Detection of antiviral activity of monoclonal antibodies, specific to Marburg virus proteins

Monoclonal antibodies (MAbs) specific to Marburg virus (MBG), Popp strain, have been previously produced and characterized by indirect ELISA. Protein specificity of MAbs was determined by immunoblotting with SDS-PAGE proteins of MBG: one to NP, four to VP40, and protein specificity of one antibody was not detected. The effect of MAb binding to protein epitopes on viral functions was investigated in vitro and in vivo. None of antibodies neutralized the virus in the neutralization test in vitro, but MAb 5G9.G11 and 5G8.H5 specific to MBG VP40 protein were active in antibody-dependent complement mediated lysis of virus-infected cells. In vivo these antibodies (5G9.G11 and 5G8.H5) protected guinea pigs from lethal MBG infection after passive inoculation. Studies of biological activity and analysis of epitope specificity of MAb-antiVP40 by competitive ELISA showed that 2 of 7 epitopes of VP40 protein of MBG induce the production of protective antibodies. Hence, MAbs 5G9.G11 and 5G8.H5 reacting with MBG VP40 protein caused lysis of virus infected cells in the presence of the complement in vitro and protected guinea pigs from MBG infection by passive inoculation.     

Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.     

Human recombinant antibodies to Ebola virus: preparation and characteristics

Human recombinant antibodies against a purified Ebola virus (EV) lysate were selected from a combinatorial library of scFv-antibodies using the phage display technique. Nine unique antibodies were identified after sequencing the Vh- and Vl-genes encoding the selected antibodies. Solid-phase enzyme immunoassay (EIA) indicated that these antibodies were able to bind both inactivated and native EV. Immunoblotting showed that 6 antibodies identified nucleoprotein (NP), one antibody did VP24 and another antibody did VP40. One of the selected antibodies reacted with two EP proteins: VP24 and VP40. Solid-phase EIA demonstrated cross-reactivity with Marburg virus (MAR) and defined VP24 MAR as a target protein for the antibody.     

Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay.

Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.     

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.     

Production of monoclonal antibodies to rotavirus suitable for detection of rotavirus in stool samples by one-step EIA.

Two stable hybridoma cell clones producing high amounts of monoclonal antibodies (MAb) to rotavirus have been selected. The MAbs were shown to recognize different epitopes of the major inner capsid rotavirus protein VP 6. The two types of MAb in question cross-reacted with human and animal group A rotaviruses. Due to their high affinity and good binding capacity to microtiter plates a simultaneous EIA was developed for detection of rotavirus in stool samples. The sensitivity and specificity of MAb one-step EIA was compared with an approved polyclonal sandwich EIA by testing 1309 stool samples.     

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes

An antigen-capture enzyme-linked immunosorbent assay (C-ELISA) was developed for detection and identification of infectious bronchitis virus (IBV) serotypes Arkansas, Connecticut, and Massachusetts using monoclonal antibodies (MAbs) specific to the S1 glycoprotein of the respective serotype. The assay (designed as a double-antibody sandwich assay) gave the best results when the S1-specific MAb, antigen, and chicken serum were of the same serotype. However, when a group-specific (M glycoprotein-specific) MAb was used for antigen capture, a distinctive pattern of cross-reactivity was observed between the antigens and heterologous chicken sera, suggesting a complex distribution of epitopes on the IBV M glycoproteins. Treatment of antigen with NP40 enhanced the ELISA signal only when the M glycoprotein-specific MAb was used for antigen capture. Although C-ELISA was inconsistent in detecting IBV in chicken tissue homogenates, it was highly effective in detecting the virus in allantoic fluid after the homogenates were given one chicken embryo passage.     

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection.     

Characterization of monoclonal antibodies to Marburg virus nucleoprotein (NP) that can be used for NP-capture enzyme-linked immunosorbent assay

After the first documented outbreak of Marburg hemorrhagic fever identified in Europe in 1967, several sporadic cases and an outbreak of Marburg hemorrhagic fever have been reported in Africa. In order to establish a diagnostic system for Marburg hemorrhagic fever by the detection of Marburg virus nucleoprotein, monoclonal antibodies to the recombinant nucleoprotein were produced. Two clones of monoclonal antibodies, MAb2A7 and MAb2H6, were efficacious in the antigen-capture enzyme-linked immunosorbent assay (ELISA). At least 40 ng/ml of the recombinant nucleoprotein of Marburg virus was detected by the antigen-capture ELISA format. The epitope of the monoclonal antibody (MAb2A7) was located in the carboxy-terminus of nucleoprotein from amino acid position 634 to 647, while that of the MAb2H6 was located on the extreme region of the carboxy-terminus of the Marburg virus nucleoprotein (amino acid position 643-695). These monoclonal antibodies strongly interacted with the conformational epitopes on the carboxy-terminus of the nucleoprotein. Furthermore, these two monoclonal antibodies were reacted with the authentic Marburg virus antigens by indirect immunofluorescence assay. These data suggest that the Marburg virus nucleoprotein-capture ELISA system using the monoclonal antibodies is a promising technique for rapid diagnosis of Marburg hemorrhagic fever.     

Mapping of two dominant sites of VP35 of Marburg virus

Five types of anti-VP35 monoclonal antibodies (MAbs), four immune sera against Marburg virus (MBGV), and 11 overlapping recombinant VP35 fragments were used to map the epitopes for VP35 of MBGV. The purified full-size recombinant VP35 was highly immunogenic and retained the B-cell epitopes that were identical to those of the viral VP35. Two major sites on VP35 and a set of truncated VP35 fragments were found by use of an enzyme immunoassay and immunoblot. Site I was located in a region between amino acids 1 and 174 of the VP35 sequence, and only polyclonal antibodies (PAbs) against MBGV recognized epitopes at this site. Site II was mapped by use of anti-VP35 MAbs to the region between amino acid residues 167 and 278 of VP35. Amino acids 252-278 of VP35 might be involved in the formation of the epitopes for MAbs. B-cell epitopes were not found on the C-terminus of VP35 by use of PAbs or MAbs.     

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.     

Generation of monoclonal antibodies against surface antigens of Moraxella bovis.

Six hybridoma lines producing monoclonal antibodies (MAbs) against Moraxella bovis were established from fusions between the SP2/0 myeloma cells and BALB/c mice splenocytes. Three antibodies were of the IgG1 isotype, two were IgG2a, and one was IgG2b. The specificity of the antibodies was determined by indirect enzyme-linked immunosorbent assay (ELISA) using whole cells of M. bovis and of other Gram-negative bacteria, and lipopolysaccharide (LPS) from M. bovis JUR2 and E. coli as antigens. Ascitic fluid produced by the six hybridoma lines inhibited hemagglutination by M. bovis GF9. One MAb (35F) reacted specifically with purified M. bovis LPS in the ELISA test. The MAb panel detected heterogeneity among the isolates recovered from different geographical regions.     

Comparison of 44/107L one-step immunocapture enzyme-immunoassay and time-resolved fluoroimmunoassay for influenza A diagnosis

One-step immunocapture enzyme-immunoassay (EIA) was compared with time-resolved fluoroimmunoassay (TR-FIA) for rapid diagnosis of influenza A infection by antigen detection. The high-affinity monoclonal antibodies (MAbs) recognising two independent epitopes on the conservative nucleoprotein were used for capture (MAb 44) and detection (MAb 107L) of antigen by both assays. The detection limit for purified recombinant influenza A virus nucleoprotein was approximately 10 pg by EIA and 5 pg by TR-FIA. The performance of the methods was evaluated by testing 43 known positive and 50 negative clinical specimens (nasopharyngeal washes and aspirates). The sensitivity and specificity was 93% and 92% for EIA and 100% and 98% for TR-FIA, respectively, in comparison to the reference A3/A1 TR-FIA. The relationship of 44/107L immunoassays has been evaluated: in comparison to 44/107L TR-FIA (100%), EIA confirmed 93% of positive and 94% of negative samples. In conclusion, the capture-detector pair of MAbs 44 and 107L can be used for the sensitive detection of influenza A viral antigen in clinical samples by both immunocapture methods. Despite the slightly lower accuracy of the EIA, widespread availability and economy of the EIA methodology makes it an advantageous alternative for the laboratory diagnosis of influenza A virus infections.     

Comparative study of the antigenic and immunogenic properties of native and recombinant Marburg virus VP35 proteins]

Antigenic structure of Marburg virus (MBG) VP35 was compared with that of the recombinant VP35 (f35) expressed in a prokaryotic system. For this purpose, a gene encoding the full-length VP35 was cloned in vector pQE31(QIAGEN) and expressed at about 70 mg/liter culture fluid in Escherichia coli JM103 as a recombinant fusion protein f35. BALB/c mice were immunized with soluble f35 or purified inactivated virions of MBG. Antibodies cross-reacting with VP35 and f35 antigens were detected by ELISA and Western Blot analysis in immune sera. Serum from a convalescent after Marburg disease and polyclonal antibodies from animals immunized with MBG recognized f35 and the MBG VP35. VP35 and its recombinant analog induced the production of specific antiviral antibodies in mice, which cross-reacted with the studied antigens. Competitive EIA showed that VP35 and f35 cross-inhibit the antigenic reactivity with polyclonal antibodies of immune sera. Antigenic structure of f35 protein corresponded to antigenic structure of MBG VP35 protein.     

Production,characterization and use of monoclonal antibodies to grapevine virus A

Summary Four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus A (GVA) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp 2/0-Ag 14. In ELISA all MAbs reacted with virus in leaf extracts fromNicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. In IEM tests, only one of the MAbs (PA 3.F 5) decorated virus particles on the entire surface. This MAb was likely induced by a surface antigenic determinant, whereas the other three MAbs (PA 3.D 11, PA 3.C 6, and PA 3.B 9) were originated by cryptotopes. Coupling polyclonal antibodies for coating protein A-sensitized plates, and monoclonal antibody conjugates for antigen detection, gave highly efficient and reproducible results for identification of GVA in field-grown grapevines.     

Induction of monoclonal antibody with predefined ELNKWA epitope specificity by epitope vaccine

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.     

Monoclonal antibodies with high virus-neutralizing activity against pandemic influenza virus A/llV-Moscow/01/2009 (H1N1)swl

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.     

Monoclonal antibodies against human basic fibroblast growth factor

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.     

Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.