ConformationalEpitopes Recognized by Protective Anti-Neisserial Surface Protein AAntibodies

Abstract

NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-stranded ␤-barrel structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein.

Comparative Proteome Analysis of Spontaneous Outer Membrane Vesicles and Purified Outer Membranes of Neisseria meningitidis

“…ELISA for quantification of NspA was performed as described previously (43), with the following exceptions. Twenty-microliter aliquots containing 1, 5, 10, 20, or 40 g of total SOMV or OM fraction protein were added to wells of flat-bottom 96-well microtiter plates (Greiner bioOne).…”

Posttranslational Modification of RecombinantPlasmodium falciparumApical Membrane Antigen 1: Impact on Functional Immune Responses to a Malaria Vaccine Candidate

“…FUNCTIONAL IMMUNE RESPONSE TO RECOMBINANT PfAMA1 3967 pressed as full-length molecules (N-terminal sequencing, reactivity with C-terminal His tag, and SDS-PAGE analysis) and in the correct conformation (shift in apparent mobility under reduced and nonreduced conditions for disulfide formation [41,42] and reactivity with conformation-specific monoclonal antibody to only the nonreduced form [21,41]). We have also produced the same allelic forms from the eukaryotic P. pastoris system (24).…”

Impaired Immunogenicity of Meningococcal Neisserial Surface Protein A in Human Complement Factor H Transgenic Mice

“…However, in our previous study, serum from mice immunized with purified unfolded NspA failed to elicit complement-mediated bactericidal activity (15), even after attempts to reconstitute the protein in liposomes (14). The E. coli microvesicle/recombinant NspA vaccine tested in the present study contained a fully functional NspA that bound human FH and, therefore, provided a convenient means to investigate the possible effect of human FH binding on NspA immunogenicity.…”

“…Further, in future studies this approach could also be used to investigate the immunogenicity of low-FH-binding mutant recombinant NspA vaccines in human FH transgenic mice (as was done for FHbp antigens [11,26,30,31]). For a vaccine intended for use in humans, the expression of NspA in N. meningitidis is more likely to retain native folding and important epitopes for eliciting bactericidal antibodies than the expression of a purified recombinant NspA in E. coli (14). Thus, it should be possible to prepare a meningococcal NOMV-NspA vaccine from a mutant N. meningitidis strain with a genetically attenuated endotoxin and overexpressed NspA by methods similar to those used to prepare meningococcal NOMV vaccines with overexpressed FHbp (27,32,33) which are intended for use in humans.…”

“…In previous studies, purified recombinant NspA vaccines failed to elicit serum bactericidal antibody responses in wild-type mice, which were thought to result from the failure of the recombinant protein to be properly folded (14,15). In contrast, microvesicles released from E. coli cells that had been transformed with the NspA gene elicited serum anti-NspA bactericidal activity (15).…”