In either case, the new mutants might be valuable in producing second-generation double mutants with even lower FH binding, or in combining two different single mutants into a FHbp vaccine that retains all of the native epitopes

In either case, the new mutants might be valuable in producing second-generation double mutants with even lower FH binding, or in combining two different single mutants into a FHbp vaccine that retains all of the native epitopes. One limitation of the library approach for discovery of FHbp mutants is that induction of FHbp expression appeared to be moderately toxic to em E /em . renamed FHbp after it was discovered to bind the human complement regulator Factor H (FH) [10]. Subsequent studies showed that binding of FH to FHbp was specific for human and chimpanzee FH [11] and for FH from a subset of rhesus macaques [12]. Vaccines made up of FHbp are immunogenic in mice [13, 14], rabbits [15], macaques [16, 17] and humans [7, 8, 18C20]; however the protective antibody responses of humans appear to be lower than those of mice [21]. Using human FH transgenic mice, we previously showed that binding of human FH decreased meningococcal FHbp vaccine immunogenicity compared with that in control mice [22]. The structure of a complex XCL1 of FHbp bound to a fragment of human FH enabled the design of FH non-binding FHbp mutants [23]. We subsequently recognized an FHbp mutant with arginine 41 replaced with serine (R41S), which retained immunogenicity in control mice in which FH does not bind FHbp, and experienced enhanced immunogenicity in R788 (Fostamatinib) human FH transgenic mice [22, 24]. Our hypothesis is usually that a mutant FHbp antigen with very low FH binding and increased immunogenicity in human FH transgenic mice will translate into greater vaccine efficacy in humans. Many FHbp mutants with decreased binding of human FH since have been identified [25C28]. However, some FHbp mutants have decreased immunogenicity compared with a wild-type FHbp vaccine in control mice in which FH does not bind to either FHbp vaccine. These data suggest that mutations that decrease FH binding can result in the loss of epitopes important for eliciting protective antibody. In the present study, we developed a novel approach to identify novel FHbp antigens using a random mutant FHbp library displayed on the surface of DNA polymerase and dNTPs at 70C for 10 min to expose dA overhangs, and were cloned into pGEM-T-Easy (Promega). The clones were transformed into DH5 (Invitrogen). Plasmid DNA was prepared (Qiagen) and 30 clones were subjected to DNA sequencing with the T7 primer. The mutation rate was 3 to 5 5 nucleotides per FHbp gene, and one to two amino acid residues per FHbp. The error-prone mutant library was digested with the restriction endonucleases C41(DE3) (Lucigen) for surface expression of the FHbp mutant library. Fluorescence-activated cell sorting of display library For sorting of cells expressing mutant FHbp, the library was produced in Luria-Bertani (LB) medium (BD Biosciences) made up of 50 g/ml of kanamycin (Sigma) to stationary phase. The cultures were diluted 1:25 into new LB medium and grown to an OD at 600 nm of 0.6. FHbp expression was induced with 1 mM IPTG for 2 h at 37 C. The bacterial cells were incubated with 50 g/ml of purified human FH (Match Technologies) for 30 min at 37C. The cells were washed twice with Dulbeccos PBS (DPBS) (Mediatech) made up of 1% BSA (Equitech). The primary antibodies were goat anti-human FH (Match Technologies; 2 g/ml), which had been affinity purified on an FH column, or a mixture of control anti-FHbp mAbs JAR R788 (Fostamatinib) 41 [29] R788 (Fostamatinib) and mAb 502 [30] (10 g/ml each), which were added and incubated for 30 min at 37C. Note that neither of these mAbs inhibited binding of FH to FHbp [31], which enabled co-immunostaining with FH. The secondary antibodies R788 (Fostamatinib) were donkey anti-goat IgG conjugated to AlexaFluor 488 (Invitrogen; 1:1000 dilution) and donkey anti-mouse IgG-AlexaFluor 647 (Invitrogen; 1:1000 dilution), which were added and incubated for 15 min at room heat. The cells were washed.