Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein

Abstract

West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIII^L297-S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1^G299-K307 and DIII-2^V371-R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1^G299-K307 and DIII-2^V371-R388 on a dot blot assay. Among them, sdAb_A1, sdAb_A6, sdAb_A9, and sdAb_A10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAb_A10 and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC₅₀) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAb_A10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAb_A10 targeting DIII-2^V371-R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV-induced neuropathogenesis.

Keywords: West Nile virus; West Nile virus-like particles; human brain microvascular endothelial cells; nanobodies; phage display; single domain antibody.

FIGURE 1

Overexpression of recombinant domain III (rDIII). (A) LDS-PAGE showing a single protein band of purified rDIII at ∼12 kDa. (B) The protein mass spectrum of rDIII showed a single peak at 12.8 kDa on MALDI-TOF MS.

FIGURE 2

Single-domanin antibodies (sdAbs) targeting rDIII and its interaction with the synthetic analogs of DIII-1^G299–K307 and DIII-2^V371–R388. (A) Western blot assay showing chemiluminescent horseradish peroxidase (HRP) signal on the lysates of sdAb producing Escherichia coli SHuffle clones at ∼15 kDa incubated with HisProbe HRP conjugate. (B–D) Dot blot assay identifying the interaction between Ni affinity-purified 96 sdAbs targeting either the synthetic analogs of DIII-1^G299–K307 (B) or DIII-2^V371–R388 (C) by the streptavidin-HRP conjugate. sdAbs possessing strong interaction with synthetic analogs are circled. (D) non-related sdAb (raised against NadA of N. meningitides) used as a negative control showed no interaction with synthetic analogs of DIII-1^G299–K307 or DIII-2^V371–R388 in the same dot blot assay—no signals for streptavidin-HRP conjugate. (E) Hyperimmune serum of horse-surviving natural WNV infection incubated with DIII-1^G299–K307 or DIII-2^V371–R388 was used as a positive control—prominent signals for streptavidin-HRP conjugate.

FIGURE 3

sdAb targeting the DIII blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) proteins. (A) NC strips transblotted with hBMEC proteins were incubated with either rDIII (positive control) or rDIII pretreated with sdAbs targeting receptor binding motifs of DIII. sdAb used to pretreat rDIII are mentioned on each NC strip. The interaction between rDIII/pre-treated rDIII and hBMEC proteins at ∼15 kDa was detected by HisProbe-HRP conjugate and chemiluminescent HRP substrate in Western blot analysis. sdAbs blocking the rDIII-hBMEC interaction showed the absence of chemiluminescent HRP signal at 15 kDa is framed. For negative control (Neg.), NC strip of hBMEC protein was precluded with the incubation of rDIII in the same Western blot assay, and no signal was detected. (B) The ability of sdAb_A1, sdAb_A6, sdAb_A9, and sdAb_A10 to block the interaction between rDIII and hBMECs was detected through cell ELISA. rDIII alone (positive control) or aforestated sdAbs-treated rDIII was incubated on paraformaldehyde-fixed hBMECs on the ELISA plate. The interaction was detected by HisProbe-HRP conjugate and UltraTMB-ELISA substrate. hBMECs precluded with the addition of either rDIII or sdAbs served as a negative control. Antigens and sdAbs added on fixed hBMECs in ELISA plate (framed) are mentioned below each bar graph. A significant difference between the compared treatments by one-way ANOVA (p < 0.05) is indicated by different alphabets.

FIGURE 4

Quality assessment and amino acid sequence of overexpressed sdAbs. (A) Overexpressed and Nickel Nitrilotriacetic Acid (Ni NTA) purified sdAbs, sdAb_A1, sdAb_A6, sdAb_A9, and sdAb_A10, subjected to LDS-PAGE showed prominent protein band at ∼15 kDa. (B) Protein mass spectrum of sdAb_A1 (1), sdAb_A6 (2) sdAb_A9 (3), and sdAb_A10 (4) showing single peak on MALDI TOF MS are presented with the corresponding molecular mass of sdAbs.

FIGURE 5

Neutralization of West Nile virus (WNV)-like particles (WNV-VLP) by sdAbs. (A) virus neutralization test used to assess the ability of sdAb_A1, sdAb_A6, and sdAb_A10 to neutralize the WNV-VLP. Hyperimmune serum of horse (positive control), sdAb_A3 (sdAb that does not block the binding of DIII to hBMEC), and non-related sdAb (negative control – VHH_F3 raised against N. meningitidis) were used in the same assay. (B) The amount of sdAb (nanograms) required to neutralize the WNV-VLP (EC₅₀) was calculated using non-linear regression (dilution of sdAb on Y-axis and quantity sdAbs in nanograms on X-axis) in Graphpad Prism 8 software. The concentration of sdAb in nanograms per well, per ml, and corresponding molar concentrations are tabulated.

FIGURE 6

Cross-reactivity, hemocompatibility, and safety of sdAb_s. (A) the interaction between sdAbs and rDIII of TBEV was assessed on ELISA to determine possible cross-reactivity. Antigen-coated (Coating), primary (primary Ab), and secondary (secondary Ab) antibodies used in the ELISA are mentioned under each bar graph of Absorbance₄₅₀ values (Mean + SE). CB, coating buffer; Anti Myc, antiMyc antibody; HisProbe, HisProbe HRP conjugate. (B) Percentage of hemolysis in sheep erythrocytes caused by 5–30 μg of sdAb_A10 was determined by measuring the absorbance of oxyhemoglobin at 414 nm over a period of 1–5 h. Even at the highest concentration of sdAb_A10, just 0.1% hemolysis was observed as compared to 0 and 100% of hemolysis in 0.9% saline (negative control) and 2% Triton X (positive control) treated erythrocytes, respectively. The entire assay was performed in triplicate, and the bar graphs represent mean ± SE. (C) XTT based proliferation assay on hBMECs treated with either 2 μg (143 pM) of sdAb_A10, 1 × PBS (negative control), and 0.1% Triton X-100 (positive control) was performed in triplicate. hBMECs treated with sdAb_A10 showed 82% viability as compared with 100% viability in negative control and 5% viability in positive control groups. The data represent mean + SE absorbance values measured at 450 nm on an ELISA plate reader. The significant difference between the compared treatments by one-way ANOVA (p < 0.05) is indicated by different alphabets.