Epitope mapping of Grapevine virus A capsid protein

Summary.  Previously characterized monoclonal antibodies (MAbs) to Grapevine virus A (GVA) showed a differential reactivity against intact or partially destabilized virus particles [2]. In the present study, this differential reactivity was confirmed and several peptides reacting with a panel of four different antibodies were identified by the PEPSCAN method of epitope mapping. Oligopeptide sequences comprised between coat protein residues 61 (V) and 72 (T) were recognized by all the antibodies tested. One of these peptides (VGPKASK) was also reactive when expressed on recombinant phage particles as a fusion protein with protein pVIII. The specificity of this sequence for antibody binding was also demonstrated by competitive-ELISA using one of the GVA MAbs. The results of this study suggest that GVA particles carry a highly structured epitope centered on a common peptide region of the coat protein sequence. Received June 5, 2001 Accepted October 26, 2001     

Bank vole monoclonal antibodies against Puumala virus envelope glycoproteins: identification of epitopes involved in neutralization

Summary Bank vole (Clethrionomys glareolus) monoclonal antibodies (MAbs) against the two envelope glycoproteins (G 1 and G 2) of the Puumala (PUU) virus were generated and characterized. Analyses of the MAbs' antigen and epitope specificities showed non-overlapping reactivities of one anti-G 1 and two anti-G 2 MAbs. A significant neutralizing activity was shown by the anti-G 1 and one of the anti-G 2 MAbs, suggesting the existence of at least one neutralizing domain on each of the two glycoproteins. The two neutralizing MAbs reacted with eight PUU-related (serotype 3) virus strains, but did not react with Hantaan, Seoul, or Prospect Hill viruses in an immunofluorescence assay, indicating reactivity with epitopes unique for PUU virus. The non-neutralizing anti-G 2 MAb also reacted with Seoul virus, revealing the presence of a conserved G 2-epitope common for PUU and Seoul viruses, not involved in neutralization. Competitive binding of the MAbs and sera from nephropathia epidemica patients indicated that the defined neutralizing and non-neutralizing epitopes of the glycoproteins were immunodominant also in humans. In another experiment, magnetic beads coated with two MAbs were bound with the virus glycoproteins and used for selective enrichment of cells secreting anti-glycoprotein antibodies.     

Enhancement of anthrax lethal toxin cytotoxicity: a subset of monoclonal antibodies against protective antigen increases lethal toxin-mediated killing of murine macrophages

We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcgamma receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.     

Identification of grapevines infected with divergent variants of Grapevine virus A using variant-specific RT-PCR

RT-PCRs, designed for the specific detection of molecular variants of Grapevine virus A (GVA), were applied to analysis of the virus from various grapevines and isolates recovered from these grapevines in Nicotiana benthamiana. Results of SSCP, cloning and sequencing revealed that the combination of these RT-PCR techniques permits reliable and rapid identification of grapevines mixed-infected with divergent variants of GVA. The results suggest that such grapevines are very common among GVA-infected grapevines in vineyards in South Africa.     

Identification of cell membrane proteins linked to susceptibility to bovine viral diarrhoea virus infection

Summary Three monoclonal antibodies directed against cell surface molecules of bovine cells inhibited subsequent infections with bovine viral diarrhoea virus (BVDV). They specifically blocked the infectivity of three non-cytopathogenic and three cytopathogenic BVDV strains. These results showed that an important mechanism for virus uptake was inhibited. The ligand of the monoclonal antibody BVD/CA 17, which blocked infectivity most efficiently, was found on leukocytes from a wide range of domestic and wild even-toed ungulates using flow cytometric analysis. In contrast, the monoclonal antibodies BVD/CA 26 and BVD/CA 27 appeared to be specific for bovine cells. Immunoprecipitation of labelled bovine cell surface proteins showed that the three monoclonal antibodies bound to proteins with identical relative molecular masses (M_r). Proteins of an apparent M_r of 93 K and 60 K were precipitated from lysates of fetal bovine kidney cells irrespectively of the MAbs used.     

A monoclonal antibody to Bacillus anthracis protective antigen defines a neutralizing epitope in domain 1

Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.     

Monoclonal IgM antibodies from cytomegalovirus-infected mice recognize the GlcNAc-containing receptor determinant of murine CMV as well as neutralizing anti-CMV IgG antibodies.

This study examines monoclonal antibodies derived from mice at different time points after infection with attenuated murine cytomegalovirus (MCMV). The antibodies obtained from mice at 3 weeks p.i. were of IgG type (designated V-antibodies) and several could neutralize the virus. Those obtained at 5 weeks p.i. were of the IgM class (designated R-antibodies), bound to uninfected (MEF, mouse embryo fibroblast) cells, and thereby blocked MCMV plaque formation. In ELISA, the IgM monoclonals (R-antibodies) bound to GalB1-3GlcNAc and GalB1-4GlcNAc, the receptor determinants for MCMV. Similarly, these GlcNAc-containing residues blocked the binding of purified IgM monoclonal antibodies (MAbs) to MEF. The R- and V-series of antibodies showed mutual binding activities; for example, IgM MAb R-2D8 bound specifically to four (V-8C4, V-1C7, V-8C7, V-9C5) of six neutralizing IgG MAbs in ELISA. The same neutralizing IgG MAbs bound to antireceptor IgM antibodies in an immunoblot assay. This suggests that the IgM monoclonals directed against the known cell surface receptor determinant are anti-idiotypic antibodies against neutralizing antiviral IgG antibodies. The neutralizing antiviral IgG MAbs bound to 60- and 66-kDa MCMV polypeptides on Western blots, suggesting that these viral polypeptides may be important in MCMV binding to this receptor. The R-series might represent anti-idiotype antibodies capable of down-regulating antiviral V-antibodies and may also represent a mechanism for the induction of IgM autoantibodies directed at cell surface glycolipids present in autoimmune CMV-associated neuropathies.     

Studies of murine malaria antigens using monoclonal antibodies

A panel of ten monoclonal antibodies made againstPlasmodium chabaudi andPlasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs toP. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted withP. chabaudi antigens. Of the MAbs toP. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus

Summary Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinantEscherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinantE. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinantE. coli.     

Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces

Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of >10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.     

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.     

Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen

The recombinant human monoclonal antibody (MAb) against hepatitis B virus (HBV) surface antigen (HBsAg) was expressed in tobacco suspension cultures. The parental CL4MAb was produced by the Epstein-Barr virus (EBV) transformed human cell line TAPC301-CL4. The CL4MAb cDNA was introduced into tobacco suspension cells by Agrobacterium mediated transformation. The monoclonal antibodies (MAbs), B294 and B303, which were derived from CL4 and subsequently produced in plant cells were selected for study. After purification on Protein A columns, B294 and B303 MAbs had anti-HBs relative affinity constants similar to the parental CL4MAb. B303 MAb interacted with cell surface HBsAgs and showed complement-dependent cytotoxicity in a manner that was similar to anti-HBs human immunoglobulins (HBIg) that are used clinically. The results of this study point to the feasibility of producing MAbs to HBsAg in plants as an alternative to HBIg.     

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.     

Characterization of neutralization epitopes of simian immunodeficiency virus (SIV) recognized by rhesus monoclonal antibodies derived from monkeys infected with an attenuated SIV strain

A major limitation in the simian immunodeficiency virus (SIV) system has been the lack of reagents with which to identify the antigenic determinants that are responsible for eliciting neutralizing antibody responses in macaques infected with attenuated SIV. Most of our information on SIV neutralization determinants has come from studies with murine monoclonal antibodies (MAbs) produced in response to purified or recombinant SIV envelope proteins or intact SIV-infected cells for relatively short periods of time. While these studies provide some basic information on the potential immunogenic determinants of SIV envelope proteins, it is unclear whether these murine MAbs identify epitopes relevant to antibody responses elicited in monkeys during infection with either wild-type or attenuated SIV strains. To accomplish maximum biological relevance, we developed a reliable method for the production of rhesus monoclonal antibodies. In the present study, we report on the production and characterization of a unique panel of monoclonal antibodies derived from four individual monkeys inoculated with SIV/17E-CL as an attenuated virus strain at a time when protective immunity from pathogenic challenge was evident. Results from these studies identified at least nine binding domains on the surface envelope glycoprotein; these included linear determinants in the V1, V2, cysteine loop (analogous to the V3 loop in human immunodeficiency virus type 1), and C5 regions, as well as conformational epitopes represented by antibodies that bind the C-terminal half of gp120 and those sensitive to defined mutations in the V4 region. More importantly, three groups of antibodies that recognize closely related, conformational epitopes exhibited potent neutralizing activity against the vaccine strain. Identification of the epitopes recognized by these neutralizing antibodies will provide insight into the antigenic determinants responsible for eliciting neutralizing antibodies in vivo that can be used in the design of effective vaccine strategies.     

Monoclonal antibodies demonstrate accessible HA2 epitopes in minor subpopulation of native influenza virus haemagglutinin molecules

Summary Haemagglutinin (HA) was detected on the surface of influenza virus infected cell with monoclonal antibodies (MAbs) against both HA glycopolypeptides, HA1 and HA2, however, the reactivity of HA2-specific MAbs was remarkably lower as compared with HA1-specific MAbs. Quantitative analysis with two MAbs, IB8 (anti-HA1) and IIF4 (anti-HA2) respectively, revealed that HA2 epitope was reachable for antibody only in minor subpopulation of the HA representing approximately 7% of all molecules (spikes). The basis of the HA heterogeneity is discussed.     

Mechanisms of transmissible gastroenteritis coronavirus neutralization

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.     

Monoclonal antibodies against the major coat protein of filamentous phage as a useful analytical tool for bacteriophage peptide/protein display

Four mouse monoclonal antibodies (MAbs) that react with filamentous M13KO7 and R408 phage were obtained. Three of these MAbs (two IgG2a and one IgG3) recognize linear sequences of the p8 main structural coat protein, and one (IgG2a) identifies a putatively conformational epitope, as suggested by Western blot. These MAbs also react with recombinant phage expressing peptide antigens fused to p8, and are though useful reagents for peptide/protein phage display screening based methods. The latter was shown in an enzyme-linked immunoadsorbent assay (ELISA) and a visual immunoassay where one of the anti-p8 MAbs was used to capture recombinant phages displaying a peptide characteristic of the Hepatitis B virus surface antigen or a Dengue virus-related peptide antigen.     

Complete genome sequence of a natural mutant of grapevine virus A (GVA)

A new genetic variant of grapevine virus A (GVA) of phylogenetic group I was identified during comparative analysis of the viruses infecting two sibling grapevines cv. Shiraz. The grapevines were propagated from a single mother plant. One of them become infected with Shiraz disease (SD), which is highly destructive on noble grapevine cultivars Shiraz and Merlot in South Africa. The new variant was not associated with SD, as it was present in both SD-affected and SD-free plants. However, unlike in an earlier study of grapevines affected by this disease, this GVA variant of group I strongly dominated over a coinfecting variant of group II associated with SD and a variant of group III. The variant, named I327-5, was mechanically transmitted from SD-affected grapevine to Nicotiana benthamiana, and its genome was fully sequenced. The sequence data revealed that the most distinctive genomic feature of variant I327-5 is the deletion of three nucleotides in the region where the ORF2 and ORF3 genes overlap. These genes of GVA encode a 19.8-kDa protein, the function of which remains unknown, and a 31-kDa protein that is indispensable for the movement of the virus in plants. An alignment of the amino acid sequences of these proteins encoded by variant I327-5 with the corresponding proteins encoded by other members of group I suggested that, as the result of mutations, a neutral threonine or alanine and a negatively charged glutamic acid, respectively, were removed from the proteins of GVA variant I327-5.     

Definition of selectable cell surface markers for human chromosomes and chromosome segments in rodent-human hybrids

A panel of 22 monoclonal antibodies (MAbs) recognizing 21 distinct human cell surface antigens was tested by mixed hemadsorption assays for reactivity with a large number of rodent-human somatic cell hybrids containing different subsets of the human chromosome complement. The serological typing results permit the assignment of six gene loci determining cell surface antigens to human chromosomes 3, 6, 11, 19, 20, and 22. In addition, analysis of hybrids retaining deleted copies (but no normal homologs) of specific human chromosomes provides regional assignments for 18 gene loci, located on seven different chromosomes. These findings extend and refine the genetic map for human cell surface antigens and identify new selectable markers for defined chromosome segments.     

Characterization of a panel of monoclonal antibodies to hepatitis C NS3 recombinant protein

Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E. coli cells and used for BALB/c mice immunization. Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained. All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3. These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous. All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein.