Genome-derived neisserial antigen 2132 (GNA2132) is a novel vaccine candidate that was identified during the Neisseria meningitidis group B strain MC58 genome-sequencing project. To assess the vaccine potential of GNA2132, we prepared antisera from mice immunized with recombinant GNA2132 (gene from strain NZ394/98). Anti-GNA2132 antibody bound to the surface of live bacteria from all 7 capsular group B or C strains tested and elicited deposition of human C3b on the bacterial surface. However, with human or infant-rat complement, anti-GNA2132 had no detectable bactericidal activity (titer, <1:4) against the nominal strain, NZ394/98, and was bactericidal against only 2 of the other 6 strains tested. These differences between strains were unrelated to GNA2132 amino acid sequence or level of protein expression. Despite lack of bactericidal activity, anti-GNA2132 antiserum passively protected infant rats against meningococcal bacteremia after challenge with all 5 resistant strains. GNA2132 is thus a promising vaccine candidate for prevention of disease caused by N. meningitidis.

A calcitonin analogue, MCT-II, having the potential to form an amphiphilic alpha-helix from residue 8 to residue 22 with a continuous surface of aliphatic leucine side chains on the hydrophobic face of the helix has been synthesized, and its physical and biological properties have been characterized. Properties exhibited by this peptide, including self-association in the micromolar concentration range with a concomitant increase in the percentage of alpha-helical structure, formation of stable monolayers at the air-water interface, and adsorption to the surface of egg lecithin single-bilayer vesicles, demonstrate that MCT-II can readily form an amphiphilic alpha-helical structure. Though MCT-II has minimal sequence homology to any particular natural analogue from residue 8 to residue 22, it has biological activity similar to that of salmon calcitonin I for receptor binding in brain and kidney membranes, for activation of adenylate cyclase, and in hypocalcemic potency in vivo. The amphiphilic alpha-helical structure of MCT-II, therefore, is important for binding to calcitonin receptors. It is also apparent that a hydrophilic residue commonly occurring on the hydrophobic face (position 15) in the natural calcitonins is not required for high biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The capsular polysaccharide of Neisseria meningitidis group B is an autoantigen, whereas noncapsular antigens are highly variable. These factors present formidable challenges for development of a broadly protective and safe group B vaccine. Mice and guinea pigs were sequentially immunized with three doses of micovesicles or outer membrane vesicles prepared from three meningococcal strains that were each antigenically heterologous with respect to the two major porin proteins, PorA and PorB, and the group capsular polysaccharide. The resulting antisera conferred passive protection against meningococcal group B bacteremia in infant rats and elicited complement-mediated bactericidal activity against genetically diverse group B strains that were either homologous or heterologous with respect to PorA of the strains used to prepare the vaccine. By using knockout strains, a portion of the bactericidal antibody was directed against the highly conserved protein, neisserial surface protein A (NspA). Further, an anti-NspA monoclonal antibody elicited by the sequential immunization was highly bactericidal against strains that were previously shown to be resistant to bacteriolysis by anti-NspA antibodies produced by immunization with recombinant NspA. Sequential immunization with heterologous vesicle preparations offers a novel approach to eliciting broadly protective immunity against N. meningitidis strains. An NspA-based vaccine prepared from protein expressed by Neisseria also may be more effective than the corresponding recombinant protein made in Escherichia coli.

Neisserial surface protein A (NspA) is currently being investigated with humans as a candidate vaccine for the prevention of meningococcal disease. Although NspA is highly conserved, the ability of anti-NspA antibodies to bind to or elicit complement-mediated bactericidal activity against diverse Neisseria meningitidis serogroup B strains is controversial. To evaluate strain differences in NspA surface accessibility and susceptibility to bactericidal activity, we prepared murine immunoglobulin G2a anti-NspA monoclonal antibodies (MAbs) and evaluated their functional activity against 10 genetically diverse N. meningitidis serogroup B strains. By colony Western blot, all 10 strains expressed NspA as detected by one or more MAbs. By flow cytometry, two MAbs were found to bind to the bacterial surface of 6 of the 10 strains. In addition, two strains showed variable NspA surface accessibility for the MAbs despite being uniformly positive for NspA expression by colony Western blotting. Only 4 of the 10 strains were susceptible to anti-NspA complement-mediated bacteriolysis. Passively administered MAb protected infant rats from developing bacteremia after challenge with N. meningitidis serogroup B strain 8047 (surface binding positive, susceptible to anti-NspA bacteriolysis), was poorly protective against strain BZ232 (surface binding variable, resistant to bacteriolysis), and did not protect against strain M986 (surface binding negative, resistant to bacteriolysis). Finally, NspA does not appear to be critical for causing bacteremia, as an NspA knockout from strain 8047 was highly virulent in infant rats. Taken together, these findings suggest that an NspA-based vaccine will need to incorporate additional antigens to elicit broad protection against N. meningitidis serogroup B.

A 12 amino acid peptide, model BB, was designed to adopt a beta-hairpin tertiary structure in water that might be stabilized by a variety of local, nonlocal, polar, and nonpolar interactions. The conformational properties of model BB with and without an intramolecular disulfide bond (BB-O and BB-R, respectively) were characterized by NMR and CD spectroscopy. The set of observed short- and medium-range nOes were consistent with the formation of stable beta-hairpin-like structures by both BB-R and BB-O. BB-O adopts two distinct conformations that differ from each other in the designed reverse turn segment. A reasonably well-defined set of structures was obtained by using restraints from the NMR data in distance geometry calculations. None of the beta-hairpin-like structures contain a beta-sheet hydrogen bonding network. The distinctive feature of intrastrand and cross-strand pairing of threonine residues observed in all of the calculated structures suggests that hydrophobic interactions between the gamma-methyl groups of threonine residues may be the structure-determining interaction in model BB. The implications of these results for the formation of beta-sheets during protein folding, the aggregation of peptides as beta-sheets, and the de novo design of independently folding beta-hairpin-like peptides are considered.