Article Figures & Data
Figures
- FIG. 1.
Concentration-dependent binding of anti-fHbp MAbs to recombinant fHbp v.2 or v.3 as measured by ELISA. JAR 10, 11, and 13 (solid lines) were from a mouse immunized with a recombinant v.2 protein. JAR 32, 33, 35, and 36 (dashed lines) were from a mouse immunized with a recombinant v.3 protein. (A) Binding to fHbp v.2 (encoded by a gene from strain 8047). (B) Binding to fHbp v.3 (encoded by a gene from strain M1239). OD, optical density.
- FIG. 2.
Inhibition of the binding of human fH to recombinant fHbp by anti-fHbp MAbs as measured by ELISA. (A) Inhibition of binding to fHbp v.1. (B) Inhibition of binding to fHbp v.2. (C) Inhibition of binding of fH to fHbp v.3. The recombinant v.1 protein is from the gene of strain MC58. Respective v.2 and v.3 recombinant proteins are those used in experiments shown in Fig. 1.
- FIG. 3.
Western blot showing residues that are important for the binding of anti-fHbp MAbs JAR 10 and JAR 33. Residues lysine (K) 180 and glutamate (E) 192 were involved in the JAR 10 epitope, and arginine (R) 180 was essential for the JAR 33 epitope. The numbering was based on the amino acid sequence of MC58 v.1 fHbp lacking the signal sequence (17) (see Materials and Methods). wt, wild type. (A) JAR 10. (B) Penta-His MAb from the same samples as those used for panel A. (C) JAR 33.
- FIG. 4.
Western blot showing residues that are important for the binding of anti-fHbp MAbs JAR 11, JAR 32, and JAR 35. Residue lysine (K) 174 was essential for the JAR 32 and JAR 35 epitopes, and an alanine (A) residue at position 174 was involved in the JAR 11 epitope. The numbering is described in the legend to Fig. 3. wt, wild type. (A) JAR 32. (B) JAR 35. (C) JAR 11. (D) Penta-His MAb.
- FIG. 5.
Western blot showing a residue that is important for the binding of anti-fHbp MAbs JAR 3 and JAR 5. The epitopes for JAR 3 and JAR 5 were eliminated by the replacement of glycine (G) by arginine (R) at position 121 (G121R) in fHbp from strain MC58 and were introduced with substitution R121G in fHbp from strain M6190. The numbering is described in the legend to Fig. 3. (A) JAR 5. (B) JAR 3. (C) Penta-His MAb.
- FIG. 6.
Model of the locations of amino acid residues affecting the expression of fHbp epitopes. Coordinates were from the solution structure of fHbp v.1 of strain MC58 (4). The B and C domains are depicted in dark and light gray, respectively. The positions of the N and C termini are indicated. (Left) Locations of amino acid residues involved in the MAb epitopes are shown in black, with their respective residue numbers shown in parentheses. (Right) Molecule rotated 120° relative to the image in the left panel. The location of the residue previously reported to be involved in the epitope of MAb 502 is shown (13). Note that not all epitopes are present on fHbp v.1 from strain MC58, but the corresponding amino acid residues involved in the epitopes expressed by fHbp v.2 and v.3 proteins are indicated. The numbering is described in the legend to Fig. 3. The figure was generated using PyMol (http://www.pymol.org ).
Tables
- TABLE 1.
Complement-mediated cooperative bactericidal activity of anti-fHbp MAbsa
JAR MAb (Ig isotype) BC50 (μg/ml) for JARb: 3c 4 5 10 11 13 32 33 35 36 Strain NZ98/254, fHbp v.1 JAR 3 (G3) >50 1 >50 >50 JAR 4 (G2a) 1 >50 4 >50 JAR 5 (G2b) >50 4 >50 >50 JAR 10 (G1) >50 >50 >50 >50 Strain 8047d, fHbp v.2 JAR 4 (G2a) >50 >50 5 4 >50 JAR 10 (G1) >50 >50 5 >50 >50 JAR 11 (G2a) 5 5 >50 >50 1 JAR 13 (G2a) 4 >50 >50 >50 1 JAR 36 (G2b) >50 >50 1 1 >50 Strain M1239, fHbp v.3 JAR 13 (G2a) >50 >50 >50 >50 1 JAR 32 (G2a) >50 >50 1 >50 >50 JAR 33 (G2a) >50 1 >50 5 >50 JAR 35 (G2b) >50 >50 5 >50 >50 JAR 36 (G2b) 1 >50 >50 >50 >50 ↵ a JAR 3, 4, and 5 were from a mouse immunized with rfHbp v.1. JAR 10, 11, and 13 were from a mouse immunized with fHbp v.2. JAR 32, 33, 35, and 36 were from a mouse immunized with rfHbp v.3.
↵ b Total concentration of two MAbs that resulted in 50% survival of bacteria after 60 min of incubation with human complement. Data shown are for MAb pairs for which the respective strain expressed epitopes recognized by both MAbs.
↵ c Data for the MAb pair JAR 3-JAR 4 were published previously (28).
↵ d Strain 8047 expressed fHbp that differed by one amino acid from that of strain 2996, which was the source of the gene for preparing the recombinant fHbp v.2 vaccine. Strain 8047 was used as the test organism, since strain 2996 expressed low amounts of fHbp.
- TABLE 2.
Alignment of amino acid sequences of selected fHbps with distinct MAb reactivity patternsa
Strain Anti-fHbp JAR MAb reactivity for group: Amino acid sequence at positionb: v.1 v.2 v.3 170 180 190 200 210 3/5 10 11 13 32/35 33 36 MC58 (v.1) 1 0 0 0 0 0 0 IDFAAKQGNG KIEHLKSPEL NVDLAAADIK PDGKRHAVIS GSVLYNQAEK NZ98/254 (v.1) 1 1 0 0 0 0 0 IDFAAKQGHG KIEHLKSPEL NVELATAYIK PDEKHHAVIS GSVLYNQDEK M3153 (v.2) 0 1 1 0 0 0 1 IDFAAKQGHG KIEHLKTPEQ NVELASAELK ADEKSHAVIL GDTRYGGEEK 8047 (v.2) 0 1 1 1 0 0 1 IDFAAKQGHG KIEHLKTPEQ NVELAAAELK ADEKSHAVIL GDTRYGSEEK RM1090 (v.2) 0 0 0 0 1 1 1 IDFTKKQGYG RIEHLKTPEQ NVELASAELK ADEKSHAVIL GDTRYGGEEK M1239 (v.3) 0 0 0 1 1 1 1 IDFTKKQGYG RIEHLKTLEQ NVELAAAELK ADEKSHAVIL GDTRYGSEEK GB988 (v.3) 0 0 0 1 0 0 1 IDFTNKQGYG RIEHLKTPEL NVDLASAELK ADEKSHAVIL GDTRYGSEEK ***: *** * :*****: * **:**:* :* .* * **** *.. *. ** ↵ a The MAb reactivity is defined by the binding of anti-fHbp MAbs with bacteria in a whole-cell ELISA. A value of 1 indicates reactivity, which is defined as an absorbance value that is >10-fold above the background level.
↵ b The alignment contains the regions of the C domain involved in the binding of six of the MAbs prepared against fHbp v.2 or v.3. Residues involved in the epitopes are shown in boldface type: JAR 10 (K180 and E192), JAR 11 (A174), JAR 13 (S216), JAR 32 (K174), JAR 33 (R180 and E192), and JAR 35 (K174). The alignment was performed with ClustalW (6). The amino acid conservation is indicated below the alignment: *, identical; :, conserved; ., semiconserved. The numbering is based on the amino acid sequence of MC58 v.1 fHbp lacking the signal sequence (17). The DNA sequences for fHbp from strains M3153, GB988 (also referred to as M01-0240988), and RM1090 have been deposited in GenBank (accession numbers EU337062 , EU337063 , and EU310268 , respectively). Accession numbers for fHbp genes from the strains described in previous studies are EU310268 , AY548375 , DQ523569 , and NC_003112 .
- TABLE 3.
Summary of Western blotting data for residues involved in MAb epitopes
Immunogen group and MAbc Reactive residue(s)a Nonreactive strain Nonreactive residue(s) Evidenceb v.1, strain MC58 JAR 3 G121 M6190 R121 KO, KI K122 03S-0408 S122 KO JAR 5 G121 M6190 R121 KO, KI K122 03S-0408 S122 KO v.2, strain 2996 JAR 10 K180 and E192 M1239 R180 or D192 KO, KI JAR 11 A174 M1239 K174 KO JAR 13 S216 RM1090 G216 KO, KI v.3, strain M1239 JAR 32 K174 8047 A174 KO, KI JAR 33 R180 and E192 8047 K180 or D192 KO, KI JAR 35 K174 8047 A174 KO, KI ↵ a Reactive residue in fHbp from the strain used as the source for immunization.
↵ b KO indicates that the KO mutation abolished reactivity in the wild-type protein, and KI indicates that the KI mutation restored reactivity in the non-reactive wild-type protein.
↵ c By ELISA, JAR 3 inhibited the binding of JAR 5 and JAR 5 inhibited the binding of JAR 3. JAR 32 inhibited the binding of JAR 35 (but not vice versa), and JAR 13 inhibited the binding of JAR 10 (Fig. 6).
- TABLE 4.
Bactericidal activity of combinations of MAbs in relation to the respective locations of the epitopes
JAR MAb pair Strain (variant) Combination BC50, in μg/mla Residues in epitopesb Approx distancec (Å) fH inhibitiond Ig isotypes 5 and 502e H44/76 <1 G121 and R204 16 ++ and ND G2b and G2a 10 and 11 8047 (v.2) 5 K180/E192 and A174 18-20 − and + G1 and G2a 33 and 32 M1239 (v.3) 1 R180/E192 and K174 18-20 − and ++ G2a and G2a 33 and 35 M1239 (v.3) 5 R180/E192 and K174 18-20 − and ++ G2a and G2b 3 and 10 NZ98/254 (v.1) >50 G121 and K180/E192 31-32 ++ and − G3 and G1 5 and 10 NZ98/254 (v.1) >50 G121 and K180/E192 31-32 ++ and − G2b and G1 13 and 35 M1239 (v.3) >50 S216 and K174 27 ++ and ++ G2a and G2b 13 and 11 8047 (v.2) >50 S216 and A174 27 ++ and + G2a and G2a 13 and 32 M1239 (v.3) >50 S216 and K174 27 ++ and ++ G2a and G2a 13 and 33 M1239 (v.3) >50 S216 and R180/E192 9-14 ++ and − G2a and G2a 13 and 10 8047 (v.2) >50 S216 and K180/E192 9-14 ++ and − G2a and G1 3 and 5 NZ98/254 (v.1) >50 G121 and G121 0 ++ and ++ G3 and G2b 32 and 35 M1239 (v.3) >50 K174 and K174 0 ++ and ++ G2a and G2b ↵ a Data are shown only for MAbs that individually were not bactericidal against the test strain (BC50 > 50 μg/ml).
↵ b For the respective MAbs in the pair. The numbering of the residues is based on the amino acid sequence of MC58 v.1 fHbp lacking the signal sequence (17).
↵ c Distances between the pairs of the MAbs were calculated between alpha-carbon positions for the respective residues using PyMol (http://www.pymol.org ). JAR 32 and JAR 35 recognize overlapping (or identical) epitopes including K174.
↵ d ++, inhibition of fH binding to rfHbp (>70%); +, partial inhibition (25 to 45%); −, no inhibition (<15%) as measured by ELISA (Fig. 2).
↵ e The amino acid affecting the expression of the MAb 502 epitope was described by Giuliani et al. (13). The inhibition of fH binding was not determined (ND).
