Structure-based design of an immunogenic, conformationally stabilized FimH antigen for a urinary tract infection vaccine

Abstract

Adhesion of E. coli to the urinary tract epithelium is a critical step in establishing urinary tract infections. FimH is an adhesin positioned on the fimbrial tip which binds to mannosylated proteins on the urinary tract epithelium via its lectin domain (FimHLD). FimH is of interest as a target of vaccines to prevent urinary tract infections (UTI). Previously, difficulties in obtaining purified recombinant FimH from E. coli along with the poor inherent immunogenicity of FimH have hindered the development of effective FimH vaccine candidates. To overcome these challenges, we have devised a novel production method using mammalian cells to produce high yields of homogeneous FimH protein with comparable biochemical and immunogenic properties to FimH produced in E. coli. Next, to optimize conformational stability and immunogenicity of FimH, we used a computational approach to design improved FimH mutants and evaluated their biophysical and biochemical properties, and murine immunogenicity using a bacterial adhesion inhibition assay. This approach identified an immunogenic FimH variant (FimH-donor-strand complemented with FimG peptide 'triple mutant', FimH-DSG TM) capable of blocking bacterial adhesion that is produced at high yields in mammalian cells. By x-ray crystallography, we confirmed that the stabilized structure of the FimHLD in FimH-DSG TM is similar to native FimH on the fimbrial tip. Characterization of monoclonal antibodies elicited by FimH-DSG that can block bacterial binding to mannosylated surfaces identified 4 non-overlapping binding sites whose epitopes were mapped via a combinatorial cryogenic electron microscopy approach. Novel inhibitory epitopes in the lectin binding FimH were identified, revealing diverse functional mechanisms of FimH-directed antibodies with relevance to FimH-targeted UTI vaccines.

Fig 1. FimH_LD produced in mammalian cells has comparable biophysical properties to material produced in E. coli.

WT or conformation stabilized FimH_LD were expressed in Expi293 cells along with mutants designed to remove non-native glycosylation on residues N7 and N70. (A) Batch purified proteins run on SDS-PAGE gel stained with Coomassie blue. C1 and C2 designate two clones of FimH_LD N7Q V27C L34C N70S and FimH_LD V27C L34C that were evaluated for expression. (B). Near-UV CD spectra of FimH_LD produced in E. coli and mammalian expression systems. Spectra of FimH_LD WT (blue) and V27C L34C (red) are shown. (C) Affinity of WT and V27C L34C FimH_LD produced in E. coli or mammalian cells for BPMP ligand by FP. (D) Thermal stability of WT and V27C L34C FimH_LD produced in E. coli or mammalian cells. (E) Mouse study design: CD-1 mice were immunized 3 times with 10 µg FimH proteins with QS21 adjuvant. (F) Sera were analyzed for the ability to block FimH-expressing E. coli binding to yeast mannan; bars represent geometric mean IC₅₀ and 95% confidence intervals. Statistical significance (p-value) of differences in responses between groups was determined using an unpaired t-test with Welch’s correction applied to log-transformed data; the bars and asterisk illustrate the significance of the difference in response for comparisons. Tabulated IC₅₀ values are shown in Table A in S1 Text.

Fig 2. Rational design of FimH mutations stabilizing the native state.

(A) Structures of FimH in unbound form (PDB structure 3JWN) and bound to mannoside ligand (PDB structure 1KLF). In pili, unbound FimH (left), complexed with the donor strand of FimG, adopts a compact conformation that binds the FimH cognate receptor, the terminal mannose moiety of glycosylated proteins, with low affinity. Upon binding a mannose moiety (right), the FimH_LD and FimH_PD separate, sidechains (colored in green) flip from protein interior to surface, and backbones of Gly residues (colored in blue) exhibit large conformational changes. Residues shown in blue and green were targeted for mutagenesis. (B) Strategies employed to stabilize FimH conformation. Unbound (grey) and bound (green) full length FimH structures with residues targeted by mutagenesis are highlighted.

Fig 3. Identification of FimH mutants with improved thermal stability and reduced mannoside ligand affinity.

(A) 64 FimH variants were screened in vitro for ability to bind mannose, thermal stability, and conformation. A subset of constructs was screened for immunogenicity in mice. (B-C) Biochemical characterization of purified FimH_LD and FimH-DSG mutants. (B) relative average binding affinities of FimH mutants to mannoside ligand. Note, the assay limit of detection was ~2000 nM. (C) filled circles display average melting temperatures of each mutant. Open circles denote melting temperature of FimH protein in the presence of mannoside ligand. Tabulated K_d and T_m values for all mutants are in Tables B and C in S1 Text respectively.

Fig 4. FimH-DSG mutants induce antibodies with superior ability to inhibit bacterial binding compared to FimH_LD mutants in mice.

(A) CD-1 mice were immunized 3 times with 10 µg FimH with QS21 adjuvant. Sera were analyzed for the ability to block FimH-expressing E. coli binding to yeast mannan. (B) Inhibitory titers were determined from serial dilution of sera from vaccinated mice and represent the reciprocal of the dilution of serum at which 50% of bacteria remain bound to the plate and are shown for post dose 2 and post dose 3 timepoints. Statistical significance (p-value) of differences in responses between groups was determined using an unpaired t-test with Welch’s correction applied to log-transformed data; the bars and asterisk illustrate the significance of the difference in response between groups. Tabulated IC₅₀ values are shown in Table D in S1 Text.

Fig 5. The lectin domain of FimH-DSG TM adopts an open conformation.

(A) Overall structure of FimH-DSG TM from X-ray crystallography data (PDB code 8V3J). (B) Superimposition of ligand binding sites of FimH-DSG TM (orange) and WT FimH from a previously published structure of native pili (PDB code 3JWN, light grey) shows remodeling of the Glycine loop due to G15A G16A mutations. The widening of the loop between Ile13 Cα-Ser17 Cα is shown in a dotted line. (C) Electron density and atomic model of Glycine loop in FimH-DSG TM. (D) Superimposition of FimH-DSG TM (orange), WT FimH in apo (PDB code 3JWN, light grey) and trimannose-bound (PDB code 6GTV, slate) forms shows that the ligand binding site of FimH-DSG TM adopts an open conformation resembling the apo state but not the ligand-bound, closed conformation.

Fig 6. Identification of novel inhibitory epitopes on FimH-DSG TM.

(A) Structure of FimH-DSG TM in complex with 329-2 and 445-3 Fabs solved by cryo-EM. Top image shows the cryo-EM map, colored by chain, as indicated. Insets show respective epitope interfaces, with each Fab shown in transparent surface representation and FimH in cartoon representation, with residues contributing to each epitope surface shown in stick representation (alpha carbons are represented by transparent spheres). (B) CryoEM structure of FimH-DSG TM in complex with 440-2 and 445-3 Fabs, colored by chain, as indicated. Inset shows the epitope interface, with the Fab shown in transparent surface representation and FimH in cartoon representation, with residues contributing to each epitope surface shown in stick representation (alpha carbons are represented by transparent spheres). (C) Sequence of FimH (with stabilizing mutations underlined) with residues contributing to each Fab’s epitope highlighted and colored by their respective Fabs as in A and B. (D) Cartoon representation of the FimH_LD in the FimH-DSG TM crystal structure. Epitopes identified in this study and in previous work by others (Mab 926, 824, 475 and 21) are highlighted, with participating residues shown as spheres and colored as indicated. Numbered gray residues indicate coincidence between two epitopes (1 = Mab 926 and Mab 475, 2 = Mab 926 and Mab 445-3, 3 = Mab 475 and Mab 824, 4 = Mab 440-2 and Mab 926).

Fig 7. Structural basis of inhibitory mechanisms of different Mabs.

(A) Superimposition of FimH WT bound to trimannose (PDB code 6GTV, blue) and FimH-DSG TM (orange) cryo-EM structure resolved from the complex with Fabs of 440-2 (green surface) and 445-3 (not shown). (B) Superimposition of FimH WT bound to trimannose (PDB code 6GTV, blue) and FimH-DSG TM (orange) cryo-EM structure resolved from the complex with Fabs of 329-2 (purple) and 445-3 (dark blue). Surface representations for the two Fabs are shown. Side chains from FimH-DSG TM that are part of the Mab epitopes are shown in stick format. In (A) and (B), shift of the clamp loop and Glycine loop from FimH-DSG TM to mannose-bound FimH WT is highlighted by dashed lines. Conformations of the important FimH epitope loops for 440-2, 329-2, and 445-3 binding are shown as orange (FimH-DSG TM structure) and blue (FimH WT-mannose bound structure) lines.