Egg yolk immunoglobulin (IgY) targeting SARS-CoV-2 S1 as potential virus entry blocker

Abstract

Aims: COVID-19 pandemic caused by SARS-CoV-2 has become a public health crisis worldwide. In this study, we aimed at demonstrating the neutralizing potential of the IgY produced after immunizing chicken with a recombinant SARS-CoV-2 spike protein S1 subunit.

Methods and results: E. coli BL21 carrying plasmid pET28a-S1 was induced with IPTG for the expression of SARS-CoV-2 S1 protein. The recombinant His-tagged S1 was purified and verified by SDS-PAGE, Western blot and biolayer interferometry (BLI) assay. Then S1 protein emulsified with Freund's adjuvant was used to immunize layer chickens. Specific IgY against S1 (S1-IgY) produced from egg yolks of these chickens exhibited a high titer (1:25,600) and a strong binding affinity to S1 (K_D = 318 nmol L^-1 ). The neutralizing ability of S1-IgY was quantified by a SARS-CoV-2 pseudotyped virus-based neutralization assay with an IC₅₀ value of 0.99 mg ml^-1 . In addition, S1-IgY exhibited a strong ability in blocking the binding of SARS-CoV-2 S1 to hACE2, and it could partially compete with hACE2 for the binding sites on S1 by BLI assays.

Conclusions: We demonstrated here that after immunization of chickens with our recombinant S1 protein, IgY neutralizing antibodies were generated against the SARS-CoV-2 spike protein S1 subunit; therefore, showing the potential use of IgY to block the entry of this virus.

Significance and impact of the study: IgY targeting S1 subunit of SARS-CoV-2 could be a promising candidate for pre- and post-exposure prophylaxis or treatment of COVID-19. Administration of IgY-based oral preparation, oral or nasal spray may have profound implications for blocking SARS-CoV-2.

Keywords: COVID-19; S1; SARS-CoV-2; antibody; egg yolk immunoglobulin Y (IgY); hACE2.

FIGURE 1

SARS‐CoV‐2 S1 protein preparation and the immunization strategy of chickens. (a) Schematic diagram of the immunization strategy to generate IgY against SARS‐CoV‐2 S1 protein. Booster immunizations of the same dose were performed at both the end of the 2nd and 4th week after the primary immunization (500‐μg protein/dose/animal) (S1 group, n = 5). The control chickens (n = 3) were similarly immunized and boosted with the mixture of PBS and adjuvant. (b) SDS‐PAGE analysis of proteins distributed between soluble and insoluble cell fraction after bacterial cell disruption. Lane 1 and 2, the total proteins of the transformed E. coli before and after induction. Lane 3 and 4, the total insoluble inclusion body proteins before and after induction; Lane 5 and 6, the total soluble protein before and after induction. (c) Analysis of SARS‐CoV‐2 S1 protein in different purification steps by SDS‐PAGE. Lane 1, inclusion body proteins solubilized in 8 mol L⁻¹ Urea; Lane 2, supernatant after passing through Ni‐NTA resin; Lane 3–5, the wash of Ni‐NTA resin; Lane 6–12, elution from Ni‐NTA resin. (d) The functional protein (folded form) was analysed by Western blotting using anti‐S antibody and developed using DAB. M, molecular weight ladder. The triangle denotes the band corresponding to the SARS‐CoV‐2 S1 protein. (e) Binding profiles of SARS‐CoV‐2 S1 to hACE2 measured by BLI in Octet K2. Kinetic constants were calculated using a minimum of five dilutions of hACE2

FIGURE 2

The strong immunization response to SARS‐CoV‐2 S1 protein in chickens. The change of S1‐specific antibody binding titers in the egg yolk (a) and the serum (b). The level of S1‐specific antibody binding titers was measured by ELISA using the purified recombinant SARS‐CoV‐2 S1 protein (OD at 450 nm) as an antigen and expressed as an ELISA value. (c) The SDS‐PAGE profile of IgY. Two IgY chains appeared on the SDS‐PAGE gel. HC, heavy chain ≈ 65 kDa; LC, light chain ≈ 27 kDa. M, molecular weight ladder; Lane 1, water‐soluble fraction (WSF); Lane 2, purified IgY. (d) Binding profiles of SARS‐CoV‐2 S1 to S1‐IgY. Binding kinetics were measured for five concentrations of S1‐IgY at twofold serial dilution ranging from 1500 to 93.75 μg ml⁻¹

FIGURE 3

Characteristics of the neutralization activity of IgY antibodies against pseudotyped SARS‐CoV‐2. Serial dilutions of the pooled egg yolk IgY or the pooled serum preparations were pre‐incubated with the pseudotyped SARS‐CoV‐2 at 37°C for 1 h before they were added to 293T‐hACE2 cells. Luciferase activity was measured 24 h later to calculate IC₅₀ of the antibody. Pseudovirus neutralization curves of IgY (a) and serum (b) from the control group (▲) and the S1 group (●) were shown. Data were expressed as mean ± SD from 3 to 5 replicates

FIGURE 4

Competition binding to SARS‐CoV‐2 S1 between S1‐IgY and hACE2. (a) The interaction between SARS‐CoV‐2 S1 protein and hACE2 was inhibited by S1‐IgY. SARS‐CoV‐2 S1 was sequentially bound by S1‐IgY, C‐IgY or N‐IgY at the indicated concentration followed by the hACE2 receptor. The legend lists the immobilized SARS‐CoV‐2 S1, IgY and receptors that correspond to each curve. (b) S1‐IgY exhibited partial competition with hACE2 for the binding sites on S1. After loading with SARS‐CoV‐2 S1, the sensors were pre‐incubated with hACE2 for 300 s and then dipped into S1‐IgY, hACE2 or the mixture of the two for 600 s. PBST served as a nonbinding control. The graph displays the time course of binding patterns. The second binding and dissociation steps revealed the competition of S1‐IgY and hACE2 with SARS‐CoV‐2 S1 (enlarged box)