Isolation and characterization of selective and potent human Fab inhibitors directed to the active-site region of the two-component NS2B-NS3 proteinase of West Nile virus

Abstract

There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes.

Figure 1. Structure and sequence alignment of the NS2B cofactor and the NS3 proteinase domain of WNV and DV2

Upper panel: sequence alignment of the NS2B–NS3pro constructs; homologous amino-acid-residue positions are shaded. The asterisks above the sequences indicate His⁵¹, Asp⁷⁵ and Ser¹³⁵ of the catalytic triad. The arrows indicate mutations (G22S, DDD/AAA, H51A, T52V and R76L). The G22S and DDD/AAA constructs also contained the K48A mutation that inactivated the Lys⁴⁸↓Gly autolytic cleavage site where glycine is the N-terminal residue of the linker. Lower panel: the structure of WNV NS2B–NS3pro (PDB accession code 2IJO) [8]. The original position of the His⁵¹, Asp⁷⁵ and Ser¹³⁵ of the catalytic triad (italicized) and the mutant positions are shown. The mutations that affect the binding of the proteinase with the antibodies are underlined. The surface is coloured by electrostatic potential (negative, red; positive, blue). The magenta ribbon shows NS2B contributing to the NS3pro active site. The positioning of a substrate-mimetic peptide (KRKARI) is shown in yellow. The model was presented using PyMol software (DeLano Scientific; http://www.pymol.org).

Figure 2. Selection process of the antibodies directed to the region proximal to the essential His⁵¹ of the catalytic triad

The purified NS2B–NS3pro K48A construct was used for selection of the phage antibody library, whereas the inert H51A mutant with the mutation of the active-site His⁵¹ was used for library blocking and counter-screening. The constructs were C-terminally tagged with a His₆ tag. The antibodies, which recognize the K48A enzyme and which do not recognize the mutant, were selected from the antibody library. BSA, ubiquitin–His₆ and CD33–His₆ were used as controls to eliminate false-positive antibodies. The AbD05323 antibody (indicated by an arrow) was selected in this particular experiment. Hisx6, His₆; ki, K_i.

Figure 3. Catalytic parameters of the inhibitory antibodies

(A) Before the addition of the Pyr-RTKR–AMC substrate (25 μM), the purified WNV proteinase (10 nM) was co-incubated for 30 min with increasing concentrations of the antibodies. The cleavage of the Pyr-RTKR–AMC peptide by the proteinase was monitored to determine the K_i values. (B) Left-hand panel: competitive inhibition of NS2B–NS3pro proteolysis of Pyr-RTKR–AMC NS2B–NS3pro by the AbD05444 antibody (0–4 μM). Right-hand panel: Lineweaver–Burk plot of the results. The inhibitory antibody was added to the reactions. The initial velocity of the Pyr-RTKR–AMC cleavage was then measured in triplicate. Note that the K_m values, but not the V_max values, are affected by the inhibitor. RFU, relative fluorescence unit; V₀ and Vss, steady-state rate.

Figure 4. The H51A, T52V and R76L WNV NS2B–NS3 mutants do not interact with the AbD05323 and AbD05444 antibodies

The purified mutant constructs were analysed by SDS/PAGE (4 μg/lane) followed by Coomassie staining (CS) and Western blotting (100 ng/lane) with a His₆-tagged antibody and the selected AbD05320, AbD05321, AbD05322, AbD05323, AbD05444, AbD05445 and AbD05446 antibodies. Note that AbD05323 and AbD05444 did not bind to the H51A, T52V and R76L constructs, which exhibit mutations in the proximity of the active site.

Figure 5. ELISA of the NS2B–NS3pro constructs

The antibodies were allowed to interact with the native K48A and mutant WNV co nstructs and with the WT DV2 constructs. The binding efficiency is expressed as a fold increase relative to the control (BSA).