Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans

Abstract

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).

Fig. 1.

GNA2132 is recognized by sera of patients after meningococcal disease. Spot intensities generated by the rGNA2132-his antigen producing a geometric mean intensity more than 2-fold higher than the his-tagged control protein (Control-his) on probing dot-blots with the 22 convalescent serum samples. All samples produced significantly higher intensities than the his-tagged control protein (P = 0.00031). The results for the his-tagged control protein (Control-his) and tetanus toxoid (TT) are included as negative and positive controls, respectively. Black bars indicate the average spot intensity for each protein.

Fig. 2.

Characterization of the recombinant 5/99 strain to assess GNA2132 immunogenicity. (A) Western blot analyses using polyclonal antibodies against NadA (panel 1) and GNA2132 (panel 2) on total cell extracts prepared from strains 5/99 (lane 1), 5/99ΔnadA (lane 2), 5/99ΔnadAΔgna2132 (lane 3), and 5/99ΔnadAΔgna2132Cgna2132 grown in the presence of 0.1 mM IPTG (lane 4). The smaller size of the GNA2132 protein in the parental 5/99 (lane 1) and 5/99ΔnadA (lane 2) strains relative to the recombinant strain reflects the larger protein size of the GNA2132 protein derived from the NZ98/254 strain (GNA2132-NZ). Western blot analysis of total cell extracts (panel 3) from strain 5/99ΔΔ (lane 1) and strain 5/99ΔΔCgna2132 grew in GC liquid broth supplemented with different concentration of IPTG (from 0.0001 to 1 mM incrementally in lanes 2–6). Arrow indicates GNA2132 protein. (B) Log2 bactericidal titers of a variety of antibodies directed against surface components and various vaccine antigens. Strains 5/99ΔΔ and 5/99ΔΔC2132 were used in the BCA using rabbit complement. Antibodies directed against capsular polysaccharide and the porins are monoclonal, whereas those used against OMV and recombinant vaccine antigens are polyclonal. Log2 titers of 2 or less are considered negative. (C) Log2 bactericidal titers from five adult subjects (subjects 1–5) receiving the recombinant MenB vaccine. Preimmune sera and sera obtained 1 month after the third dose were analyzed using human complement. Log2 titers no greater than 1 are considered not protective. Expression of GNA2132 in 5/99ΔΔC2132 strain was induced by addiction of 1 mM IPTG.

Fig. 3.

GNA2132 protein binds heparin in vitro. (A and B) Heparin affinity binding of different recombinant GNA2132 proteins. The molarity (mM of NaCl) of the elution peak is indicated for each construct (0 mM indicates that the protein is present exclusively in the flow-through fraction, FT). The full-length protein (rGNA2132^MC58-his) binds the column (red line) and is eluted with a salt gradient (green line). The Arg-rich region mutants are recovered in the unbound fraction (blue and pink lines). (C) Native PAGE gels of rGNA2132^MC58-his in the presence of heparin or HSPG. (D) Sensorgrams obtained by SPR binding analysis of rGNA2132^MC58-his at concentrations ranging from 0 μM to 0.43 μM. BSA (0.4 μM) was used as negative control.

Fig. 4.

Heparin increases serum resistance of unencapsulated N. meningitidis expressing GNA2132. (A) Nm strains grown in the presence of 1 mM IPTG were incubated with either 50 U of heparin or buffer for 30 min before performing the bactericidal assay. (B) Bactericidal assays were performed in the presence of 50 U of heparin using strains grown in the presence or absence of 1 mM IPTG. The x axis denotes the strain and the y axis indicates the percent survival. Values plotted are the average survival calculated from three or more independently performed experiments. Error bars denote standard deviations. Asterisks indicate statistically significant differences (A, P = 0.011; B, P = 0.0021). (C) Expression levels of GNA2132 correlated with serum resistance in experiments in which the concentration of IPTG was varied. Nm strain YΔ2132-C2132 was grown at different concentrations of IPTG and subjected to a serum bactericidal assay in the presence of heparin (50 U).

Fig. 5.

GNA2132 is processed by NalP and hLf. (A) Western blot analysis of OMP and supernatants of a selected panel of Nm strains. (B) Western blot analysis of OMP and supernatants of MCΔ2132, MC58, NZ98/254, and MCΔ2132-CNZ strains. All of the Western blots were performed using a mouse polyclonal antibody generated with the rGNA2132^MC58-his protein. (C) Western blot analysis of OMP and supernatants preparations of MCΔ2132, MC58, and MCΔnalP strains using a mouse polyclonal antibody against GNA2132. In all of the Western blots, the full-length protein (GNA2132), the N-terminal region (N) and the C-terminal region (C) are indicated by arrows. (D) SDS/PAGE analysis and Coomassie staining showing the effect of hLf on rGNA2132^MC58-his, C-his, and C1-his. hLf, GNA2132, and fragments (N1, C1, and C) are indicated by arrows. (E) Schematic representation of the GNA2132 protein (strain MC58). The Arg-rich region at position 296 is indicated by a black box. NalP and hLf cleavage sites are indicated by a black and a white arrow, respectively.