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ABSTRACTHomologous recombination was used to generate a number of mutants of serogroup B Neisseria meningitidisB16B6 with the following characteristics: (i) an inability to bind human or porcine transferrin because of loss of both transferrin binding proteins (Tbp) A and B [strain B16B6(Strr)/tbpA−B−] and (ii) an ability to bind porcine transferrin but not human transferrin [strain B16B6(Strr)/tbpAapBap] due to replacement of the meningococcal Tbp with the Tbp ofActinobacillus pleuropneumoniae. During construction of the B16B6(Strr)/tbpAapBapstrain, transformants expressing only TbpA or TbpB of A. pleuropneumoniaewere isolated [strains B16B6(Strr)/tbpAapB−and B16B6(Strr)/tbpA−Bap]. Expression of the A. pleuropneumoniaeTbp in N. meningitidisB16B6 was iron regulated and expressed under the control of the meningococcal promoter. The relative abilities of the meningococcal transformants to bind porcine transferrin were in the order B16B6(Strr)/tbpAapBap> B16B6(Strr)/tbpAapB−> B16B6(Strr)/tbpA−Bap. Of these transformants, only B16B6(Strr)/tbpAapBapcould grow in the presence of porcine transferrin as the sole iron source, achieving a growth rate similar to that of the B16B6 parent strain in the presence of human transferrin.

ABSTRACTIn search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis(strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing fromN. meningitidisinfection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients’ T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2,778 bp). The strong CD4+T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients’ serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspAfailed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeaegenome sequences, suggesting that tspAis unique to the genusNeisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimuriumandEscherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4+T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.

ABSTRACTClostridium difficiletoxin A binds nonspecifically to a mouse monoclonal antibody (MAb) immunoglobulin G3 λ chain [IgG3(λ)], through the Fab component. This binding, which is retained even after boiling the MAb, is temperature dependent, with more toxin bound at 4 than 37°C (P= 0.0024). The nonspecific binding was decreased by incubation of the IgG3 λ MAb with α- or β-galactosidase (P= 0.0001 and 0.029, respectively), indicating that toxin A binds to a carbohydrate moiety on the Fab. However, binding was not blocked by the Bandeiraea simplicifolialectin BS-1, indicating that a terminal α-galactose may not be involved. Binding was also not affected by competitive assays with Lewis X antigen. The dependence on carbohydrate moieties in nonspecific binding was also shown for two other MAbs, IgA(κ) and IgM(λ), with demonstration of a significant reduction in binding with α-galactosidase (P= 0.0001 and 0.0002, respectively) but not β-galactosidase (P= 0.27 and 0.25, respectively).