Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein

Abstract

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

Figure 1. Ghfp is not expressed on the surface of N. gonorrhoeae.

(A) Western blot analysis of Ghfp expression by N. gonorrhoeae strains F62, F62Δghfp and FA1090, and fHbp V3.28 expressed by N. meningitidis strains M1239 and M1239Δfhbp using anti-Ghfp serum. (B) Western blot analysis of Ghfp expressed by a panel of clinical N. gonorrhoeae isolates (GC1-11, inclusive). Surface expression of Ghfp (C) and fHbp V3.28 (D) was assessed by flow cytometry analysis using anti-Ghfp serum. Error bars, SEM of three separate experiments; ** p<0.05 and NS (not significant, Student's t-test). Representative flow cytometry overlays are shown below the graphs. Bacteria incubated without anti-Ghfp serum are shown as the grey filled areas. (E) Bacteria were exposed to proteinase K (3 ng/ml and serial three-fold dilutions) and the effect on proteins (shown) determined by Western blot analysis using anti-Ghfp, anti-RecA and anti- α-Lst serum.

Figure 2. fH binding capacity of Ghfp.

(A-C) fH binding to wild type and modified Ghfp and fHbp V3.45 was assessed by far Western analysis using normal human serum as the source of fH. Western blots are representatives of three separate experiments. Molecular mass is shown in kDa. (D) Typical equilibrium fit for binding of fH_6–7 to Ghfp^M4–5. (E) SPR was performed with Ghfp, Ghfp^M4 (R288H), Ghfp^M5 (D318G) and Ghfp^M4–5 (R288H/D318G); NBD, no binding detected. (F) Detection of full length fH (5 nM) binding to wild-type and modified Ghfp by ELISA. Error bars, SEM of three separate experiments; *** p<0.01 and * p<0.1 (Student's t-test). (G) Cartoon presentation of the predicted structure of Ghfp (Yellow) and fH (Blue). Residues M1 (R176), M2 (D199) and M3 (D212) are shown in green while those amino acids that are important for fH binding, M4 (R288) and M5 (D318G), are shown in red.

Figure 3. fH binding to modified fHbp V3.45.

(A) Analysis of fH binding to modified V3.45 fHbp by far Western using normal human serum as the source of fH. Molecular mass is shown in kDa. (B) SPR values of fH_6–7 binding to wild type and modified V3.45 fHbp; NBD, no binding detected. (C) Detection of full length fH (5 nM) binding to wild-type and modified V3.45 fHbp by ELISA. Data represents the mean ± SEM of three different experiments.

Figure 4. Immunogenicity of Ghfp.

(A) Detection of fHbp variants in whole cell lysates of N. meningitidis by Western blot analysis using anti-Ghfp serum. Recognition of recombinant V1 (B), and V2and V3 (C) fHbps by anti-Ghfp serum by ELISA. (D) SBA responses for anti-Ghfp serum.

Figure 5. Immune responses against isogenic N. meningitidis strains.

(A) Western blot analysis of whole cell lysates of fHbp expressed by isogenic MC58Δfhbp strains detected by anti-Ghfp serum. (B) Surface expression of different fHbps in the isogenic MC58Δfhbp strains detected by flow cytometry. Graph shows the mean ± SEM of three separate experiments. (C) Representative corresponding flow cytometry overlay of MC58 (grey hatched area), MC58Δfhbp and MC58Δfhbp+fhbp V1.1 detected by anti-Ghfp by flow cytometry. (D) SBA responses against isogenic MC58 strains for anti-Ghfp, fHbp V1.1, and fHbp V3.45 serum using rabbit complement; NK, no killing. (E) SBA responses against isogenic H44/76 strains with anti-Ghfp serum using rabbit complement.