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Microbial Immunity and Vaccines

Fine Antigenic Specificity and Cooperative Bactericidal Activity of Monoclonal Antibodies Directed at the Meningococcal Vaccine Candidate Factor H-Binding Protein

Peter T. Beernink, Jo Anne Welsch, Michal Bar-Lev, Oliver Koeberling, Maurizio Comanducci, Dan M. Granoff
Peter T. Beernink
1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California
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Jo Anne Welsch
1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California
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Michal Bar-Lev
1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California
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Oliver Koeberling
1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California
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Maurizio Comanducci
2Novartis Vaccines, Siena, Italy
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Dan M. Granoff
1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California
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  • For correspondence: dgranoff@chori.org
DOI: 10.1128/IAI.00367-08
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  • FIG. 1.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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

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  • TABLE 1.

    Complement-mediated cooperative bactericidal activity of anti-fHbp MAbsa

    JAR MAb (Ig isotype)BC50 (μg/ml) for JARb:
    3c4510111332333536
    Strain NZ98/254, fHbp v.1
        JAR 3 (G3)>501>50>50
        JAR 4 (G2a)1>504>50
        JAR 5 (G2b)>504>50>50
        JAR 10 (G1)>50>50>50>50
    Strain 8047d, fHbp v.2
        JAR 4 (G2a)>50>5054>50
        JAR 10 (G1)>50>505>50>50
        JAR 11 (G2a)55>50>501
        JAR 13 (G2a)4>50>50>501
        JAR 36 (G2b)>50>5011>50
    Strain M1239, fHbp v.3
        JAR 13 (G2a)>50>50>50>501
        JAR 32 (G2a)>50>501>50>50
        JAR 33 (G2a)>501>505>50
        JAR 35 (G2b)>50>505>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

    StrainAnti-fHbp JAR MAb reactivity for group:Amino acid sequence at positionb:
    v.1v.2v.3170180190200210
    3/510111332/353336
    MC58 (v.1)1000000 IDFAAKQGNG KIEHLKSPEL NVDLAAADIK PDGKRHAVIS GSVLYNQAEK
    NZ98/254 (v.1)1100000 IDFAAKQGHG KIEHLKSPEL NVELATAYIK PDEKHHAVIS GSVLYNQDEK
    M3153 (v.2)0110001 IDFAAKQGHG KIEHLKTPEQ NVELASAELK ADEKSHAVIL GDTRYGGEEK
    8047 (v.2)0111001 IDFAAKQGHG KIEHLKTPEQ NVELAAAELK ADEKSHAVIL GDTRYGSEEK
    RM1090 (v.2)0000111 IDFTKKQGYG RIEHLKTPEQ NVELASAELK ADEKSHAVIL GDTRYGGEEK
    M1239 (v.3)0001111 IDFTKKQGYG RIEHLKTLEQ NVELAAAELK ADEKSHAVIL GDTRYGSEEK
    GB988 (v.3)0001001 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 MAbcReactive residue(s)aNonreactive strainNonreactive residue(s)Evidenceb
    v.1, strain MC58
        JAR 3G121M6190R121KO, KI
        K12203S-0408S122KO
        JAR 5G121M6190R121KO, KI
    K12203S-0408S122KO
    v.2, strain 2996
        JAR 10K180 and E192M1239R180 or D192KO, KI
        JAR 11A174M1239K174KO
        JAR 13S216RM1090G216KO, KI
    v.3, strain M1239
        JAR 32K1748047A174KO, KI
        JAR 33R180 and E1928047K180 or D192KO, KI
        JAR 35K1748047A174KO, 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 pairStrain (variant)Combination BC50, in μg/mlaResidues in epitopesbApprox distancec (Å)fH inhibitiondIg isotypes
    5 and 502eH44/76<1G121 and R20416++ and NDG2b and G2a
    10 and 118047 (v.2)5K180/E192 and A17418-20− and +G1 and G2a
    33 and 32M1239 (v.3)1R180/E192 and K17418-20− and ++G2a and G2a
    33 and 35M1239 (v.3)5R180/E192 and K17418-20− and ++G2a and G2b
    3 and 10NZ98/254 (v.1)>50G121 and K180/E19231-32++ and −G3 and G1
    5 and 10NZ98/254 (v.1)>50G121 and K180/E19231-32++ and −G2b and G1
    13 and 35M1239 (v.3)>50S216 and K17427++ and ++G2a and G2b
    13 and 118047 (v.2)>50S216 and A17427++ and +G2a and G2a
    13 and 32M1239 (v.3)>50S216 and K17427++ and ++G2a and G2a
    13 and 33M1239 (v.3)>50S216 and R180/E1929-14++ and −G2a and G2a
    13 and 108047 (v.2)>50S216 and K180/E1929-14++ and −G2a and G1
    3 and 5NZ98/254 (v.1)>50G121 and G1210++ and ++G3 and G2b
    32 and 35M1239 (v.3)>50K174 and K1740++ 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).

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Fine Antigenic Specificity and Cooperative Bactericidal Activity of Monoclonal Antibodies Directed at the Meningococcal Vaccine Candidate Factor H-Binding Protein
Peter T. Beernink, Jo Anne Welsch, Michal Bar-Lev, Oliver Koeberling, Maurizio Comanducci, Dan M. Granoff
Infection and Immunity Aug 2008, 76 (9) 4232-4240; DOI: 10.1128/IAI.00367-08

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Fine Antigenic Specificity and Cooperative Bactericidal Activity of Monoclonal Antibodies Directed at the Meningococcal Vaccine Candidate Factor H-Binding Protein
Peter T. Beernink, Jo Anne Welsch, Michal Bar-Lev, Oliver Koeberling, Maurizio Comanducci, Dan M. Granoff
Infection and Immunity Aug 2008, 76 (9) 4232-4240; DOI: 10.1128/IAI.00367-08
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KEYWORDS

Antibodies, Bacterial
Antibodies, Monoclonal
Antibody Specificity
Antigens, Bacterial
Bacterial Proteins
Complement System Proteins
Microbial Viability

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