Characterization of a human-mouse chimeric antibody reactive with a human melanoma associated antigen.

The data presented demonstrate that a human-murine chimeric antibody has been generated that retains the immunoreactivity and has similar pharmacokinetic properties of its parent murine monoclonal antibody NRML-05. A competitive ELISA assay demonstrated that antigen reactivity of both parent and chimera were nearly identical. In a direct cell binding assay, NRML-05 and chimeric antibodies were immunoreactive, 81% and 83%, respectively. Scatchard Analysis of the antibodies indicate very similar affinities for NRML-05 (4.4 x 10(9) M-1) and chimera (2.7 x 10(9) M-1). Localization of the two antibodies to tumor xenografts in nude mice were very similar in biodistribution studies. The chimeric antibody is not as well recognized by antiglobulin from patients who have responded to non-idiotypic murine antibody determinants. Although this data does not predict how immunogenic a chimera would be in the clinical setting, it does suggest that patients without significant anti-idiotypic antiglobulin response could benefit from second and subsequent administrations with chimeric antibody. Even though anti-idiotypic responses may occur with the chimera, these may be reduced as a result of the presentation of these epitopes on the less immunogenic human constant domains.

Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli.

The genes encoding the S1 and S2 subunits of pertussis toxin were expressed in Escherichia coli under lac operon transcription and translation control with pUC8 and pUC18 as the expression vectors. Various versions of the subunits were detected with anti-S1 or anti-S2 monoclonal antibodies. Recombinant S1, but not S2, subunit contained the enzymatic NAD-glycohydrolase and NAD:Gi ADP-ribosyltransferase activities. Both activities were also expressed by a truncated version of the S1 subunit in which the 48 carboxy-terminal amino acid residues, including a predicted Rossman structure and one of the two cysteines, had been deleted. The epitope for an anti-S2 monoclonal antibody was localized to the N-terminal 40-amino-acid region of the S2 subunit. Both the S1 and S2 subunits expressed in E. coli reacted with human hyperimmune serum. The full length and the truncated recombinant S1 subunit also reacted in Western blots with a neutralizing and protective monoclonal anti-S1 antibody. The different versions of S1 and S2 subunits expressed in E. coli are useful for mapping active sites, epitopes, and regions that interact with receptors or the other subunits in the holotoxin. These recombinant subunits will also facilitate the development of a safer, new-generation vaccine against whooping cough.

Detection of subunits of pertussis toxin in Tn5-induced Bordetella mutants deficient in toxin biological activity.

Monoclonal antibodies with specificity for pertussis toxin subunits S1, S2, and S4 were used in Western blots to show that the subunits were not secreted into culture medium from Tn5 insertion mutants. The mutants are deficient in toxin biological activities due to an insertion in the S3 subunit structural gene. The Western blots demonstrated that each of the respective subunits was exported in a wild-type strain. Anti-S1 and anti-S2 monoclonal antibodies were capable of detecting subunits in solubilized whole-cell material from a wild-type strain and from the Tn5 mutants lacking only in biologically active toxin (Tox-). Another Tn5 insertion mutant, lacking all known B. pertussis virulence factors (Vir-), did not produce any of the subunits either in whole cellular extracts or in culture supernatants. The data demonstrate that Tn5 Tox- insertion mutants, though defective in toxin activity, synthesize some toxin subunits. The presence of the S3 subunit is most likely a necessity for transport of the toxin from cells. Alternatively, a nonstructural gene coding for a protein involved in transport of the toxin across the membrane may be affected by the Tn5 mutation.

Nucleotide sequence homology to pertussis toxin gene in Bordetella bronchiseptica and Bordetella parapertussis.

Multiple strains of Bordetella parapertussis and B. bronchiseptica were examined for the presence of nucleotide sequences which hybridized with a cloned 4.5-kilobase (kb) fragment of B. pertussis DNA containing the genes responsible for pertussis toxin expression. All six B. parapertussis strains tested had nucleic acid sequences that hybridized with the cloned 4.5-kb fragment in Southern blot analyses. When the B. parapertussis DNA was digested with restriction endonuclease PstI, the pattern of hybridization was identical to that obtained with B. pertussis. Only five of the seven B. bronchiseptica strains tested had sequences that hybridized with the 4.5-kb fragment. Three of these B. bronchiseptica strains had a hybridization pattern identical to B. pertussis upon PstI digestion and Southern blot analyses. Two B. bronchiseptica strains were shown to lack a PstI cleavage site downstream from the region analogous to that coding for the pertussis toxin structural genes. Monoclonal antibody analyses were unable to detect pertussis toxin subunits S1 and S2 in Western blots with cellular material or culture supernatant from several B. bronchiseptica and B. parapertussis strains that possessed the DNA homologies. In addition, preliminary Northern hybridizations with RNA isolated from B. bronchiseptica and B. parapertussis strains suggested that the homologous regions were not transcribed. The data show that the gene coding for the toxic component of B. pertussis is common in other Bordetella species, though the gene probably is not expressed.

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans.

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.

Phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum in virulent, tissue-cultured treponemes.

Specific monoclonal antibody and Western blot analysis were used to examine the phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum among organisms cultivated in vitro. Tissue-cultured treponemes synthesized the 47 kDa immunogen as well as, or better than, organisms cultivated in vivo (rabbit testicles).

Molecular specificities of monoclonal antibodies directed against virulent Treponema pallidum.

Radioimmunoprecipitation (RIP) and Western blot analyses with specific anti-Treponema pallidum subsp. pallidum monoclonal antibodies were used to identify antigens with apparent masses of 102, 84, 54, 53, 52, 47, 32, 29, and 24 kilodaltons (kDa). Cross-reactivity of these antibodies with T. pallidum subsp. pertenue antigens and lack of cross-reactivity with T. phagedenis biotype Reiter, T. vincentii, T. refringens, T. scoliodontum, and T. denticola were also demonstrated by RIP and Western blot analyses. Reactivities in the T. pallidum immobilization test, along with the RIP of lactoperoxidase-catalyzed iodination products, suggested that the identified antigens were surface associated. The abundance and surface association of the 47- and 84-kDa antigens were supported by reactivity in the microhemagglutination test for T. pallidum and by strong reactivity of monoclonal antibodies upon indirect immunofluorescence assays with rabbit-cultivated T. pallidum subsp. pallidum, respectively, but not with T. phagedenis biotype Reiter. Anti-47-kDa and anti-84-kDa monoclonal antibodies were also reactive in indirect immunofluorescence assays using treponemes found in dark-field-positive smears of human genital ulcers.

The metabolism of 5'-methylthioadenosine and 5-methylthioribose 1-phosphate in Saccharomyces cerevisiae.

Cordycepin sensitive mutants of Saccharomyces cerevisiae, which are permeable to 5'-deoxy-5'-methylthioadenosine (MTA), were used to study the fate of the methylthioribose carbons of this purine nucleoside. Evidence is presented for the recycling of the methylthio group and part of the ribose portion of MTA in a biosynthetic pathway which leads to the synthesis of methionine. The main pathway involves the phosphorylytic cleavage of MTA by MTA phosphorylase yielding 5-methylthioribose 1-phosphate and adenine as products. Loss of the phosphate group of 5-methylthioribose 1-phosphate, concurrent with the rearrangement of the ribose carbons, leads to the synthesis of 2-keto-4-methylthiobutyric acid. In the final step of the sequence, 2-keto-4-methylthiobutyric acid is converted to methionine via transamination. Several compounds not directly associated with the biosynthesis of methionine were also isolated. These compounds, which may arise through the degradation of intermediates in the pathway, were: 5'-methylthioinosine, a deaminated catabolite of MTA; 5-methylthioribose, a result of the phosphorylysis of 5-methylthioribose 1-phosphate, and 3-methylthiopropionaldehyde, 3-methylthiopropionic acid and 2-hydroxy-4-methylthiobutyric acid, all arising from the catabolism of 2-keto-4-methylthiobutyric acid.

Monoclonal antibody with hemagglutination, immobilization, and neutralization activities defines an immunodominant, 47,000 mol wt, surface-exposed immunogen of Treponema pallidum (Nichols).

Radioimmunoprecipitation (RIP) analyses performed on 125I-surface-labeled Treponema pallidum cells using various immune sera revealed the presence of six major surface antigens (immunogens) with apparent molecular weights of 47 K, 36 K, 34 K, 32 K, 29 K, and 13 K. Among these, the 47 K surface antigen was most abundant. Radioimmunoprecipitation assays using 125I-labeled T. phagedenis biotype Reiter or immunoblot analyses using the same strain, failed to reveal the presence of the 47 K mol wt antigen in the representative nonpathogenic treponeme. Preabsorption of anti-T. pallidum immune rabbit serum (IRS) with the Reiter organism did not remove anti-T. pallidum antibodies from immune serum that reacted with the 47 K mol wt immunogen or other immunogens of T. pallidum present in the characteristic antigenic profile. Monoclonal antibodies (mAb) directed specifically against the 47 K mol wt immunogen of T. pallidum also failed to react with an analogous 47 K mol wt component in Treponema phagedenis biotype Reiter, further suggesting the unique presence of this antigen in pathogenic treponemes. The presence of the 47 K mol wt surface immunogen in pathogenic treponemes other than T. pallidum subspecies pallidum was also observed (43). Anti-47 K immunogen mAb was nonreactive against rabbit IgG or IgM. mAb directed specifically against the 47 K mol wt immunogen of T. pallidum was examined for strategic functional activities. It was found to be reactive in the microhemagglutination assay for T. pallidum antibodies, the T. pallidum immobilization test, and was found to be capable of significant blockage of attachment of virulent T. pallidum to host cells in tissue culture. Additional significant biological activity for the anti-47 K mol wt immunogen mAb was revealed through results of the in vitro-in vivo neutralization test of Bishop and Miller, in which a 99% or 100% neutralizing activity was demonstrated. The combined data of this study suggest that the 47 K mol wt immunogen of T. pallidum represents an abundant, immunodominant, surface-exposed immunogen possessing potential biological importance in the pathogenesis and immunology of T. pallidum infection. These studies serve to establish the first functionally defined immunogen for T. pallidum, which may represent the major immunogen of the organism.

Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponema pallidum subspecies.

Murine monoclonal antibodies directed against a 47,000-dalton immunodominant surface-exposed antigen of Treponema pallidum subsp. pallidum (Nichols) were isolated. These monoclonal antibodies cross-reacted with analogous 47,000-dalton antigens of two other virulent treponemes, T. pallidum subsp. pertenue and T. pallidum subsp. endemicum (Bosnia A), as determined by radioimmunoassay and immunoblot analyses. Immunoelectron microscopy confirmed that the 47,000-dalton antigen of T. pallidum subsp. pallidum was a surface-associated cellular component. Surface binding assays and immunoelectron microscopic studies also suggested that the analogous 47,000-dalton antigenic component of T. pallidum subsp. pertenue may not have been oriented toward the bacterial surface in the same way as the T. pallidum subsp. pallidum antigen or that the relevant antigenic determinant(s) may not have been exposed to the outer surface in the same way. The significance of this antigen relative to its apparent conservation among pathogenic treponemes and its possible diagnostic and vaccinogenic potentials are discussed.