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 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.

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.

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.

Sensitivity and specificity of enzyme immunofiltration and DNA hybridization for the detection of HCMV-infected cells.

The sensitivity and specificity of enzyme immunofiltration and DNA hybridization were compared in human cytomegalovirus (HCMV) (AD 169)-infected MRC-5 cells. The enzyme immunofiltration was carried out on glass fiber filters in microplates, using an HCMV (AD 169) monoclonal antibody and a peroxidase conjugate. The DNA hybridization was carried out with a microfiltration apparatus, using a 32P-labelled HCMV (AD 169) Eco R1 D fragment probe. The sensitivities of enzyme immunofiltration and DNA hybridization were 1.82 X 10(3) and 1.13 X 10(3) infected cells, respectively. Both methods were highly specific, but enzyme immunofiltration was faster and simpler.

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.

Polioviruses as indicator of virological quality of water.

Viruses were found in 47 out of 53 samples of raw domestic sewage collected over a 13-month period from a high socioeconomic level community. At least 39 out of these 47 samples contained polioviruses as was expected in a population where children were receiving Sabin live-attenuated vaccine. Polioviruses could then be used as a suitable indicator of the virological quality of water in communities where Sabin vaccine is used.

Polioviruses and other enteroviruses in urban sewage from Laval (Canada): presence of non-vaccinal strains of poliovirus.

During a 13-month period (August 1, 1977-August 31, 1978) 55 samples of 2-4 L of raw sewage from an urban residential area were collected and studied for the presence of human enteroviruses. Viruses were recovered from 47 (90.5%) of the samples. Of the 201 viral isolates propagated, 180 (89.5%) were identified by the serum neutralization test as poliovirus types 1,2, or 3; 16 (8%) were identified as coxsackie B viruses; and 5 (2.5%) could not be identified by the methods used. While all polioviruses, types 2 and 3 isolates, were vaccine-like (rct-/d-) or intermediate strains, 14 poliovires, type 1 out of 55 selected isolates, were found to be non-vaccine-like (rct+/d+), 8 were rct-/d-), and 33 were intermediate strains. Out of nine samples submitted to the serodifferentiation (McBride) test, six poliovires, type 1 rct+/d+ and three rct+/d-, were related to wild strains.

Human monoclonal antibodies to cytomegalovirus recognize viral epitopes on the surface of virus-infected cells.

Four human monoclonal antibodies directed against human cytomegalovirus were produced by fusing Sp2/HPT heteromyeloma cells with peripheral blood lymphocytes, after stimulation in vitro for 6 days. The human hybridomas have been maintained in culture for one year and secrete, when cultured in serum-free medium, between 3.1 and 8.1 micrograms/ml of antibodies/10(6) cells/24 hours. HCV-1 and HCV-2 are IgG kappa, while HCV-3 and HCV-4 are IgG3 lambda. The four monoclonal antibodies immunoprecipitate a viral protein of 64 kD. Kinetic studies using indirect immunofluorescence indicate that this antigen appears late in the viral infectious cycle. All four monoclonal antibodies recognize human cytomegalovirus prototype strains AD-169, Davis and Towne, and 14 clinical isolates collected between 1984 and 1987. No reactivity was observed with other human herpesviruses. While no neutralizing activity could be observed with the human monoclonal antibodies, binding assays on unfixed infected cells showed that they recognized viral epitopes located on the cell surface membrane. These hybridomas may be useful for future therapy of immunocompromised patients.

Heterohybridomas secreting human monoclonal antibodies against Haemophilus influenzae type b.

Seven human monoclonal antibodies (HmAb) directed against outer membrane antigens of Haemophilus influenzae type b (Hib) were produced by fusing Sp2/HPT heteromyeloma cells with human tonsillar lymphocytes sensitized in vitro for 6 days. The heterohybridomas were maintained in culture for at least one year and secreted, when cultured in Dulbecco's modified Eagle's medium without fetal calf serum, between 1 and 15 micrograms/10(6) cells/ml/24 h. All of the HmAb were IgGs except HiH-12 which is an IgM. Antibodies directed against the lipopolysaccharide and proteins of apparent molecular masses of 43, 37 and 27 kDa were identified by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer membrane. Binding radioimmunoassay with live bacteria showed that five out of seven HmAb adsorbed to cell surface-exposed antigenic determinants. HmAb HiH-6, HiH-7 and HiH-10 reacted with a surface-accessible determinant on the 43-kDa outer membrane protein. In a dot enzyme immunoassay, these HmAb recognized 103 out of 111 Hib strains isolated worldwide. The strains were selected to represent the most common genotypic variations among Hib. None of these HmAb reacted with other bacterial species tested. These HmAb may serve to study the bacterial surface antigens implicated in the human humoral response and protection to Hib infections.

Anti-idiotypic mimicry of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus.

Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody.

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.

Mouse models of infection for Neisseria meningitidis B,2b and Haemophilus influenzae type b diseases.

A disseminated and fatal infection was established in C57BL mice, injected intraperitoneally with either Neisseria meningitidis B,2b or Haemophilus influenzae type b bacteria plus enhancement factors. The effects of mucin, hemoglobin, and iron dextran as enhancement of bacterial infectivity in mice were evaluated individually and in combination. A mixture of mucin and hemoglobin was most effective in enhancing the virulence of the pathogens. Inbred mouse lines were more susceptible than outbred ones. Relative virulence of a number of bacterial strains was also compared in one selected mouse line. Neisseria meningitidis B,2b and Haemophilus influenzae type b strains were more virulent than non-B,2b and nontypable strains. Finally, the course of bacteremia for the two infections in mice was followed by quantitative blood cultures. The animals succumbed to the generalized condition within 72 h. In the case of Neisseria meningitidis B,2b, 10 organisms with 4% mucin and 1.6% hemoglobin were sufficient to kill 50% of the animals. For Haemophilus influenzae type b, 300 bacteria with 5% mucin and 2% hemoglobin were necessary to obtain similar effects.