examined the irreversible and specific inactivation of antibodies (Abs) to Factor VIII (FVIII) responsible for failure of FVIII replacement therapy in hemophilia A (HA) patients. Electrophilic analogs of FVIII (E-FVIII) and its C2 domain (E-C2) were prepared by placing the strongly electrophilic phosphonate groups at surface-exposed Lys side chains of diverse antigenic epitopes. IgG Abs to FVIII from HA patients formed stable immune complexes with E-FVIII and E-C2 that were refractory to dissociation by SDS treatment and boiling, procedures that dissociate noncovalent Ab-antigen complexes. The rate-limiting step in the reaction was formation of the initial noncovalent complexes. Conversion of the initial complexes to the irreversible state occurred rapidly. The antigenic epitopes of E-FVIII were largely intact, and most of the Abs were consumed covalently. E-FVIII expressed poor FVIII cofactor activity in clotting factor assays. Nonspecific interference by E-FVIII in clotting factor function was not evident. Treatment with E-FVIII, and to a lesser extent E-C2, irreversibly relieved the FVIII inhibitory effect of HA IgG in clotting factor assays. Small FVIII peptides did not display useful reactivity, highlighting the diverse epitope specificities of the Abs and the conformational character of FVIII epitopes. E-FVIII is a prototype reagent able to attain irreversible and specific inactivation of pathogenic Abs.     

Monoclonal antibody-defined epitope map of expressed rubella virus protein domains.

An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.     

Conserved epitopes on the hemagglutinin-neuraminidase proteins of human and bovine parainfluenza type 3 viruses: nucleotide sequence analysis of variants selected with monoclonal antibodies.

We have previously identified 11 epitopes located in two topologically nonoverlapping antigenic sites (A and B) and a third bridging site (C) on the human type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) glycoprotein by using monoclonal antibodies (MAbs) which inhibit hemagglutination and virus infectivity (K. L. Coelingh, C. C. Winter, and B. R. Murphy, Virology 143:569-582, 1985). We have identified three additional antigenic sites (D, E, and F) on the HN molecule by competitive-binding assays of anti-HN MAbs which have no known biological activity. Epitopes in sites A, D, and F are conserved on the bovine PIV3 HN glycoprotein and also among a wide range of human isolates. The dideoxy method was used to identify nucleotide substitutions in the HN genes of antigenic variants selected with neutralizing MAbs representing epitopes in site A which are shared by human and bovine PIV3. The deduced amino acid substitutions in the variants were located in separate hydrophilic stretches of HN residues which are conserved in the primary structures of the HN proteins of both human and bovine PIV3 strains.     

Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion.

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles.     

Computational analysis of antigenic epitopes of avian influenza A (H7N9) viruses

Influenza virus can rapidly change its antigenicity, via mutation in the hemagglutinin (HA) protein, to evade host immunity. The emergence of the novel human-infecting avian H7N9 virus in China has caused widespread concern. However, evolution of the antigenicity of this virus is not well understood. Here, we inferred the antigenic epitopes of the HA protein from all H7 viruses, based on the five well-characterized HA epitopes of the human H3N2 virus. By comparing the two major H7 phylogenetic lineages, i.e., the Eurasian lineage and the North American lineage, we found that epitopes A and B are more frequently mutated in the Eurasian lineage, while epitopes B and C are more frequently mutated in the North American lineage. Furthermore, we found that the novel H7N9 virus (derived from the Eurasian lineage) isolated in China in the year 2013, contains six frequently mutated sites on epitopes that include site 135, which is located in the receptor binding domain. This indicates that the novel H7N9 virus that infects human may already have been subjected to gradual immune pressure and receptor-binding variation. Our results not only provide insights into the antigenic evolution of the H7 virus but may also help in the selection of suitable vaccine strains.