Highly conserved Neisseria meningitidis surface protein confers protection against experimental infection.

A new surface protein, named NspA, which is distinct from the previously described Neisseria meningitidis outer membrane proteins was identified. An NspA-specific mAb, named Me-1, reacted with 99% of the meningococcal strains tested indicating that the epitope recognized by this particular mAb is widely distributed and highly conserved. Western immunoblotting experiments indicated that mAb Me-1 is directed against a protein band with an approximate molecular mass of 22,000, but also recognized a minor protein band with an approximate molecular mass of 18,000. This mAb exhibited bactericidal activity against four meningococcal strains, two isolates of serogroup B, and one isolate from each serogroup A and C, and passively protected mice against an experimental infection. To further characterize the NspA protein and to evaluate the protective potential of recombinant NspA protein, the nspA gene was identified and cloned into a low copy expression vector. Nucleotide sequencing of the meningococcal insert revealed an ORF of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a predicted molecular weight of 18,404 and a isoelectric point of 9.93. Three injections of either 10 or 20 microg of the affinity-purified recombinant NspA protein efficiently protected 80% of the mice against a meningococcal deadly challenge comparatively to the 20% observed in the control groups. The fact that the NspA protein can elicit the production of bactericidal and protective antibodies emphasize its potential as a vaccine candidate.

Molecular characterization of human antibodies to bacterial antigens: utilization of the less frequently expressed VH2 and VH6 heavy chain variable region gene families.

Structural analysis of the human immunoglobulin repertoire holds promise for determining the basis of variable region gene usage in response to a variety of auto and exogenous antigens. Here we report the nucleotide sequences of the heavy and light chain variable regions expressed by three human monoclonal antibodies specific for two clinically relevant bacterial pathogens, Bordetella pertussis and Haemophilus influenzae type b. The cell lines were derived by in vitro stimulation of lymphocytes from spleen or tonsillar tissue, respectively, and bind to different antigens from the two organisms. The single B. pertussis antibody is of the IgM lambda isotype and utilizes the single VH6 gene segment in combination with a V lambda 2 gene and demonstrates limited somatic mutation, yet is highly indicative of an antigen-driven immune response. One H. influenzae antibody is of the IgG2 lambda isotype and expresses a VH3 gene segment with a V lambda 1 gene, while the second cell line produces an IgG3 lambda antibody expressing a combination of VH2/V lambda 3. Both molecules show evidence of somatic mutation. The D gene segments of the heavy chains vary in length and display limited sequence homology with known germline D segments. As demonstrated previously, JH4 predominates (two JH4 and one JH3) and all three utilize the J lambda 3 gene segment. In addition, we have isolated and sequenced a number of germline VH2 gene segments in an attempt to better understand the nature of the VH2 germline repertoire. In addition to contributing to the understanding of the human antibody repertoire, such clinically relevant molecules may prove to be a source of passive immunotherapy for those at risk to developing disease.

Structural characteristics of four human hybridoma antibodies specific for the pp65 protein of the human cytomegalovirus and their relationship to human rheumatoid factors.

Four human hybridoma antibodies directed against the human cytomegalovirus (CMV) were characterized with respect to their immunoglobulin gene usage and expression of rheumatoid factor (RF) associated idiotypes and variable region epitopes. The aims of these experiments were: (1) to characterize the immunoglobulin gene usage of four antibodies directed against a single protein of a human pathogen; and (2) to examine how this humoral response may be linked to the production of RFs, autoantibodies found in the majority of patients with rheumatoid arthritis (RA). All four anti-CMV antibodies were of the gamma heavy chain isotype and were specific for the immunodominant 65 kDa viral matrix phosphoprotein (pp65). The four anti-pp65 antibodies expressed different light (L) and heavy (H) chain variable region gene combinations. These were: VkIII/VH3, V lambda 1/VH3, V lambda 1/VH4 and V lambda 3/VH3, respectively for the HCV-2, HCV-3, HCV-63 and HCV-65 hybridoma cell lines. Although none had RF activity, each of these antibodies expressed a unique set of RF-associated determinants, implying different three-dimensional configurations of the variable regions of these antibodies. The HCV-2 antibody, however, had the most extensive similarities to human RFs since it not only expressed the greatest number of RF-associated determinants but also had a protein sequence that was very homologous to RFs of the "Po" idiotypic family. Furthermore, predicted germline gene usage by anti-CMV antibodies and RFs suggest that some are encoded by identical or similar genes and that the different specificities are achieved by somatic mutations in the L and H chain complementarity determining regions (CDRs) and genetic diversity in the H chain CDR3.

Parameters affecting in vitro immunization of human lymphocytes.

A method for the in vitro generation of antibody-secreting cells from human tonsil lymphocytes is described. Several parameters affecting such in vitro immunization have been studied in order to define optimal conditions for the production of specific antibody. In the presence of 15% of T-cell growth factors, lymphocytes prepared by Ficoll-Paque centrifugation increased 2.5 times over a period of 6 days. The major factors triggering specific antibody production against Haemophilus influenzae type b outer membrane preparations were: the use of tonsil lymphocyte cultures instead of peripheral blood lymphocytes, foetal calf serum instead of human serum, T-cell growth factors as a non-specific lymphocyte activator, low concentrations of antigen ranging from 1 to 10 ng/ml, a cell concentration of 4 X 10(6) cells/culture, and finally the removal of OKT8 positive suppressor T-cells. Under these conditions, a maximum antibody production was achieved after 7 days of culture and specific antibody response was obtained after in vitro immunization of tonsil lymphocytes from 10 out of 11 individuals tested.

High yield monoclonal antibody production in ascites.

BALB/c males were mated with Swiss Webster/HPB females to produce a first generation cross. Hybridoma cells derived from fusing SP2/0 myeloma cells and histocompatible spleen cells were injected intraperitoneally into these mice to induce formation of ascites tumors and production of ascitic fluid containing large quantities of monoclonal antibody. Mice, 80 days old weighing between 28 g to 35 g, were treated with 0.5 ml of pristane 18 days before inoculation of 3.2 X 10(6) hybridoma cells. The first generation crosses, (BALB/c male X SW/HPB female)F1, produced up to 4 times more ascitic fluid of equally high antibody level over a longer period compared to the BALB/c parent. This first generation cross is a cost effective means for monoclonal antibody production.

Parameters affecting ascites tumour formation in mice and monoclonal antibody production.

Hybridoma cells injected intraperitoneally into mice induce formation of ascites tumours and production of ascites fluid containing high levels of monoclonal antibody. Several parameters affecting the growth of the immunoglobulin-producing tumours have been studied in order to define optimal conditions for ascitic fluid formation and monoclonal antibody production. Using hybridomas produced by fusing SP2/0 myeloma cells with immunized mouse spleen cells we have shown: (1) that the optimal number of hybridoma cells required to induce an ascites tumour was between 6 and 32 X 10(5) cells; (2) that each mouse should be treated with a maximum of 0.5 ml of pristane; (3) that the priming period for pristane should be 14 days prior to the injection of cells; (4) that ascites formation and monoclonal antibody production is significantly better in males; and finally (5) that the age of mice used should range between 43 and 78 days. Under these conditions each mouse produces on average 7-10 ml of ascites fluid, containing a high level of antibody, over a maximum period of 6 days. The animals should start producing between the 5th and 9th day and usually survive 11-16 days after being injected with the tumour cells.

Development and application of monoclonal antibodies for the detection of cell infecting mycoplasmas.

Two hybridoma cell lines producing monoclonal antibodies (MAbs) to mycoplasmas were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with whole cell Mycoplasma pneumoniae antigens. The MAbs designated as MYC-4 and MYC-9 bound to a single M. pneumoniae protein band with approximate molecular weights of 150,000. In dot-EIA both MAbs reacted with ten Mycoplasma spp., two serovars of Ureaplasma urealyticum and Acholeplasma laidlawii. The MAbs have great potential to be used as an antibody probe for the rapid screening of cell cultures and hybridomas for mycoplasmal contamination.

A novel approach to the laboratory diagnosis of Chlamydia trachomatis infections using monoclonal anti-idiotypic antibodies.

We have developed a novel enzyme immunoassay (EIA) for the specific detection of Chlamydia trachomatis utilizing a monoclonal anti-idiotypic antibody to an antibody directed against a chlamydia specific epitope on 60 kDa heat-shock protein (HSP60). The basis of the assay is the inhibition of the binding of idiotype to anti-idiotype by antigen present in test samples. Two configurations of the assay were developed: a blocking EIA and a competition EIA. Greater sensitivity was observed using the competition EIA, with the assay detecting purified recombinant HSP60 and purified chlamydia in a concentration-dependent manner from 0.01 to 10 micrograms protein and from 0.5 to 12 micrograms total protein, respectively. The assay is highly specific and offers several potential advantages over currently available EIAs for the detection of this pathogen.

Monoclonal antibody characterization of Jamestown Canyon (California serogroup) virus topotypes isolated in Canada.

Jamestown Canyon (JC) virus of the California (CAL) serogroup has been isolated in 12 American states and 6 Canadian provinces. A study was undertaken to produce monoclonal antibodies (MAbs) to JC virus and to use these MAbs to assay for possible heterogeneity among naturally occurring JC topotypes in Canada. MAbs were produced to the prototype strain of JC virus using BALB/c mice. Twenty-seven secreting MAbs were obtained and three of these MAbs were propagated and studied. All three MAbs, M1 (IgG1), M2 (IgG2b), and M3 (IgG2a), were reactive by immunofluorescent antibody assay against JC-infected vero cells and by ELISA against JC antigen. MAb M2 reacted with all members of the Melao complex, MAb M1 reacted only with Keystone virus, while MAb M3 exhibited no reactivity with other CAL serogroup viruses. Only MAb M3 possessed neutralization and hemagglutination inhibition activities against JC virus. The MAbs were also tested by ELISA and for neutralizing activity against 13 JC topotypes isolated in 5 provinces from Newfoundland to Saskatchewan. ELISA confirmed closer identity of the Canadian topotypes to JC as opposed to the closely related South River virus. The MAbs verified all Canadian topotypes to be JC virus but revealed different patterns of reactivity between these topotypes and prototype JC virus.

New monoclonal antibodies for the detection of immediate early antigens of cytomegalovirus.

Two new monoclonal antibodies, CIE-1 and CIE-2, were developed for the rapid detection of human cytomegalovirus (HCMV) infection. They were found to be reactive with immediate early protein of HCMV in the nuclei of infected fibroblasts, as early as 3 hours post-infection. By radioimmunoprecipitation, CIE-1 was found to react with a protein with an apparent molecular weight of 70,000, whereas CIE-2 precipitated 2 proteins of 70,000 and 72,000 daltons, respectively. Both monoclonal antibodies recognized three prototype strains of HCMV: AD-169, Towne, and Davis, and did not cross-react with other human herpesviruses. CIE-1 and CIE-2 were compared with four commercial anti-HCMV monoclonal antibodies (Clonab, Dupont, Sera-Lab and Syva) by testing 88 clinical isolates. Culture confirmation tests and shell vial assays showed that CIE-1 and CIE-2 were more sensitive than several of these reagents and equally sensitive to the Dupont reagent. Moreover, CIE-1 and CIE-2 produced a bright, sharp staining of the nuclei of infected cells. These monoclonal antibodies should thus be valuable in rapid diagnosis of HCMV.

Neutralizing monoclonal antibody against enterovirus-70 reacts with viral proteins 1C and 1D.

A library of murine monoclonal antibodies against the prototype Enterovirus-70 (EV-70) strain, J670/71, was made for the purpose of studying the immunologically reactive determinants of the virus. Each of the monoclonal antibodies reacted with several other strains of Enterovirus-70 when tested by immunofluorescence. However, none of these monoclonal antibodies reacted with any other picornavirus tested. It was found that all of the monoclonal antibodies precipitated EV-70 viral proteins 1C and 1D in radio-immunoprecipitation assays. However, only one of these monoclonal antibodies, an IgG3 kappa, was capable of neutralizing the virus.

Non-specific immunoprecipitation of rotavirus Vp6 protein with Staphylococcus aureus.

The specificity of Staphylococcus aureus and protein A-Sepharose (PA-S) were compared in the radioimmunoprecipitation assay for the characterization of monoclonal antibodies (mAbs) against rotavirus proteins. Five mAbs directed against bovine rotavirus Q17 proteins Vp6 and Vp7 and one mAb directed against human rotavirus protein Vp4 were used in this study. mAbs directed against other viruses, NS-1 culture supernatant and ascitic fluid, were used as control reagents. A non-specific immunoprecipitation of the viral protein Vp6 was always found with S. aureus, but not with PA-S. mAb 74 reacted with rotavirus antigens in ELISA and in indirect immunofluorescence assay but did not immunoprecipitate a viral protein with PA-S. This mAb immunoprecipitated the viral protein Vp6 when S. aureus reagent was used. This false positive reaction was always present and could lead to confusing results in the analysis and characterization of mAbs against rotavirus.

Enzyme-linked immunosorbent assay with polyvalent gonococcal antigen.

An indirect enzyme-linked immunosorbent assay (ELISA) with a mixture of eight different gonococcal outer-membrane proteins (OMP) as coating antigen was evaluated for detection of gonococcal antibody in 507 sera obtained from patients selected from high-risk and low-risk populations. The indirect ELISA method was more specific and sensitive when the polyvalent antigen was used than when OMP from only one serotype was used. Past episodes of the gonorrhoea had a significant influence on the seropositivity of the test. In a selected low-risk population the specificity of the assay was 94% and in a selected high-risk population the sensitivity was 78%. When sera from 24 asymptomatic individuals were tested the sensitivity was 83%. The ELISA polyvalent-antigen test should be useful as an aid for the detection of gonorrhoea in populations with a low prevalence.

A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b.

Monoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (10(3)) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 X 10(3)-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype-specific surface-exposed OMP of H. influenzae type b.

Monoclonal anti-idiotypes for the rapid detection of human cytomegalovirus.

A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVB1, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVB1 to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10(3) PFU/ml, with 50% inhibition at approx. 3 x 10(3) PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents.

Candidate Neisseria meningitidis NspA vaccine.

The highly conserved NspA protein has been found in the outer membrane of every Neisseria meningitidis strain tested so far. Two monoclonal antibodies (MAbs) directed against this protein were used to demonstrate that biologically important epitopes of the NspA protein are exposed at the surface of serologically distinct meningococcal strains. Analysis of sera collected from mice that survived a deadly meningococcal challenge following immunization with recombinant NspA protein (rNspA) revealed the presence of cross-reactive antibodies which efficiently attached to and killed the four serogroup B strains tested. These data are additional proof that the NspA protein is exposed at the surface of intact meningococcal cells, which is an important characteristic for a vaccine candidate.

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.