Monoclonal antibodies to Marburg virus proteins and their immunochemical characteristics]

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus proteins are prepared. Positive hybridomas were selected by solid-phase enzyme immunoassay (EIA) by specificity of their immunoglobulin reaction with Marburg virus antigen. Virus proteins reacting with MAbs were identified by immunoblotting. Out of 20 examined hybridoma antibodies, 5 reacted with protein VP35, 5 with VP40, 3 with NP, 1 with protein complex VP35-VP40, MAb 7H10 detected 2 proteins (VP40 and NP), and 5 MAbs did not bind virus proteins in this assay. Marburg virus antigen adsorbed on the surface of plates were detected by indirect EIA with biotin-treated MAbs (PEIA-MAb) and indirect EIA (IEIA-MAb). The sensitivity of both methods differed with different hybridoma antibodies and was the maximum with MAb 5F1 specific to Marburg virus nucleoprotein: 5-10 and 1-2 ng/ml for the direct and indirect methods, respectively. Purified MAbs 7C4, 7D8, and 5F1 were used as antigen captures in EIA for detecting immunoglobulins to Marburg virus in a serum from convalescent after Marburg fever. The results recommend the above MAbs for use in test systems for the diagnosis of the disease and detecting virus antigen.     

人源抗EV71病毒基因工程抗体的研究

目的 研制人源抗EV71病毒基因工程抗体.方法 采集多个患儿外周血淋巴细胞,构建人源抗EV71单链（scFv）噬菌体抗体基因文库.用纯化的EV71 VPI蛋白对抗体库进行筛选,将筛出抗体的轻链和重链通过序列测定确定抗体轻重链型别后分别克隆入全抗体表达载体pAC-LCH3R后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.结果 成功地获得了1株抗EV71病毒VPI蛋白的人源单克隆抗体,利用ELISA、IFA对所获人源单克隆抗体的功能特性进行鉴定,表达成分泌型IgG全抗体在体外与A型及C4亚型EV71病毒的中和反应结果 显示为阴性.结论 从免疫库中获得了1株人源非中和单抗,为EV71病毒引起的手足口病的鉴别诊断奠定了基础.     

Antigenic structure of Ebola virus VP35 protein]

Antigenic structure of Ebola virus (EV) (strain Mayinga) nucleocapsid protein VP35 was analyzed using monoclonal antibodies to EV VP35 and polyclonal antibodies to EV. EV protein VP35 was shown to have antigenic sites inducing the production of antibodies in animals. For better characterization of protein VP35 antigenic structure. EV gene encoding the full-length VP35 was cloned in vector pQE31 as a recombinant fusion protein (rec.VP35). The antigenic and immunogenic properties of rec.VP35 and EV VP35 were compared by ELISA and Western blot analysis with polyclonal and monoclonal antibodies. Antibodies of positive sera and VP35 MAbs cross reacted with the analyzed antigens. The topography of epitopes on EV VP35 and rec.VP35 was studied using MAbs and polyclonal antibodies to rec.VP35 in a competitive antibody binding assay. Two epitopes of one site were identified on these proteins. These epitopes are present on infectious virion protein VP35 and are stable during physicochemical exposures.     

Detection of immunoglobulin G, M, and A antibodies to enterovirus structural proteins by immunoblot technique in echovirus type 4-infected patients

Paired serum specimens from 24 patients with echovirus (EV) type 4 infection by virus isolation were tested by the immunoblot technique for the presence of IgG, IgM, and IgA antibodies to EV4 structural proteins. Single sera from 20 patients without neutralizing enterovirus IgM were used as controls. All the sera from EV4-infected patients had IgG antibodies to VP1 of EV4 but also 13 out of the 20 controls. 23 out of 24 EV4-infected patients elicited IgM and IgA specific antibodies to VP1, a pattern highly significant as compared with controls (3/20 for IgM and 8/20 for IgA). In 16 out of the 24 EV4-infected patients, the IgM antibodies were also directed against VP2 (versus 2 out of 20 in the control group). Anti-VP2 IgA were detected in 4 out of the 24 EV4 patients (versus 0 in controls). The 24 paired sera from EV4-infected subjects were also tested by immunoblot technique against three other enteroviruses: EV21, coxsackievirus A9 and poliovirus 1. Cross-reactivities were observed to a large extent against VP1 and VP2 proteins with the three classes of antibodies. These results confirm the data of previous studies on the reactivity of IgM antibodies to various structural proteins that IgG antibodies react exclusively to VP1. Furthermore, this study demonstrates the occurrence of circulating IgA antibodies directed to VP1 and sometimes VP2 in the course of enterovirus infection. The potential interest of this latter finding for diagnosis requires further investigation.     

Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus

A comparison of antibody titers to JC virus (JCV) or BK virus (BKV) was made by hemagglutination inhibition (HI) and enzyme immunoassay (EIA) with 114 human plasma samples. Antibody titers to JCV or BKV determined by HI were lower than those determined by EIA. Nevertheless, as HI titers increased so did EIA titers. When antibody data were compared by the Spearman rank correlation test, highly significant correlations were found between HI and EIA titers. Results obtained by plotting EIA antibody titers for JCV against those for BKV generally showed a reciprocal relationship, i.e., samples with high antibody titers to JCV had lower antibody titers to BKV and vice versa. Some samples, however, had antibody titers to both viruses. Of the samples tested, 25.4% (25 of 114) had HI and EIA antibody titers to JCV and BKV which were identical or closely related. This is not the scenario one would expect for cross-reactive epitopes shared by the two viruses, but one suggesting that these samples were from individuals who had experienced infections by both viruses. Adsorption with concentrated JCV or BKV antigen of sera with high antibody titers to both JCV and BKV and testing by JCV and BKV EIA gave results which support this conclusion. Although 52.6% (51 of 97) of the samples from the Japanese population tested had very high antibody titers (>/=40,960) to either JCV or BKV, none of the samples were found by a dot blot immunoassay to have antibodies which cross-reacted with simian virus 40. The results from this study, in agreement with those of others, suggest that humans infected by JCV or BKV produce antibodies to species-specific epitopes on their VP1 capsid protein, which is associated with hemagglutination and cellular binding.     

Location of neutralizing epitopes defined by monoclonal antibodies generated against the outer capsid polypeptide, VP1, of foot-and-mouth disease virus A12

The epitopes of six monoclonal antibodies generated against type A12 foot-and-mouth disease virus (FMDV) VP1 or its largest cyanogen bromide fragment (13 kd) were characterized. Five of these monoclonal antibodies neutralized viral infectivity. Solid-phase and competitive antigen binding assays using virion-derived antigens or a biosynthetic VP1 polypeptide identified two distinct neutralizing epitopes. One epitope was located between amino acid residues 145-168 of VP1 and the other between amino acids 169-179. The results indicate that antibodies reacting with two distinct areas of the VP1 polypeptide are capable of neutralizing FMD virus.     

Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization

Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. Rabbit sera selected from positive AGID test data were used to standardize the method. Briefly, wells of flexible microtitre plates coated with antigen were incubated with antiserum followed by a secondary labelled antibody. The radioactivity remaining in the wells after washing provided a measure of the amount of specific antibodies in the serum. When testing a group of rabbit sera, negative for EIA virus antibodies by the AGID test, in the SPRIA a range of positive reactivities was noted. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody.     

Development of a quantitative enzyme linked immunosorbent assay for monitoring the Enterovirus 71 vaccine manufacturing process

Enterovirus 71 (EV71), the etiologic agent causes outbreaks with significant mortality in young children in Asia and currently there is no vaccine available. In this study, we report a quantitative enzyme linked immunosorbent assay (Q-ELISA) to determine the concentration of the EV71 VP2 antigen. EV71 virus-like particles (VLPs) were produced in the baculovirus expression system and used as the EV71 antigen reference standard. Antisera from both EV71-immunized chickens and rabbits were very efficient and useful as capture antibodies to bind various forms of EV71 antigens, whereas a commercial VP2-specific virus neutralizing monoclonal antibody MAB979 was found to be suitable for quantifying the amount of VP2 antigen. This Q-ELISA was used successfully to determine VP2 content at each stage of EV71 vaccine manufacturing process, particularly during the upstream harvest, downstream purification and viral inactivation steps. The amount of VP2 antigen and the magnitude of neutralizing titers were found to be dose-dependent in mice immunized with vaccine candidates. These results indicate that Q-ELISA could provide off-line timely quantitative measurements of VP2 antigen throughout the production cycle to evaluate critical attributes and conditions that may affect virus yields in culture media, the quality of purification methods, the stability and potency of final vaccine formulations.     

Multiple amino acid substitutions in the HN protein of the paramyxovirus,SV 5, are selected for in monoclonal antibody resistant mutants

Summary Monoclonal antibody resistant (MAR) mutants (which escaped antibody-mediated neutralization) were selected from simian (W 3) and human (LN) isolates of simian virus 5 (SV 5), using monoclonal antibodies (MAbs) specific for antigenic sites 4 and 5 on the HN glycoprotein. Resistance correlated with an inability of the selecting antibody to bind with the respective MAR mutants. Sequence comparisons between parental and mutant HN proteins revealed multiple non-adjacent amino acid substitutions in the majority of MAR mutants. The same multiple substitutions were identified in mutants selected from both the LN and W 3 isolates of SV 5. Furthermore, different mutations on the primary sequence of the HN protein conferred resistance to the same MAb.     

Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs

Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein's upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.     

Comparison of a baculovirus-based VP2 enzyme immunoassay (EIA) to an Escherichia coli-based VP1 EIA for detection of human parvovirus B19 immunoglobulin M and immunoglobulin G in sera of pregnant women

A split-sample study was conducted to evaluate the clinical performance of an enzyme immunoassay that detects the human parvovirus B19 virus (B19V) immunoglobulin M (IgM) or IgG in the sera of pregnant women. The initial study compared a baculovirus-expressed VP2 enzyme immunoassay (BVP2 EIA) (Biotrin International Inc., Dublin, Ireland) with the currently available and commonly used Escherichia coli-expressed VP1 enzyme immunoassay (EVP1 EIA) (MRL Diagnostics, Cypress, Calif.). There was a high degree of agreement between the two assays in the detection of IgM antibodies (283 of 307 [92.2%]) or IgG antibodies (279 of 311 [89. 7%]), with the majority of discrepancies (IgM, 17 of 24 [71%]; IgG, 16 of 31 [50%]) being due to equivocal data obtained with the EVP1 EIA. Specimens with discordant BVP2 EIA and EVP1 EIA results (23 of 24 IgM and 32 of 32 IgG results) were analyzed further by baculovirus-based VP1 immunofluorescence assays (BVP1 IFAs) (Biotrin International). The BVP2 EIA and BVP1 IFA results for 20 of 23 and 28 of 32 specimens for IgM and IgG, respectively, were concordant. In contrast, the EVP1 EIA and BVP1 IFA data for only 3 of 23 and 4 of 32 specimens for IgM and IgG, respectively, were in agreement, despite the fact that the same capsid antigen was used. Both the BVP2 EIAs and BVP1 IFAs utilize a conformational viral capsid antigen, while the EVP1 EIA uses a denatured viral capsid antigen. In conclusion, the BVP2 EIAs produced far fewer equivocal results for IgM and IgG, correlating more closely to the confirmatory BVP IFAs, than did the EVP1 EIAs and proved to be more accurate for detecting B19V antibodies in the sera of pregnant women.     

Antigenic characteristics of the complete and truncated capsid protein VP1 of enterovirus 71

The complete VP1 protein of enterovirus 71 (EV71) and a series of truncations were expressed in Escherichia coli and their antigenic characteristics were studied. Immunoblot analysis showed the major immunoreactive region of the VP1 protein was located in the N-terminal portion at position of amino acid (aa) 1-100. The complete VP1 possessed strong cross-reactivity with antisera against coxsackievirus A16 (CA16) and echovirus 6 (Echo6), while the truncated fragment at position 1-100 aa only had weak cross-reactivity. Moreover, an EV71-specific linear epitope at position 94-105 aa was identified using two EV71-specific mAbs (2B9 and 5B7) with indirect ELISA, but could not be recognized by antibodies against EV71 virus particles. The complete and all of truncated VP1 proteins except His-VP1(202-297) and GST-VP1(202-248) failed to elicit a significant neutralizing antibody response in mice. His-VP1(202-297) and GST-VP1(202-248) containing neutralizing epitope(s) could be recognized only by anti-EV71 mouse sera but not rabbit or human sera. These findings may contribute to a further understanding of antigenic characteristics of the capsid protein VP1 and may be helpful to the development of diagnostic reagents and vaccines.     

Reactivity of genotype-specific recombinant proteins of human erythrovirus B19 with plasmas from areas where genotype 1 or 3 is endemic

Human erythrovirus (parvovirus) B19 (B19) is a common human pathogen. The recent discovery of three genotypes, 1 to 3, raised issues related to the ability of genotype-specific antigens to cross-react with antibodies elicited by other genotypes. This study assessed antibody capture and immunoglobulin G (IgG) cross-reactivity between genotype 1 and genotype 3 recombinant antigens and analyzed the potential gain of adding VP1 protein to VP2 capsid antigen. Plasma samples from genotype 1- or genotype 3-infected populations were blindly tested with blindly prepared reagents. The IgG reactivity was assessed with baculovirus-expressed VP2 or VP1 and VP2 recombinant genotype 1 or genotype 3 proteins in a standardized enzyme immunoassay (EIA). A high degree of agreement (>95%) between EIA results was observed, with Spearman correlation coefficient and kappa reliability coefficient results of >/=0.95 for samples from the United Kingdom and >/=0.77 for samples from Ghanaian children, respectively. Most discrepant results were related to equivocal reactivity. The addition of VP1 to VP2 capsids did not significantly impact antibody detection. These data suggest that the currently available genotype-1-based IgG EIA is suitable to detect antibody to B19 genotype 3 in Ghanaian children. However, samples from the Ghanaian adult population exhibited atypical results in the assay, possibly due to the high levels of nonspecific IgG antibodies found in adults living in this region. Within these limitations, the study demonstrates that genotype 1 and genotype 3 antigens are equally effective in detecting either antibody species.     

Generation of murine monoclonal antibodies which cross-neutralize human enterovirus genogroup B isolates

A live enterovirus 71 (EV71) isolate designated, EV71/E59, with genotype B4 produced in Vero cells and purified over a sucrose gradient was used as the immunogen to generate EV71-specific murine monoclonal antibodies. Four hybridoma clones derived from the fusion of splenocytes of EV71/E59-preimmunized BALB/c (H-2^d) mice and the NS-1 myeloma cells that exhibit stable growth were selected for detailed characterization. The proof that the hybridomas produced are indeed true independent clones was based on the obervations that they expressed different complementarity-determining regions (CDRs) in their κ light chain genes. Purified ascitic fluids produced by the individual clones reacted against the viral capsid protein, VP1, in Western blot; and recognized distinct sites of a common epitope localized at the C-terminal half of VP1. Each of the monoclonal antibodies exhibited potent neutralizing activities against the immunizing virus strain, as well as two other isolates namely, N0781-TW-01, and N2838, of subgenogroups B4 and B5, respectively, that were found commonly in recent outbreaks in Taiwan. It was also observed the monoclonal antibodies acted cooperatively in neutralizing the EV71/E59 virus.     

Evaluation of immunoassays for detection of antibodies to human herpesvirus 7.

An enzyme immunoassay (EIA), an immunoblot assay (IB), and an indirect immunofluorescence assay were developed for detection of human herpesvirus 7 (HHV-7) antibodies in human serum. Cross-absorption studies with EIA or IFA using HHV-7 and human herpesvirus 6 (HHV-6) antigens indicated that most human sera contain cross-reactive HHV-6 and HHV-7 antibodies and that the degree of cross-reactivity varies between individual serum specimens. Inhibition of homologous antibody activity by absorption with heterologous virus ranged from 0 to 57% by EIA. However, for every sample tested, absorption with homologous virus removed more activity than did heterologous virus. An 89-kDa protein was identified as an HHV-7-specific serologic marker by IB. Activity to this protein was not removed by absorption with HHV-6 antigen. Of the three assays, the EIA was the most sensitive (94%), while the IB was the most specific (94%). Approximately 80% of specimens collected from German adults and children older than 2 years were positive for HHV-7 antibodies by these assays.     

Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides

Enterovirus 71 (EV71) is the main causative agent of Hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortalities. Currently, there is no vaccine available and treatment is limited to palliative care. In this study, antisera were raised in mice against 95 overlapping synthetic peptides spanning the VP1 capsid protein of EV71. Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, respectively, are capable of eliciting neutralizing antibodies against EV71 in the in vitro microneutralization assay. SP70 was identified to be particularly potent in eliciting a neutralizing antibody titer comparable to that obtained with a whole virion-immune serum. Immunization of mice with either SP55 or SP70 triggered an EV71-specific IgG response as high as that obtained with the whole virion as immunogen. The IgG sub-typing revealed that the neutralizing antibodies elicited by both synthetic peptides are likely belonging to the IgG1 sub-type. Alignment with databases showed that the amino acid residues of SP70 are highly conserved amongst the VP1 sequences of EV71 strains from various sub-genogroups. Altogether, these data indicate that SP70 represents a promising candidate for an effective synthetic peptide-based vaccine against EV71.     

EV71 VP1人源单克隆抗体筛选

目的 通过建立新型人源化单克隆抗体制备技术平台,筛选抗EV71 VP1单克隆抗体.方法 使用耦联EV71 VP1抗原的磁珠对EV71感染者外周血淋巴细胞进行分选,得到分泌EV71VP1特异性抗体的B淋巴细胞.有限稀释后,低渗裂解细胞,采用逆转录和巢式PCR扩增免疫球蛋白重链和轻链基因,经测序鉴定后克隆到真核表达载体Cloning vector AbVec-hIgG1、Cloning vectorAbVec-hIgKappa、Cloning vector AbVec-hIgLambda中.通过瞬时转染293T细胞得到重组抗体.结果 成功筛选到1对抗EV71 VP1的人源单克隆抗体基因.结论 本研究初步成功地建立了人源化单克隆抗体的筛选方法,为手足口病早期诊断、治疗以及筛选其他人源化单克隆抗体奠定了基础.     

Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis

The efficacy of enzyme immunoassay (EIA) in detecting diagnostic antibody rises to influenza A and B viruses was compared with complement fixation (CF) and hemagglutination inhibition (HI) tests in 455 patients with an acute respiratory infection. EIA and HI detected significantly more diagnostic antibody rises against influenza A than the CF method (96 and 87 vs. 47, respectively). In the case of influenza B significantly more diagnostic influenza B antibody rises were observed by EIA than by CF or HI (59 vs. 37 and 40, respectively). In most of the cases antibody rises in EIA were found in both IgG and IgA isotypes whereas increases in IgM antibodies were seen less frequently. Purified hemagglutinins (HA) were prepared from influenza A HI- and H3-subtypes and from influenza B viruses and used as antigens in EIA and the results were compared with those of HI. Infections caused by influenza A HI-subtype showed good homologous antibody responses in EIA but heterologous antibody responses to H3-subtype and influenza B HAs were frequently observed. Heterologous responses were clearly less frequent in patients with infections caused by the H3-subtype. Influenza B infections occasionally raised HA antibodies against influenza A H1-subtype but not to the H3-subtype. Interestingly, HI detected these heterologous responses at least as frequently as EIA. When whole viruses were used as antigens in EIA, subtype specificity was not observed and cross-reactions between influenza A and B virus antibodies were found. These observations suggest that, although EIA can show greater diagnostic efficacy over HI and CF methods, HI is still the serological method of choice in determining the causative subtype of influenza A virus infection.     

Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus

The gene 9 nucleotide sequence was determined for rhesus rotavirus and each of 14 viral variants selected for their resistance to neutralizing monoclonal antibodies. Each variant contains a single gene 9, VP7, mutation which permits viral growth in the presence of the antibody. Variant mutations were identified in two distinct neutralization regions. Region A was identified by monoclonal antibodies that are involved in both serotype-specific and serotype cross-reactive neutralization. Region C was identified by serotype-specific neutralizing monoclonal antibodies. Heterotypic neutralizing monoclonal antibody 57-8 selected variants with a mutation at amino acid 94 in the A region, the same amino acid location selected by serotype-specific monoclonal antibodies. Monoclonal antibody 3 selected a VP7 mutation at amino acid 99 resulting in additional N-linked glycosylation of the VP7 protein. Despite the added VP7 glycosylation, variant v3 was not broadly resistant to additional VP7-specific neutralizing monoclonal antibodies.     

Identification of amino acids located in the antibody binding sites of human hepatitis A virus.

Antigenic mutants of human hepatitis A virus (human-HAV) were isolated by their resistance to neutralizing monoclonal antibodies raised to human-HAV. The nucleotide sequence determined for the capsid regions of 12 mutants identified amino acid changes that clustered in three non-overlapping sites; one in VP3 and two in VP1. All mutants had a change at amino acid residue 70 in VP3, indicating its primary importance for antibody binding. Ten mutants had two amino acid changes occurring in the VP3 site as well as one in one of the two VP1 sites. These data suggest that both sites in VP1 interact with the single VP3 site to form the immunodominant epitope of HAV. The amino acid changes found in the antigenic mutants of human-HAV selected in this study were located in the same positions as changes found in strains of HAV isolated from Old World monkeys. These simian strains of HAV are not recognized by most monoclonal antibodies raised to human-HAV, suggesting that the observed amino acid changes are part of the antibody binding site.