Porcine reproductive and respiratory syndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing new insights into the antigenic properties of the virus

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Despite great efforts of pig holders, veterinarians, researchers and vaccine developers, the virus still causes major production losses. It is clear that efficient and correct monitoring and rational development of vaccines are crucial in the combat against this pathogen. PRRSV-specific monoclonal antibodies (mAbs) are essential tools for both diagnostic and research purposes. This study describes the production of PRRSV GP3-, GP5- and N-specific hybridomas and an extensive characterization of the mAbs. The N-specific mAbs generated in this study appear to be useful tools for diagnostics, as they were found to react with genetically very different PRRSV isolates and may serve to discriminate between European and American type PRRSV isolates. These mAbs also allowed detection of the PRRSV N protein in both formalin-fixed, paraffin-embedded tissue sections and frozen tissue sections of PRRSV-infected lungs, further illustrating their diagnostic value. Different neutralization assays pointed out that none of the GP3- and GP5-specific mAbs tested shows virus-neutralizing capacity. This is noteworthy, as these mAbs recognize epitopes in the predicted ectodomains of their target protein and since the GP5-specific antibodies specifically react with the antigenic region that corresponds to the "major neutralizing epitope" suggested for American type PRRSV. The current findings argue against an important role of the identified antigenic regions in direct antibody-mediated neutralization of European type PRRSV in vivo. However, it is also clear that findings concerning a specific PRRSV epitope cannot always be generalized, as the antigenic determinants and their biological properties may differ radically between different virus isolates.     

Induction of antibodies to canine herpesvirus in mice by immunization with anti-idiotypic antibodies.

Anti-idiotypic antibodies (anti-Id Abs) were produced in rabbits after inoculation with two mouse monoclonal antibodies (mAbs) directed against canine herpesvirus (CHV) glycoproteins (gps). One of the mAbs, 12H11, was directed against an epitope on gp 145/112 of CHV which induced virus neutralizing (VN) antibodies and against a cross-reacting epitope on the gp 143/108 of feline herpes-virus type 1 (FHV-1). The other mAb, 11F7, was directed against epitopes on CHV gp47 which induce VN and hemagglutination-inhibition (HAI) antibodies. Using VN-inhibition and HAI-inhibition assays with CHV and FHV-1, the anti-Id Abs obviously inhibited the activities of autologous mAbs, suggesting that anti-Id Abs mimic the epitopes of CHV gp 145/112 or FHV-1 gp 143/108 and CHV gp47 by binding the anti-combining site of the mAbs. These anti-Id Abs, when injected into mice, elicited specific CHV-neutralizing and HAI antibody responses, and one of them also elicited a specific FHV-1-neutralizing antibody response. These data supported the idea that immunization with anti-Id Ab can induce specific VN antibody response, as has been theorized by other workers.     

Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen.

Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.     

病毒糖基化作用对免疫应答的影响

病毒蛋白糖基化较为常见,但糖基化的类型各不相同。病毒的糖基化蛋白具有识别宿主细胞,介导病毒囊膜与宿主细胞融合以及引起病毒免疫逃避现象等多种生物学特性。人类免疫缺陷病毒的GP41与猪呼吸与繁殖综合征病毒GP5蛋白上存在诱骗表位,诱骗表位抑制临近中和表位的识别。GP5蛋白膜外区上非中和表位A表位会降低临近中和表位B表位的免疫原性,使针对B表位中和抗体延缓产生。除了两个表位位置临近外,寡糖链对B表位的遮蔽或许是造成该现象的主要因素。本文就糖基化的类型,病毒糖基化蛋白的生物学特性以及与糖基化作用相关的猪繁殖与呼吸综合征病毒GP5蛋白诱骗表位进行了介绍以期阐明糖基化作用与免疫应答现象之间的联系。     

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.     

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain.

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.     

Characterization of Env antigenicity of feline foamy virus (FeFV) using FeFV-infected cat sera and a monoclonal antibody

To characterize neutralizing antigenicity in relation to env genotypes of feline foamy virus (FeFV), serological analyses were performed using FeFV-infected cat sera and several field isolates including two env genotypes (F17- and FUV-types). Since three cats from which FeFV were isolated were found to have undetectable titers of virus neutralization (VN) antibodies, even to the homologous virus, VN antibodies were further examined with complement supplementation as an enhancement factor. With the presence of complement, the VN titers of FeFV-infected cat sera increased drastically. Although most of serum samples neutralized strains of either env genotype, sera sampled from two cats neutralized all the strains examined at similar titers, suggesting that superinfection with both env genotypes of FeFV might have occurred in the two cats. Further, we produced a monoclonal antibody (mAb) specifically neutralizing FeFV strains of FUV-type. The mAb was shown to have higher affinity to an epitope on Env of FUV-type than that of F17-type by immunoprecipitation assay. This study supplies basic information important for studies on FeFV vector development as well as on the relationship between the virus and the host immune response.     

Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP5. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP5 protein.     

A transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is now considered to be one of the most important diseases in countries with intensive swine industries. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In this study, we designed 5 of the small hairpin RNAs (shRNAs) targeting the GP5 and M gene of PRRSV respectively, and investigated their inhibition to the production of PRRSV. The highest activity displayed in shRNAs of the ORF6e sequence (nts 261-279), which the inhibition rate reached was 99.09%. The result suggests that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore, the transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against PRRS virus was established. It presented stable inhibition to the replication and amplification of PRRS. The work implied that shRNAs targeting the GP5 and M gene of PRRSV may be used as potential RNA vaccines in vivo, and supplied the screening methods of transformed pig embryonic fibroblast which are prerequisite for the disease-resistant transgenic pigs to PRRS.     

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.     

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.     

中和抗体在抗猪繁殖与呼吸综合征感染过程中的作用

中和抗体在抗猪繁殖与呼吸综合征病毒(PRRSV)感染过程中起重要作用,被动输入PRRSV的中和抗体可预防PRRSV感染妊娠母猪,并且对母猪和仔猪都可起到消除性免疫作用。然而在自然感染PRRSV的过程中,这种中和抗体产生较晚,为病毒在宿主体内大量复制并感染其他易感动物提供了充裕的时间。PRRSV的主要中和位点位于糖蛋白GP5,核心区域称作B位点,GP5的另一个显性表位称作A位点,是非中和位点。A位点与B位点的同时出现将会抑制机体对B位点的免疫反应。当PRRSV发生中和位点突变时,产生无位点A的PRRSV,这种类型的PRRSV可诱导中和抗体的产生。文章通过对PRRSV的免疫反应、中和抗体的作用以及中和位点等三方面的阐述,综述了中和抗体在抗PRRSV感染过程中的作用。     

PRRSV GP5和M抗原中和表位的融合表达与纯化以及间接ELISA检测方法的建立

为建立检测猪血清中猪繁殖与呼吸综合征病毒(PRRSV)中和抗体的间接ELISA方法,分析了PRRSV结构蛋白GP5和M的二级结构抗原指数、抗原性、亲水性、表面可及性以及潜在中和表位等参数,选取GP5的47～61、143～156和186～199位氨基酸,M蛋白的63～70、133～146和156～169位氨基酸作为免疫原...     

Epitope specificity of monoclonal antibodies to the N-protein of porcine reproductive and respiratory syndrome virus determined by ELISA with synthetic peptides

In an attempt to develop an alternate to ELISAs using recombinant N-proteins as antigen for the sero-diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infections of pigs I have measured the binding of nine anti-N-protein mAbs, which had been previously generated by various investigators, to overlapping peptides encompassing amino acids 19-70 of the N-proteins of the North American prototype (VR2332) and the European prototype (Lelystad virus, LV) of PRRSV. I also measured the binding of the mAbs to HerdChek ELISA plates coated with recombinant N-protein. All mAbs bound in an indirect ELISA to some of the peptides whether the mAbs had previously been reported to recognize continuous or discontinuous epitopes, but with different specificity and titer. Three mAbs bound with high titer to different linear epitopes located in amino acid segments 23-33, 31-50 and 43-56 and also with similar high titers to HerdChek plates. mAb SDOW17 bound with high titer to HerdChek plates but poorly to any of the peptides. In contrast, four mAbs bound with broad specificity to peptides containing an epitope(s) in amino acid segment 30-48, but poorly, or not at all, to HerdChek ELISA plates. Thus, this epitope is missing on the antigens of the HerdChek ELISA or is destroyed during immobilization of the antigens on the plate. A mAb to the N-protein of the closely related mouse arterivirus lactate dehydrogenase-elevating virus bound to the same epitope. Abs that bound with broad specificity to an epitope(s) in the 30-50 amino acid segment were also detected by the peptide ELISA in sera of 25 field sera that were sero-positive in the HerdChek ELISA, but also in sera of pigs from two out of three herds tested that were sero-negative by this test.     

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains six structural proteins the roles of which are not completely understood. In a preceding study, immunization with the dutch isolate I10 of PRRSV had led to the development of MAbs against four structural proteins [Wieczorek-Krohmer, M., 1994. Herstellung und Charakterisierung von monoklonalen Antik?rpern gegen das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms (PRRSV). Inaugural-Dissertation, Ludwig-Maximilians-Universit?t, München] here finally identified by reaction with individual plasmid-expressed PRRSV proteins as products of ORFs 3 (GP3), 4 (GP4), 5 (GP5) and 7 (N). Surprisingly, the MAbs against GP5 revealed the presence of two antigenically distinct virus populations in the isolate I10, the population PRRSV-'PPV', isolated from plaques and the PRRSV-'EPV', gained by end point dilution. MAbs against GP3, GP4 and N reacted with both I10 populations as well as with natural PRRSV isolates. However, the anti-GP5 MAbs exclusively recognized PRRSV-'PPV'. In this study immunization of mice with both separated I10 populations confirmed that solely PRRSV-'PPV' possesses the property to induce an immune response ultimately leading to the establishment of MAbs against GP5. Whereas the 15 anti-GP5 MAbs (derived from four independent fusions) reacted exclusively with PRRSV-'PPV' of the isolate I10, anti-GP4 MAbs detected their target antigen on various isolates of European origin and were able to neutralize them. As indicated by competition assays and selection of neutralization-resistant virus mutants, all GP5 MAbs are directed against a single antigenic site on the ORF 5 protein. Both groups of neutralizing antibodies bound to the surface of purified virions demonstrating that the recognized epitopes represent surface structures of the virion envelope. However, anti-GP5 MAbs mediated the binding of more gold granules than anti-GP4 MAbs. Comparison of the neutralizing effect of anti-GP4 and anti-GP5 MAbs revealed the anti-GP5 MAbs as the more efficient antibodies. For the complete neutralization of about 100 ID50 of PRRSV-'PPV' anti-GP5 culture supernatant was effective up to a dilution of 1:1280 whereas the most effective anti-GP4 antibodies exhibited a comparable effect only up to 1:64. These results indicate that PRRSV GP5 in principle is a major target for neutralizing antibodies, as is found for other arteriviruses, but that in nature 'ORF 5 escape mutants' may develop as easily as in vitro.     

Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture

A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established. One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF). This mAb is very well suited for diagnosis of PEV infections. However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM). Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM. Both mAbs presumably recognize conformation-dependent epitopes of the virus.     

Antibody and cellular immune responses of swine following immunisation with plasmid DNA encoding the PRRS virus ORF's 4, 5, 6 and 7.

The objectives of this study was to investigate the role of DNA vaccines in the generation of an immune response and that elicited against individually encoded proteins of PRRSV. The genomic regions encoding ORF s 4, 5, 6 and 7 of the PRRS virus vaccine strain were cloned into the mammalian expression vector pc DNA 3.1 (+). Inoculations with the recombinant plasmids resulted in detection of PRRS virus-specific antibodies in 71 per cent of the immunized animals by ELISA, virus neutralization and/or Western blotting assays. In addition, cellular immune responses were detected in 86 per cent of the immunized pigs by interferon gamma assay and/or proliferation assay. Pigs in the control group had no detectable immune response to PRRS virus. The results obtained demonstrated that DNA immunization against PRRS virus results in the production of both humoral and cell mediated immune responses in pigs. The results also indicate that neutralization epitopes for PRRS virus are present on the viral envelope glycoproteins encoded by ORF 4 and ORF 5.     

Antigenic variation among foot-and-mouth disease virus type A field isolates of 1997-1999 from Iran

The sequences of the antigenically relevant capsid proteins VP1-3 of 10 isolates obtained during an epizootic of serotype A foot-and-mouth disease virus in Iran, and collected within two and a half years, were found to be highly similar. However, each isolate differed by at least one amino acid from all others. This prompted us to analyze the immunological reactivity of the isolates. To this end, monoclonal antibodies (mAbs) against one isolate were generated and characterized with regard to neutralizing activity and reactivity with trypsinized virus. These mAbs as well as others raised against A22 virus were used for antigen profiling. This distinguished four antigenic conditions among the isolates and 16 reactivities among the mAbs. These findings, together with the observed sequence differences indicated the location of several epitopes. Many mAbs recognized the minor antigenic sites on VP2 and 3 and some the major site, the GH-loop of VP1. One epitope was composed of residues of the capsid proteins VP1 and 2.     

猪繁殖与呼吸综合征病毒持续感染与保护性免疫应答机制

对猪繁殖与呼吸综合征病毒持续感染与保护性免疫应答机制目前尚未完全阐明,有报道认为病毒中和抗体对抗感染不起保护性作用,甚至起负作用。近年来的研究结果表明,中和抗体在保护性免疫中起重要作用。文章针对猪繁殖与呼吸综合征病毒持续感染与清除,病毒抗原表位与中和抗体的产生机制,病毒感染动物免疫应答反应模型,病毒中和抗体在保护性免疫中的作用等内容做一综述。