Egg-Derived Anti-SARS-CoV-2 Immunoglobulin Y (IgY) With Broad Variant Activity as Intranasal Prophylaxis Against COVID-19

Abstract

COVID-19 emergency use authorizations and approvals for vaccines were achieved in record time. However, there remains a need to develop additional safe, effective, easy-to-produce, and inexpensive prevention to reduce the risk of acquiring SARS-CoV-2 infection. This need is due to difficulties in vaccine manufacturing and distribution, vaccine hesitancy, and, critically, the increased prevalence of SARS-CoV-2 variants with greater contagiousness or reduced sensitivity to immunity. Antibodies from eggs of hens (immunoglobulin Y; IgY) that were administered the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were developed for use as nasal drops to capture the virus on the nasal mucosa. Although initially raised against the 2019 novel coronavirus index strain (2019-nCoV), these anti-SARS-CoV-2 RBD IgY surprisingly had indistinguishable enzyme-linked immunosorbent assay binding against variants of concern that have emerged, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529). This is different from sera of immunized or convalescent patients. Culture neutralization titers against available Alpha, Beta, and Delta were also indistinguishable from the index SARS-CoV-2 strain. Efforts to develop these IgY for clinical use demonstrated that the intranasal anti-SARS-CoV-2 RBD IgY preparation showed no binding (cross-reactivity) to a variety of human tissues and had an excellent safety profile in rats following 28-day intranasal delivery of the formulated IgY. A double-blind, randomized, placebo-controlled phase 1 study evaluating single-ascending and multiple doses of anti-SARS-CoV-2 RBD IgY administered intranasally for 14 days in 48 healthy adults also demonstrated an excellent safety and tolerability profile, and no evidence of systemic absorption. As these antiviral IgY have broad selectivity against many variants of concern, are fast to produce, and are a low-cost product, their use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation.

Clinical trial registration: https://www.clinicaltrials.gov/ct2/show/NCT04567810, identifier NCT04567810.

Keywords: COVID-19; IgY; SARS-CoV-2; chicken immunoglobulin; clinical trial body; immunoglobulin Y; infectious diseases.

Figure 1

Design of phase 1 human single-ascending and multiple-dose study.

Figure 2

RBD and IgY preparation. (A) Workflow of the study. IgY preparation for intranasal drops as antiviral prophylaxis. (B) Cell-free expressed RBD derived from the Spike protein on the viral envelope of SARS-CoV-2. (C) Characterization of the recombinant protein RBD by ELISA and HPLC. (D) Determination of the affinity of the cell-free expressed RBD (amino acids 328-533) and mammalian-expressed full-length S1 to the hACE2 using Biacore.

Figure 3

IgY purification and characterization. (A) Western blot analysis of the IgY preparation. (B) HPLC profile of the IgY preparation. (C) Western blot analysis of anti-SARS-CoV-2 IgY against RBD fragment and full S1 recombinant protein. (D) IgY yield for various batches derived from 100 eggs each. (E) Western blot data of different lots of anti-SARS-CoV-2 RBD IgY (Y0120-Y0199). Pools of 100 eggs laid by 9 hens over 2 weeks were used for each pool of IgY preparation between May 2020 and March 2021. IgY lot samples were diluted 1:500 followed by a 1:3000 dilution of rabbit anti-IgY HRP conjugate. First left lane shows the Coomassie stain of the same gels. (F) Time-dependent ELISA titers of sera from 3 individual hens following continual immunization (left); arrows indicate immunization timing. Time-dependent ELISA titer of 3 hens after immunization was stopped for up to 12 weeks (right). (G) Neutralization of pseudovirus SARS-CoV-2 by various lots of anti-SARS-CoV-2 RBD IgY (conducted at RetroVirox). (H) Neutralization of live index SARS-CoV-2 virus by anti-SARS-CoV-2 RBD IgY (Y0180, conducted at USAMRIID).

Figure 4

Common SARS-CoV-2 variants and anti-SARS-CoV-2 IgY interaction with them (A) A scheme depicting locations of mutated amino acids in Alpha through Mu variants of SARS-CoV-2, focusing on the RBD domain only. Each color bar indicates the amino acid in the index virus that was mutated in the variant. (B) Spike protein of SARS-CoV-2 Alpha, Beta, Delta, and Omicron are shown from left to right. Molecular Operating Environment was used to create the figure (35). The location of mutations in the structure of the S protein trimer of SARS-CoV-2 (PDB ID: 7A98) for 4 of the common variants are indicated in red and glycosylation sites are indicated in pink throughout the S protein. Blue ribbon indicates RBD (amino acids 328-533), and the orange ribbon indicates receptor binding motif (amino acids 437-508). (C) Binding of anti-SARS-CoV-2 RBD IgY to recombinant S1 full length (FL) of the index virus, the RBD of the Alpha and Beta variants, and the immunizing RBD of the index virus by ELISA. (D) Neutralization of pseudovirus (VSV-S) SARS-CoV-2 carrying S protein of index virus, Alpha, or Beta variants by anti-RBD IgY. (E) Neutralization of live index or Delta viruses by anti-SARS-CoV-2 IgY against the RBD. (F) Neutralization of pseudoviruses listed in (D) by human serum of immunized individuals. (G) Neutralization of live D614G vs. Delta variants by human serum of immunized individual or by anti-SARS CoV-2 IgY. Microscopic evaluation of monolayers of Vero E6 cells after 96 hours infection with the indicated authentic (live) SARS-CoV-2 variant. Images from infected cells are shown after 4 days of infection with SARS-CoV-2 variants in the absence or presence of test items. Top three panels: Infection in the presence of MEX-BC2/2020 and bottom three panels: infection with the Delta variant each in the presence of vehicle alone, serum of a person immunized twice with the Moderna (mRNA-1273) vaccine or anti-SARS CoV-2 RBD IgY, as indicated, all at the indicated concentration (neutralization experiments in panels D-H & I were conducted by RetroVirox using pseudovirus or live virus, as indicated). (H) Binding of anti-SARS-CoV-2 RBD IgY to the index virus and Omicron variant (B.1.1.529) RBD domain using ELISA. (I) Neutralization of live index or Omicron variant of SARS-CoV-2 by anti-SARS-CoV-2 RBD IgY. Except when indicated, the studies were done over several months; therefore, the absolute titers in the ELISA and neutralization studies were not identical. However, each experiment included the same positive control; index RBD for ELISA and index virus for neutralization assays.

Figure 5

Preclinical toxicity of 28-day treatment with anti-SARS-CoV-2 RBD IgY in rats. (A) Study design. (B) Summary of findings. (C) Serum levels of 27 cytokines over time in rats treated with IgY (Tx) or vehicle/placebo (PL). Data are provided for Day 1 before treatment (D1H0); Day 1, 4 hours after the treatment (D1H4); 24 hours after the two treatments, 6 hours apart, at 24 hours after the first treatment (D1H24); 28 days of twice-daily treatments and 4 hours of the treatment of that day (D28H4); and 28 days of twice-daily treatments and 24 hours of the treatment of that day (D28H24). Red indicates a statistical difference with a false discovery rate (FDR) significant p-value (p < 0.05).

Figure 6

Lack of cross-reactivity of anti-SARS-CoV-2 RBD IgY with human tissues. Immunohistochemical testing of anti-SARS-CoV-2 RBD IgY (top row), control IgY (middle row; negative control), and anti-human macroglobulin antibodies (bottom row; positive control) with human nasal mucosa (left two panels) and human lungs (right two panels). Bars provide a magnification scale.