A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes

Abstract

Factor H binding protein (FHbp) is part of two vaccines recently licensed for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp is classified in three phylogenic variant groups that have limited antigenic cross-reactivity, and FHbp variants in one of the groups have low thermal stability. In the present study, we replaced two amino acid residues, R130 and D133, in a stable FHbp variant with their counterparts (L and G) from a less stable variant. The single and double mutants decreased thermal stability of the amino- (N-) terminal domain compared with the wild-type protein as measured by scanning calorimetry. We introduced the converse substitutions, L130R and G133D, in a less stable wild-type FHbp variant, which increased the transition midpoint (Tm) for the N-terminal domain by 8 and 12 °C; together the substitutions increased the Tm by 21 °C. We determined the crystal structure of the double mutant FHbp to 1.6 Å resolution, which showed that R130 and D133 mediated multiple electrostatic interactions. Monoclonal antibodies specific for FHbp epitopes in the N-terminal domain had higher binding affinity for the recombinant double mutant by surface plasmon resonance and to the mutant expressed on meningococci by flow cytometry. The double mutant also had decreased binding of human complement Factor H, which in previous studies increased the protective antibody responses. The stabilized mutant FHbp thus has the potential to stabilize protective epitopes and increase the protective antibody responses to recombinant FHbp vaccines or native outer membrane vesicle vaccines with overexpressed FHbp.

Meningococcal Factor H Binding Protein Vaccine Antigens with Increased Thermal Stability and Decreased Binding of Human Factor H

“…The two substitutions in FHbp ID 22 that increased the thermal stability in our previous study also decreased binding of human FH by 7-fold compared with the wild-type FHbp (22). Since previous studies of FHbp mutants suggested that at least 30-to 100-fold decreases in FH binding were needed to achieve optimal immunogenicity in the presence of human FH (17,18), in the present study, we sought additional FHbp substitutions to decrease FH binding further.…”

“…As described in the introduction, we previously engineered a double mutant, L130R G133D, of FHbp ID 22 in variant group 2 that had 21°C higher thermal stability of the N-terminal structural domain (22). In the present study, to evaluate whether the mutations that increased the thermal stability of FHbp also affected its immunogenicity in the absence of binding of human FH, we immunized groups of wild-type CD-1 mice with two doses of the recombinant FHbp ID 22 wild-type or double-mutant protein.…”

“…The oligonucleotide primers had first been phosphorylated with polynucleotide kinase (New England BioLabs). The L130R G133D double mutant, which was engineered to increase thermal stability, was described previously (22). Triple mutants were constructed using the pET21b-FHbp ID 22 L130R G133D mutant as the plasmid template and the appropriate primers.…”

“…Additional studies showed that FHbp antigens in variant group 2 were less thermally stable than those in variant group 1 or 3 (16,18). We recently stabilized an FHbp variant group 2 protein by replacement of two amino acid residues, L130 and G133, with their counterparts, R130 and D133, from a variant group 1 protein (22). The L130R G133D double mutant had 21°C-higher thermal stability of the N-terminal domain and 7-fold-decreased binding of human FH compared to the wild-type FHbp ID 22 (22).…”

“…In the present study, we combined the amino acid substitutions previously reported to increase FHbp stability (22) with new, additional single-amino-acid substitutions to decrease further binding of human FH to FHbp antigens. Collectively the data show the vaccine potential of several novel FHbp mutants in variant group 2, which could be combined with other FHbp sequence variants or other meningococcal antigens to increase the protective antibody responses to maximize the breadth of protection against meningococci.…”