No abstract

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N-and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N-and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N-and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the Nand C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.

The distribution of the two isotypes of tbpB in a collection of 108 serogroup B meningococcal strains belonging to the four major clonal groups associated with epidemic and hyperendemic disease (the ET-37 complex, the ET-5 complex, lineage III, and cluster A4) was determined. Isotype I strains (with a 1.8-kb tbpB gene) was less represented than isotype II strains (19.4 versus 80.6%). Isotype I was restricted to the ET-37 complex strains, while isotype II was found in all four clonal complexes. The extent of the allelic diversity of tbpB in these two groups was studied by PCR restriction analysis and sequencing of 10 new tbpB genes. Four major tbpB gene variants were characterized: B16B6 (representative of isotype I) and M982, BZ83, and 8680 (representative of isotype II). The relevance of these variants was assessed at the antigenic level by the determination of crossbactericidal activity of purified immunoglobulin G preparations raised to the corresponding recombinant TbpB (rTbpB) protein against a panel of 27 strains (5 of isotype I and 22 of isotype II). The results indicated that rTbpB corresponding to each variant was able to induce cross-bactericidal antibodies. However, the number of strains killed with an anti-rTbpB serum was slightly lower than that obtained with an anti-TbpA ؉ B complex. None of the sera tested raised against an isotype I strain was able to kill an isotype II strain and vice versa. None of the specific antisera tested (anti-rTbpB or anti-TbpA ؉ B complex) was able to kill all of the 22 isotype II strains tested. Moreover, using sera raised against the C-terminus domain of TbpB M982 (amino acids 352 to 691) or BZ83 (amino acids 329 to 669) fused to the maltose-binding protein, cross-bactericidal activity was detected against 12 and 7 isotype II strains, respectively, of the 22 tested. These results suggest surface accessibility of the C-terminal end of TbpB. Altogether, these results show that although more than one rTbpB will be required in the composition of a TbpB-based vaccine to achieve a fully cross-bactericidal activity, rTbpB and its C terminus were able by themselves to induce cross-bactericidal antibodies.

Because the meningococcal transferrin receptor was shown to elicit bactericidal and protective antibodies in laboratory animals, we undertook a study of the protective role of each of the polypeptides within the Tbp1-Tbp2 complex. We developed a procedure to purify from Neisseria meningitidis B16B6 the two proteins in milligram amounts and raised specific antisera in rabbits and mice. Only antisera specific for Tbp2 displayed bactericidal activity against the parent strain. Mice immunized with purified Tbp2 survived a lethal challenge to a similar degree as animals immunized with the Tbp1-Tbp2 complex, demonstrating that Tbp2 played an important role in the protective activity observed with the complex. Both Tbp1-and Tbp2-specific antisera inhibited transferrin binding to the purified receptor in a solid-phase binding assay, suggesting that the antibodies were able to interact with the Tbp1 molecule only when it was removed from its membrane environment. Finally, Tbp2-specific immunoglobulins were able to lower the growth rate of the meningococci when human transferrin was their sole iron source. Therefore, in all four different systems tested, Tbp2 or antibodies specific for Tbp2 displayed biological characteristics close to those of the Tbp1-Tbp2 complex. This suggests that Tbp2 plays an important role in the protective activity of the complex, eliciting antibodies that are not only bactericidal but also inhibitory for meningococcal growth.

Transferrin-binding protein B (TbpB) is a surface-exposed protein, variable among strains of Neisseria meningitidis, that has been considered as a vaccine candidate. To define a TbpB molecule that would give rise to broadly cross-reactive antibodies with TbpB of many strains, specific antisera were produced against three recombinant TbpB variants from strain M982: one corresponding to the full-length TbpB; one in which stretches of amino acids located in the central part of the molecule, described as hypervariable, have been deleted; and one corresponding to the N-terminal half of the molecule, described as the human transferrin binding domain. The reactivity of these antisera against 58 serogroup B strains with a 2.1-kb tbpB gene representing different genotypes, serotypes, and subtypes and different geographic origins was tested on intact meningococcal cells. In parallel, the bactericidal activity of the antisera was evaluated against 15 of the 58 strains studied. Of the 58 strains, 56 (98%) reacted with the antiserum specific for the N-terminal half of TbpB M982; this antiserum was bactericidal against 9 of 15 strains (60%). On the other hand, 43 of 58 strains reacted with the antiserum raised to full-length TbpB while 12 of 15 (80%) were killed with this antiserum. The antiserum specific to TbpB deleted of its central domain gave intermediate results, with 53 of 58 strains (91.3%) recognized and 10 of 15 (66.6%) killed. These results indicate that the N-terminal half of TbpB was sufficient to induce cross-reactive antibodies reacting with the protein on meningococcal cells but that the presence of the C-terminal half of the protein is necessary for the induction of cross-bactericidal antibodies.