Avian antibodies (IgY) targeting spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibit receptor binding and viral replication

Abstract

Background: The global pandemic of Coronavirus infectious disease 2019 (COVID-19), caused by SARS-CoV-2, has plunged the world into both social and economic disarray, with vaccines still emerging and a continued paucity of personal protective equipment; the pandemic has also highlighted the potential for rapid emergence of aggressive respiratory pathogens and the need for preparedness. Avian immunoglobulins (IgY) have been previously shown in animal models to protect against new infection and mitigate established infection when applied intranasally. We carried out a proof-of-concept study to address the feasibility of using such antibodies as mucosally-applied prophylaxis against SARS-CoV-2.

Methods: Hens were immunized with recombinant S1 spike glycoprotein of the virus, and the resulting IgY was evaluated for binding specificity, inhibition of glycoprotein binding to angiotensin converting enzyme-2 (ACE2) protein (the requisite binding site for the virus), and inhibition of viral replication in Vero cell culture.

Results: Titers of anti-S1 glycoprotein IgY were evident in yolks at 14 days post-immunization, peaking at 21 days, and at peak concentrations of 16.8 mg/ml. IgY showed strong and significant inhibition of S1/ACE2 binding interactions, and significantly inhibited viral replication at a concentration of 16.8 mg/ml. Four weeks' collection from eggs of two hens produced a total of 1.55 grams of IgY.

Conclusions: In this proof-of-concept study we showed that avian immunoglobulins (IgY) raised against a key virulence factor of the SARS-CoV-2 virus successfully inhibited the critical initial adhesion of viral spike glycoproteins to human ACE2 protein receptors and inhibited viral replication in vitro, in a short period using only two laying hens. We conclude that production of large amounts of IgY inhibiting viral binding and replication of SARS-CoV-2 is feasible, and that incorporation of this or similar material into an intranasal spray and/or other mucosal protecting products may be effective at reducing infection and spread of COVID-19.

Fig 1. SDS-PAGE analysis of SARS-CoV-2 S1 spike-specific IgY carried out under reducing conditions.

Lanes 4–7 show IgY extracted from yolks at indicated time intervals; lanes 8–10 show IgY in serum at Week 4; “A” and “B” indicate serum from each hen. IgY heavy and light chains show characteristic bands at 65 and 27 kDa, respectively. “Control” indicates material from unimmunized hens.

Fig 2. Titers of anti-SARS-CoV-2 S1 spike IgY in egg yolks over time.

Eggs were collected from immunized hens weekly over 6 weeks following the first immunization and analyzed by ELISA against the S1 antigen. Stars indicate dates of immunization injections.

Fig 3. Box and whisker plots of SARS-CoV-2 spike glycoprotein (S1) Receptor Binding Domain (RBD) percent binding to angiotensin converting enzyme-2 protein in a cell-free system.

Relative binding proportion (Y-axis) was determined by the kit-provided standard binding inhibitor. Boxes represent interquartile range (IQR) with median shown as center bar of each sample group. Whiskers represent 1.5 times the IQR. P-value, by two-sample t-test method, and 95% confidence interval (CI) was calculated using R software package EnvStats (v.2.3.1). [19] Undiluted (16.8 mg/ml), and logarithmic dilutions (1.68, 0.168, and 0.0168 mg/ml) are shown. Coral, control IgY, teal, anti-SARS-CoV-2 spike glycoprotein IgY.

Fig 4. Box and whisker plots of SARS-CoV-2 spike glycoprotein (S1) percent binding to angiotensin converting enzyme-2 protein in a cell-free system.

Relative binding proportion (Y-axis) was determined by the kit-provided standard binding inhibitor. Boxes represent interquartile range (IQR) with median shown as center bar of each sample group. Whiskers represent 1.5 times the IQR. P-value, by two-sample t-test method, and 95% confidence interval (CI) was calculated using R software package EnvStats (v.2.3.1) [19]. Undiluted (16.8 mg/ml), and logarithmic dilutions (1.68, 0.168, and 0.0168 mg/ml) are shown. Coral, control IgY, teal, anti-SARS-CoV-2 spike glycoprotein IgY.

Fig 5. Box and whisker plots of SARS-CoV-2 viral replication patterns in vitro.

Boxes represent interquartile range (IQR) with median shown as center bar of each sample group. Whiskers represent 1.5 times the IQR. P-value, by two-sample t-test method, and 95% confidence interval (CI) was calculated using R software package EnvStats (v.2.3.1) (Millard, 2013). Undiluted (16.8 mg/ml), and logarithmic dilutions (1.68, 0.168, and 0.0168 mg/ml) are shown. P-values less than 0.05 are shown. Coral, control IgY; gold, undiluted S1 IgY; green, S1 IgY diluted at 1:10; blue, S1 IgY diluted at 1:100; purple, S1 IgY diluted at 1:1000.