Novel blocking human IgG directed against the pentapeptide repeat motifs of Neisseria meningitidis Lip/H.8 and Laz lipoproteins

Abstract

Ab-initiated, complement-dependent killing contributes to host defenses against invasive meningococcal disease. Sera from nonimmunized individuals vary widely in their bactericidal activity against group B meningococci. We show that IgG isolated from select individuals can block killing of group B meningococci by human sera that are otherwise bactericidal. This IgG also reduced the bactericidal efficacy of Abs directed against the group B meningococcal protein vaccine candidates factor H-binding protein currently undergoing clinical trials and Neisserial surface protein A. Immunoblots revealed that the blocking IgG was directed against a meningococcal Ag called H.8. Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when binding of blocking Ab to meningococci was inhibited using either synthetic peptides corresponding to H.8 or a nonblocking mAb against H.8. Furthermore, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab')(2) fragments were ineffective. Blocking required IgG glycosylation because deglycosylation with peptide:N-glycanase eliminated blocking. C4b deposition mediated by an anti-factor H-binding protein mAb was reduced by intact blocking IgG, but not by peptide:N-glycanase-treated blocking IgG, suggesting that blocking resulted from inhibition of classical pathway of complement. In conclusion, we have identified H.8 as a meningococcal target for novel blocking Abs in human serum. Such blocking Abs may reduce the efficacy of select antigroup B meningococcal protein vaccines. We also propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.

Figure 1. Bactericidal activity of individual human sera against strain H44/76 and binding of IgG and IgM binding in select killing and non-killing sera to H44/76

A. Hemolytically active sera obtained from 19 normal individuals were tested for complement dependent bactericidal activity against wild-type N. meningitidis serogroup B strain H44/76. The final concentration of each serum in the bactericidal reaction mixture was 20%. Sera that killed ≥50% (percent survival ≤50%) of bacteria were designated killing (K) and those that killed <50% (percent survival > 50%) as non-killing (NK) sera. B. Serogroup B strain H44/76 was incubated with each of four sera that had displayed maximal bactericidal activity (K1 through K4) or each of four sera with the least bactericidal activity (NK3 through NK6). The amount of IgG and IgM binding was quantified by flow cytometry. Grey shaded histograms represent binding of IgG or IgM in killing (K) sera, while histograms depicted by the solid lines binding of Ig in NK sera. The x-axis represents fluorescence on a log₁₀ scale and the y-axis the number of events. The secondary antibody control (labeled “sec. Ab ctrl”; broken lines) reaction did not contain serum.

Figure 2. Select non-killing (NK) sera possess blocking activity and bactericidal activity of certain sera can be blocked

A. Blocking potential of four non-killing (NK) sera. The final concentration of heat-inactivated NK sera in the reaction mixture was 20% (v/v) and concentration of K sera (complement active) was 10%. Blocking (≥50% decrease in killing compared to killing serum alone using the formula: [% killing (K) − % killing (K + heat inactive NK)] / % killing (K)] x 100) is indicated by dark gray boxes. Combinations of sera that yielded 25 to 50% increase in survival (blocking) compared to baseline are indicated by the light grey shaded boxes. B. Blocking of K-mediated bacterial killing of H44/76 by NK is dose responsive. Wild-type group B strain H44/76 was incubated with 10% K1 serum either alone or in the presence of 10% and 20% heat-inactivated NK3 serum. In the converse experiment, 10% NK3 serum (complement active) was incubated with 10% and 20% heat-inactivated K1 serum. The y-axis indicates percent survival and each column represents the mean ±SEM of 3 separate experiments.

Figure 3. IgG in NK3 mediates blocking

A. IgG purified from NK3 blocks killing by K1 serum. IgG purified from NK3 was added incrementally (final concentrations 1 and 2 mg/ml) to a bactericidal reaction mixture containing strain H44/76 and serum K1 (10% (v/v)). IgG purified from K1 served as a control. B. Depletion of IgG from NK3 abrogates blocking activity. Heat inactivated IgG-depleted NK3 was added to the bactericidal reaction mixture described in A. The control used heat-inactivated NK3 serum in the bactericidal reaction mixture. C. IgM purified from NK3 does not possess blocking activity. Incremental doses of IgM purified from NK3 failed to block killing by K1. In all experiments (A, B and C) the y-axis indicates percent survival and each column represents the mean (±SEM) of 3 separate experiments.

Figure 4. Blocking potential depends on the specificity of killing antibody

A. Bactericidal activity of an anti-fHbp mAb is attenuated by IgG in NK3. N. meningitidis strain H44/76 was incubated with increasing concentrations (0.25, 0.5, 1, and 2 µg/ml) of JAR 3 in a reaction mixture that contained either 20% (v/v) of blocking serum NK3 (Ab and complement intact; solid circle) or 40% (v/v) IgG-depleted NK3 (solid box). The higher concentration of IgG-depleted NK3 was used to normalize for the amount of hemolytic complement activity in the bactericidal reaction mixture (the process of IgG depletion resulted in loss of ~50% of hemolytic activity). B. IgG depletion of NK3 does not affect bactericidal activity of anti-PorA mAb P1.7. Serial dilutions (1/1000, 1/500, 1/250 and 1/125) of ascitic fluid containing murine anti-PorA mAb P1.7 were each incubated with strain H44/76 and the bactericidal assay performed as described above in A. C. The bactericidal activity of anti-group B capsular mAb SEAM 12 is not significantly blocked by NK3 IgG. Strain H44/76 was incubated with mAb SEAM 12 and complement (40% (v/v) normal human serum depleted of IgG and IgM), either in the absence or presence of IgG purified from NK3 (2 mg/ml) and survival at 30 min was measured. The black bar represents survival in the presence of complement alone. Survival in the presence or absence of NK3 IgG for each concentration of SEAM 12 were not significantly different (p>0.05). D. The bactericidal activity of murine polyclonal anti-fHbp antiserum against H44/76 is blocked by NK3 IgG. Strain H44/76 was incubated with polyclonal anti-fHbp at antiserum dilutions of 1:3000 and 1:6000 either in the presence or absence of IgG purified from NK3 (2 mg/ml) and complement (40% (v/v) normal human serum depleted of IgG and IgM). Survival was measured at 30 min in a serum bactericidal assay. Survival in the presence of NK3 IgG was significantly higher than the corresponding reaction mixture that lacked NK3 IgG (p<0.05 in each instance). In all experiments the y-axis represents percent survival and each data point represents the mean (±SEM) of 3 separate experiments.

Figure 5. Lip/H.8 is a meningococcal target for IgG in blocking serum

Bacterial lysates of strain MC58 and its isogenic lipoprotein Lip/H.8 knockout (KO) mutant were electrophoresed on a 12% Bis-Tris gel followed by western blotting. Parallel blots were incubated with a 1:100 dilution of NK3 serum (nonbactericidal and possessing blocking activity), NK6 (nonbactericidal and without blocking activity), K1 (bactericidal activity that could be blocked by NK3) and K3 (bactericidal activity that could not be blocked by NK3). Human IgG-reactive bands were disclosed with alkaline phosphatase conjugated anti-human IgG. The black asterisk (on the left) indicates the Lip/H.8-reactive band. The location of PorA and lipooligosaccharide (LOS) reactive bands are indicated with arrows on the right. The identity of each serum in terms of its bactericidal activity (SBA) and blocking activity is indicated in the labels below the gel pictures. Sera that lack SBA or blocking activity are labeled as n.a. (not applicable) for that particular category.

Figure 6. Expression of both Lip/H.8 and Laz are required for maximal blocking

A. Alignment of amino acid sequences of Lip/H.8 and Laz of serogroup B N. meningitidis MC58. Lip/H.8 is a 98 amino acid protein composed almost entirely of 12–15 pentapeptide repeats (over half are perfect ‘AAEAP’ sequences and the remainder represent modifications (‘imperfect’ repeats)) depending on the strain. Laz is a 183 amino acid protein composed of 7 mostly imperfect AAEAP pentameric repeats that comprises the Lip/H.8-like domain at its N-terminus. The pentameric repeats are highlighted as bold letters. Each pentameric repeat is marked with a bar overhead labeled with the repeat number. The C-terminal portion of Laz (amino acid residues 57 to 183) is homologous to bacterial copper binding proteins called azurin. Both proteins are lipid modified at the cystine residue following cleaveage of the N-terminal 17 amino acid signal sequence. Conserved amino acids of the Lip/H.8 domain within both proteins are shown. B. Bacterial lysates of strain MC58 and its isogenic Lip/H.8 and Laz single knockout mutants (L to R) were electrophoresed on a 12% Bis-Tris gel followed by blotting and incubation with mouse monoclonal antibody (mAb) anti-Lip/H.8 antibody, 2C3. Bound 2C3 was disclosed using alkaline phosphatase conjugated goat anti-mouse IgG. Bands specific for Lip/H.8 (Lip) and Laz are indicated. C. Loss of either Lip/H.8 or Laz decreases blocking by NK3. Wild-type MC58 (black columns) and its Lip/H.8 (light grey columns) and Laz mutants (dark grey columns) were incubated with 10% (v/v) killing serum K1 and 20% (v/v) heat inactivated blocking serum NK3. D. Deleting either Lip/H.8 or Laz from strain MC58 increases killing of bacteria by anti-fHbp mAb JAR 3 in the presence of NK3. Wild-type MC58 (black columns) and its Lip/H.8 (light grey columns) and Laz mutants (dark grey columns) were incubated with anti-fHbp mAb JAR 3 (0.25 µg/ml) and NK3 (20% (v/v). E. Killing of H44/76 is restored by diverting blocking human anti-LipH.8 IgG from the bacterial surface using F(ab)₂ fragments of murine anti-Lip/H.8 mAb 2C3. Strain H44/76 was incubated with 0.25 µg/ml of anti-fHbp mAb JAR 3 in the presence of increasing dose s (2.5, 5 and 10 µg/ml) of F(ab)₂ fragments of murine anti-Lip/H.8 mAb 2C3, , followed by addition of 20% (v/v) of NK3 (complement active). F. Synthetic peptides corresponding to N- and C- terminal Lip/H.8 sequences (singly or together) divert NK3 IgG from the bacterial surface and restore JAR 3-mediated killing of H44/76. Strain H44/76 was incubated with increasing amounts (2 and 4µM) of synthetic 30-mer peptides corresponding to the N- and C-terminal regions of Lip/H.8, individually or as an equimolar mix to a bactericidal reaction mixture that contained JAR 3 (0.25 µg/ml) and NK3 (20% (v/v); complement active) (grey shaded bars). Controls where bacteria were incubated with the peptides, IgG-depleted NK3 (lacks blocking Ab) and JAR 3 are shown by the open bars. G. Recombinant His-tagged Laz (contains the N-terminal imperfect Lip/H.8-like repeats or H. recombinant His-tagged azurin (contains only the azurin domain of Laz), at concentrations 5 and 10 µg/ml were used to divert blocking IgG and restore killing of JAR3. Samples containing NK3 serum and IgG depleted NK3 serum as complement source are represented by solid grey and white columns, respectively. Bars in Figures 6C, 6D, 6F, 6G and 6H represent mean percent survival ±SEM of 3 separate experiments, while Fig 6E shows the mean (range) of 2 independent experiments.

Figure 7. Blocking IgG requires Fc N-linked glycan for its activity

A. F(ab)₂ fragments of NK3 IgG do not mediate blocking. F(ab)₂ fragments prepared by pepsin digestion of NK3 IgG were added in increasing concentrations (0.5 or 0.1 mg/ml) to a bactericidal reaction mixture containing strain H44/76, mAb JAR3 and IgG depleted serum NK3, 20% (v/v) used as the complement source. The corresponding doses of mock-digested NK3 IgG served as a control. Each bar shows the mean percent survival (± range) of 2 independent experiments. B. Intact and deglycosylated NK3 IgG bind similarly to strain H44/76. Intact (buffer-treated) and deglycosylated (PNGase treated) NK3 IgG were incubated (separately) with strain H44/76 and the IgG binding was determined by flow cytometry using polyclonal anti-human IgG FITC. Bacteria stained with the secondary antibody alone was used as a background control. C. Loss of Fc glycan is associated with loss of blocking activity. Increasing (0.5 and 1 mg/ml) concentrations of deglycosylated (PNGase-treated) NK3 IgG was added to a bactericidal reaction mixture containing strain H44/76, mAb JAR3 and IgG depleted serum NK3 (20% (v/v)) used as the complement source. The corresponding concentrations of buffer-treated (intact) NK3 IgG served as a control. The y-axis indicates mean percent survival (±SEM) of 3 separate experiments.

Figure 8. Blocking IgG inhibits the classical pathway activation of complement by anti-fHbp mAb JAR 3

N. meningitidis serogroup B strain H44/76 was incubated with mAb JAR3, IgG depleted NK3 serum used as the source of complement and 100 µg of either buffer-treated (intact)- or PNGase-treated NK3 IgG. A and B represent binding of JAR 3 to and C4b deposition on bacteria measured by flow cytometry. FACS histograms represent the following: grey shaded histograms, IgG-depleted NK3 (complement (C′) control) only; solid black line, JAR 3 + C′; broken line, JAR 3 + C′ + intact NK3 IgG; solid grey line, JAR 3 + C′ + PNGase-treated NK3 IgG. The numbers accompanying the C4b deposition histograms represent the average of the geometric mean fluorescence of the entire bacterial population of two separate experiments. Similarly, the effect of NK3 IgG on C4b deposition mediated by anti-PorA mAb 1.7 was also examined. C and D represent mAb P1.7 binding to, and C4b deposition on H44/76, respectively. FACS histograms are coded as described for A and B, with the exception that PNGase-treated NK3 IgG was not tested (no solid grey line). Numbers alongside the C4b histograms are as described for panel C.