2013. immunogenicity in wild-type mice. From these mutants, we selected two, K219N and G220S, to combine with the stabilized double-mutant FHbp antigen. The two triple mutants decreased FH binding >200-fold, increased the thermal stability of the N-terminal domain name by 21C, and bound better to an anti-FHbp MAb than the wild-type FHbp. In human-FH-transgenic mice, the FHbp triple mutants elicited 8- to 15-fold-higher protective antibody responses than the wild-type FHbp antigen. Collectively, the data suggest that mutations to eliminate binding of human FH and to promote conformational stability act synergistically to optimize FHbp immunogenicity. INTRODUCTION serogroup B is one of the leading causes of bacterial meningitis and sepsis in North America and the European Union (1, 2). The disease burden is usually highest in infants (2), who have not yet developed natural immunity, and in young adults living under crowded housing conditions, such as dormitories and military barracks. Two protein-based vaccines were recently developed to protect against meningococcal serogroup B disease. One of the vaccines, MenB-FHbp (Pfizer), is usually licensed in the United States; the second, MenB-4C (Bexsero; GSK), is usually licensed in the United States, the European Union, Australia, and Canada. MenB-4C is now part of the routine immunization program in the United Kingdom. In the United States, both vaccines are recommended for persons at increased risk of acquiring meningococcal serogroup B disease, including those with persistent complement deficiencies, those potentially exposed during serogroup B outbreaks, GW 7647 and microbiologists with routine exposure to (3). Both of the licensed serogroup B vaccines include factor H binding protein (FHbp), which is a highly sequence-variable surface antigen; more than 930 GW 7647 amino acid sequence variants have been identified to date (http://pubmlst.org/neisseria/fHbp). Based on amino acid sequence identity, FHbp variants can be classified in two subfamilies (4), three variant groups (5), or 10 modular groups (6). The two licensed vaccines contain divergent FHbp sequence variants in variant group 1, which corresponds to subfamily B. In addition, the MenB-4C vaccine containing nonlipidated FHbp uses aluminum as an adjuvant, whereas the MenB-FHbp vaccine relies on aluminum and the adjuvant properties of the lipid moieties of the two FHbp variants. The MenB-FHbp vaccine includes an FHbp sequence variant from each of the two subfamilies (7), whereas the MenB-4C vaccine includes FHbp and three GW 7647 other protective antigens (8, 9). Thus, two different strategies Acta2 were used to expand the cross-protection by antibodies elicited by the licensed vaccines against diverse meningococcal strains. Meningococci recruit the complement regulator factor H (FH), using FHbp (10) and several alternative ligands, including neisserial surface protein A (NspA) (11) and porin B2 (PorB2) (12). By binding FH using one or more of these ligands, meningococci downregulate complement alternative pathway amplification, which renders the bacteria more resistant to complement-mediated killing. Antibodies to FHbp elicit complement-mediated bactericidal activity and can inhibit binding of FH to FHbp, which defeats this bacterial evasion mechanism. However, in human-FH-transgenic mice, binding of FH to the FHbp vaccine antigen decreases protective antibody responses, possibly by interfering with antigen uptake, processing, or presentation. To GW 7647 overcome this limitation of the FHbp antigen, considerable effort has been devoted to identifying mutant FHbp antigens with decreased binding of FH, including structure-based (13,C16) and mutant library (17) approaches. Candidate mutants have been identified in variant groups 1 (13, 14, 16), 2 (15, 16, 18), and 3 (16), and a subset of these mutants have been evaluated in human-FH-transgenic-mouse immunogenicity models. In previous studies, we investigated the vaccine potential of FHbp ID 22 in variant group 2, since this sequence variant is prevalent in serogroup W strains in sub-Saharan Africa (19, 20) and the same or similar sequence variants are present in serogroup B strains in the United States and the European Union (1, 21). 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 GW 7647 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 21C-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.