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. 2021 May 28;16(5):e0252399. doi: 10.1371/journal.pone.0252399

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

Chad Artman 1,#, Kyle D Brumfield 2,3,#, Sahil Khanna 4, Julius Goepp 1,*
Editor: Paulo Lee Ho5
PMCID: PMC8162713  PMID: 34048457

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.

Introduction

The global pandemic of Coronavirus infectious disease 2019 (COVID-19), caused by several variants of the SARS-CoV-2 virus, has plunged the world into both social and economic disarray. The recent emergence of several efficacious vaccines will mitigate this impact, but full population-level coverage remains in the future. Emergence of new strains may further complicate attainment of widespread immunity to this virus and its variants. Finally, the absence of effective non-vaccine countermeasures highlights concerns about future outbreaks of emerging viral respiratory illnesses. Under these circumstances, an effective, rapidly-deployable, non-vaccine prophylactic approach, especially one that could be stockpiled ahead of new outbreaks, may add new tools to the public-health preparedness armamentarium.

Passive immunization using pathogen-specific antibodies is a potential means of preventing disease by various infectious agents in both humans and animals [1–3]. A critical necessity in this approach, however, is the production and availability of large quantities of inexpensive antibodies. Therefore, polyclonal antibodies may be advantageous over monoclonal antibodies, which, while highly epitope-specific, may not offer full clinical protection and are costly to produce in bulk [4].

Immunoglobulin Y (IgY), the primary circulating antibody in the serum of avians, reptiles, and amphibians, is transferred to yolks of developing eggs and provides passive immunity to the newly-hatched young [4, 5]. Readily harvested at high yields from immunized hens’ eggs, IgY has been shown to be safe and effective at both prophylaxis and treatment of bacterial and viral infections in laboratory and agricultural animals [1, 4]. A small number of human studies have shown IgY to be safe and effective in human therapeutic applications [2, 3, 6–8].

With regard to respiratory viral infections, intranasal application of IgY targeting whole, inactivated influenza virus has been shown to prevent murine influenza viral infection when applied prior to viral challenge, and to mitigate influenza disease when applied within a limited period following challenge [9, 10]. Human studies have demonstrated safety and efficacy of prophylactic intra-oral IgY against Pseudomonas aeruginosa in cystic fibrosis patients, and nonspecific intranasal polyclonal IgA in upper respiratory disease [11–14].

Given high transmissibility, lack of either a vaccine or an effective treatment, high mortality with the current COVID-19 crisis, and the threat to economic wellbeing posed by extended closures of businesses and public services, we conducted a proof-of-concept study of IgY targeting SARS-CoV-2 as a potential mucosal prophylaxis against infection. Results showed inhibition of binding to Angiotensin-2 (ACE2) receptor protein and reduced viral replication in laboratory studies, and feasibility studies of mucosal prophylactic formulation in vivo are in progress.

Materials and methods

Immunization of laying hens

Recombinant SARS-CoV-2 spike glycoprotein (S1) with sheep Fc-tag (HEK293; The Native Antigen Company, Oxford, United Kingdom) was reconstituted in phosphate-buffered saline (PBS) to 10% w/v and mixed under high-shear conditions with the poultry adjuvant Montanide ISA 70 VG (30% antigen in PBS, 70% adjuvant, v/v), following manufacturer’s specifications (Seppic Inc, Fairfield, NJ USA). The resulting emulsion, containing 100 μg spike glycoprotein/ml, was filter-sterilized using a 0.22 μm pore size polycarbonate filter membrane (VWR International, Radnor, PA, USA). Commercial White Rock and Rhode Island cross-bred, sexlink hens (Pinola Hatchery, Shippensburg, PA, USA) were maintained segregated in three pairs and acclimated for two weeks prior to immunization in a protected coop at ambient temperatures on a 12-hour light/dark cycle on ad libitum water and a commercial diet (Martin’s Layer Mash 16%, Martin’s Elevator, Inc., Hagerstown, MD, USA).

On immunization day 1, 0.5 ml (50 mcg antigen) of the vaccine/adjuvant preparation was injected intramuscularly into each breast of each of one hen pair (test pair). For control, two hens were injected with a PBS/adjuvant (30%/70% v/v) mixture and an additional pair of hens remained unimmunized. On immunization day 14, booster immunizations were performed identically to immunization day 1 for each hen pair. Protocols for hen maintenance, immunization, and phlebotomy were approved by the Scaled Microbiomics, LLC Animal Use and Care Committee (approval number 2020–001).

Serum IgY antibody determination

To detect circulating anti-S1 Spike antibody levels, 4 mL whole blood was drawn by brachial venipuncture from individual hens at 3 and 4 weeks after the first immunization, using 23G x ¾” SecureTouch safety scalp vein butterfly sets (Exel International, Quebec, Canada). To allow for clot formation, blood samples were incubated at room temperature (23°C– 25°C) for 30 minutes and stored at 4°C overnight (16 h). Blood serum was separated from cells by centrifugation at 5,000g for 5 minutes, and IgY titer (from both immunized and unimmunized hens) was determined by enzyme-linked immunosorbent assay (ELISA) against the S1 antigen as described below. Samples were only obtained at Weeks 3 and 4; serum collection was discontinued after Week 4 when yolk IgY levels were detected.

IgY extraction and concentration

For IgY egg yolk extraction, two eggs were collected weekly from each pair of hens beginning the first day prior to first immunization. IgY was extracted from yolks using polyethylene glycol (PEG) as described elsewhere [15], with the following modifications. Briefly, yolks were pooled, and lipid content was removed by centrifugation (13,000 x g for 20 min at 4°C) using PEG 6000 (Alfa Aesar, Haverhill, MA, USA) at 12% w/v. The resulting precipitate was resuspended in PBS and dialyzed against dilute sodium chloride (0.1% w/v) overnight (16 h) and then against PBS for three hours.

Total protein concentration of egg yolk and serum was determined by bicinchoninic acid method kit, following manufacturer’s specifications (Thermo Fisher Scientific, Rockford, IL, USA), using S1-coated 96-well plates. Absorbance values were read at 490 nm, and standard curve showed linear behavior over seven serial 1:2 dilutions (R2 = 0.99; S1 Fig) using bovine gamma-globulin (Bio-Rad, Hercules, CA, USA) as the protein standard set. Resulting IgY was stored at -20°C until further analysis (< 2 weeks).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

To determine purity of both yolk- and serum-derived IgY, SDS-PAGE was conducted under reducing conditions using 12% polyacrylamide gel (NuSep Inc., Germantown, MD, USA) with a Novex Mini-Cell (Invitrogen, Carlsbad, CA, USA). Briefly, the sample was mixed with sample buffer and incubated for 5 min at 100°C. A total of 10 μl of sample was loaded into each well. Precision Plus Dual Color Pre-stained protein standard (10-250kDa) (Bio-Rad, Hercules, CA, USA) was used as a molecular weight marker. The protein bands were visualized with Protein Fixative (Ward’s Science, Rochester, NY, USA). The gel was imaged using a standard camera; the original gel image can be found in the S1 Raw image. Specificity of IgY binding to purified S1 glycoprotein was demonstrated by dot-blot (S2 Fig).

Enzyme-linked immunosorbent assay

IgY titer against SARS-CoV-2 S1 glycoprotein subunit was measured by an indirect noncompetitive ELISA as reported previously with slight modifications [4, 16, 17]. Briefly, a 96-well microtiter plate was coated with S1 antigen at 4 ng/μl protein in carbonate coating solution (BioLegend, San Diego, California) using 100 μl/well. Plates were blocked using skim milk in PBS (5% w/v) overnight (16 h) at 4°C. Serial dilutions (1:100, 1:1,000, 1:10,000) of anti-S1 IgY, obtained from both yolk and serum, were incubated in blocked plates for one hour at room temperature (23°C-25°C). Bound anti-S1 IgY was detected with 100 μl per well horseradish peroxidase-conjugated goat anti-chicken IgY (1:2,500) (ImmunoReagents, Inc., Raleigh, NC, USA). After incubation for 1 hour at room temperature (23°C-25°C), the plate was washed five times using commercial ELISA wash buffer (Thermo Fisher, Rockford, IL, USA). Next, 100 μl per well 3,3’-5,5’-tetramethylbenzidine (VWR International, Radnor, PA, USA) one-component substrate was added and incubated for 15 min at room temperature (23°C-25°C). The color development was stopped with 2N sulfuric acid (VWR International, Radnor, PA, USA) (100 μl per well), and the optical density (OD) was measured on a microtiter plate reader (Molecular Devices, Sunnyvale, California, USA) at 450 nm. Experiment reproducibility and assay calibration was established by including triplicate wells coated with S1 antigen (background from antigen noise) which received a blank IgY treatment (PBS with 5% w/v skim milk; No IgY), triplicate non-coated wells (background from IgY noise) which received a blank antigen treatment (carbonate coating buffer; No S1 antigen), and two negative unimmunized IgY experimental controls obtained from eggs (S1-vaccinated hen pre-immunization IgY and unimmunized control hen IgY) in each plate. SARS-CoV-2 S1-specific IgY titer was defined as the maximum dilution multiple of the sample with an OD value that was ≥ 2.1 times that of both negative controls.

Binding inhibition of S1 to ACE2

Binding interactions of S1 to ACE2 protein were analyzed using the SARS-CoV-2 Inhibitor Screening Kit (Acro Biosystems, Newark, DE), according to the manufacturer’s instructions [18]. This kit provides the receptor binding domain (RBD) peptide of the S1 glycoprotein spike, and a standardized inhibitor of S1/ACE2 binding, which provides a positive control. Method Verification was carried out per the included kit instructions (S1 File) [18].

Additionally, to determine inhibitory capacity of anti-SARS-CoV-2 S1 IgY against the entire S1 glycoprotein, the assay was repeated with the following modifications. Briefly, recombinant SARS-CoV-2 spike glycoprotein (S1) with sheep Fc-tag (HEK293; The Native Antigen Company, Oxford, United Kingdom) was used in place of the S1 receptor binding domain protein provided by the kit.

Plaque reduction assay

To evaluate the ability of S1-specific IgY to prevent viral replication in vitro, a plaque reduction assay was carried out. Briefly, anti-S1 and control (unimmunized) IgY were incubated for 1 hour at 37°C with SARS-Cov2 virus (BEI Resources, Manassas, VA, USA) at a 1:1 ratio (50 μl of undiluted antibody with 50 μl of virus with a titer of 3 x 105 pfu/ml). Vero cells (ATCC) were seeded at a density of 3 x 105 cells/ml and incubated overnight at 37°C until confluent. Antibody-viral mixtures were serially diluted (undiluted, 1:10, 1:100, 1:1000) in Dulbecco’s modified Eagle’s medium and used to infect Vero cells. Infected plates were incubated at 37°C for 1 hour, followed by addition of an agar overlay (2X Eagle’s Minimum Essential medium and 0.06% agarose at a 1:1 ratio) into each well. Infected cells with no IgY treatment and cells treated with antibody from unimmunized hens’ eggs were used as controls. Plates were incubated at 37°C in a 5% CO2 cell culture incubator for 48 hours. Cells were then fixed with formaldehyde (10% v/v), added to plates with agar plugs, and incubated at room temperature (23°C-25°C) for 45 min. Plates were rinsed with deionized water, and agar plugs removed. Wells were stained using crystal violet (1% v/v) and washed with ethanol (20% v/v) twice. Plaques were counted to determine viral titer as plaque forming units (pfu/ml); raw data are available in S2 File.

Results

Isolation and purification of IgY

Avian IgY was successfully isolated from yolks, yielding on Day 28 following first immunization, up to 16.8l mg/ml (min. 10 mg/ml, avg. 12.4 mg/ml), and from serum, yielding, on Day 21 following first immunization, up to 23.1mg/ml (min. 20.9 mg/ml, avg. 22.1mg/ml). SDS-PAGE showed characteristic IgY banding patterns, with molecular weights of 68 kDa and 25 kDa, representing the IgY heavy and light chains, respectively (Fig 1). By the end of the 6-week study period, a total of 125 ml of IgY in PBS solution had been produced, yielding a total of 1.55 g IgY from two immunized hens.

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

Fig 1

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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.

Titer of anti-S1 glycoprotein IgY in egg yolks

Titers of IgY against the S1 glycoprotein rose following two immunizations to a peak value of 1:10,000 by Week 3, falling to 1:1000 by Week 6 (Fig 2), similar to our observations in work with other viral and bacterial antigens (S3 Fig). Serum anti-S1 glycoprotein antibody levels were detected at weeks 3 and 4 at titers of 1:10,000, after which serum collection was discontinued.

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

Fig 2

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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.

Inhibition of S1 spike binding to ACE2 protein by S1-specific IgY

Using the kit-provided S1 spike glycoprotein RBD peptide as substrate against the target ACE2 protein, binding was inhibited significantly in a concentration-dependent manner at IgY dilutions through 1:10 (undiluted, p = 1.2 x 10−5; 1:10, p = 1.3 x 10−4; Fig 3). The undiluted material used in this and subsequent analyses contained 16.8 mg IgY/ml.

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.

Fig 3

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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.

When the binding inhibition assay was employed using the full S1 spike glycoprotein, binding of the spike glycoprotein to the target ACE2 protein was significantly inhibited in a concentration-dependent manner at all IgY dilutions through 1:100 (undiluted, p = 4.2 x 10−6; 1:10, p = 1.5 x 10−6; 1:100, p = 8.6 x 10−4; Fig 4).

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.

Fig 4

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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.

Inhibition of SARS-CoV-2 virus replication by IgY

To further evaluate the antiviral activity of the S1-specific IgY, a plaque reduction assay was performed. The undiluted IgY at a protein concentration of 16.8 mg/ml produced a significant 38.3% reduction in plaque-forming units compared with control IgY (Fig 5).

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

Fig 5

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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.

Discussion

SARS-CoV-2, the causative viral agent of COVID-19, is transmitted person-to-person primarily through close contact and respiratory droplets [20]. After entry to the human respiratory tract, the virus must bind to ACE2 protein receptors on respiratory epithelial cells [21]. These features of viral infection offer an opportunity for prevention of active COVID-19 by intercepting incoming viral particles and preventing adhesion, which is required for cellular invasion. While masks and social distancing play important roles in slowing individual infection and person-to-person spread, protection is incomplete, and these methods have substantial social and economic impact. An alternate, or additional, means of protection might be offered by prophylactic treatment of mucosal surfaces.

In this proof-of-concept study we demonstrated that IgY targeting the functionally-important S1 spike glycoprotein of SARS-CoV-2 can be produced rapidly after only two injections of laying hens with the intact glycoprotein as antigen, yielding titers of IgY antibody of 1:10,000 by 3 weeks following the first immunization (Fig 2). Titers fell by 6 weeks to levels consistent with those in prior studies of influenza antigens in which up to 5 immunizations were performed [4]. The resulting IgY was strongly inhibitory of the essential S1 spike/ACE2 protein binding interaction required for initiation of infection (Figs 3 and 4), and demonstrated significant viral neutralization in a plaque reduction assay carried out with active SARS-CoV-2 viral particles (Fig 5).

In the four-week egg collection period of this study, we collected a total of 1,550 mg polyclonal IgY produced by two immunized hens, using a simple physicochemical extraction PEG extraction method. Flocks of 100,000 hens are commonly used in commercial egg production facilities, suggesting that in excess of 1,000 kg of this material could be produced annually by one such flock in large-scale production.

IgY has seen use in studies of respiratory infections, both viral and bacterial. Orally administered IgY showed high safety and efficacy in preventing colonization with Pseudomonas aeruginosa in human cystic fibrosis patients [2, 3]. IgY raised in ostrich eggs against pandemic strains of influenza (A/H1N1) was shown to inhibit hemagglutination and viral replication of the cognate strain in vitro [22].

Studies by Wallach, Wen, and others have demonstrated that intranasally-applied aqueous solutions of IgY targeting influenza viruses (A and B) is completely protective against murine infection when applied up to 6 hours prior to lethal viral exposure, and to prevent mortality when administered up to 16 hours post-exposure to a lethal dose of multiple viral strains [9, 10, 23]. Pathology examination of lung tissue from animals treated post-exposure showed significant reduction in lung pathology and viral replication compared with untreated mice [4, 23].

These findings, taken together with the work on IgY against SARS-CoV-2 presented here, suggest that an intranasal formulation of IgY targeting the SARS-CoV-2 virus may demonstrate similar prophylactic, and possibly therapeutic, results.

Given the large abundance of target-specific IgY in yolks of immunized hens’ eggs, the speed at which large quantities can be produced, the history of safety and efficacy of intranasal IgY in prophylaxis and treatment of serious respiratory infections, the present lack of a vaccine against SARS-CoV-2, and the imperative to prevent individual infections and person-to-person spread, we believe that further study of anti-SARS-CoV-2 IgY is warranted. Future studies should examine generation of IgY against RBD epitopes of the S1 glycoprotein, to reduce production costs and increase target specificity.

Animal studies are underway to determine in vivo efficacy; human studies to establish safety and tolerability of a final formulation will also be required, and regulatory pathways should also be explored. SARS-CoV-2 is not exclusively transmitted by nasal inhalation; the active IgY antibodies studied here are readily incorporated into oral lozenges or throat sprays, and can be formulated into sterile ophthalmic solutions as needed.

This proof-of-concept study has certain limitations. The number of hens used to generate anti-SARS-CoV-2 spike IgY was small, which may limit generalizability. Our data, however, show only minimal differences in IgY levels between hens (data not shown). We used ordinary agricultural laying hens, rather than specific pathogen-free birds, leaving open the small possibility of inadvertent inclusions of potential pathogens that could be transmitted to human subjects; this can be addressed in future studies seeking evidence of pathogens in the IgY material collected. This study was designed as a brief proof-of-concept, so that we collected eggs for a limited period, and performed only two immunizations, limiting our ability to accurately predict commercial production levels. In our hands, a more typical immunization schedule of up to five injections at two-week intervals produces sustained high-level titers.

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.

Supporting information

S1 Fig. Standard curve for bicinchoninic acid (BCA).

Total protein concentration was determined by BCA method targeting SARS-CoV-2 S1. Curve shows linearity over eight serial dilutions (R2 = 0.99).

(TIF)

Click here for additional data file. (537.7KB, tif)
S2 Fig. Dot-blot image showing IgY binding specificity against purified S1 glycoprotein.

Hens were immunized using norovirus virus-like particles (NVLP), and IgY was prepared as mentioned in the “Materials and methods” section. IgY solutions (NVLP, S1, and unimmunized) were spotted onto a nitrocellulose membrane (Azure Biosystems, Dublin, CA, USA) and allowed to dry at room temperature (RT) for 30 minutes. Each membrane was blocked in tris-buffered saline and Tween 20 (TBST) supplemented with 5% (v/v) skim milk for 1 hr at RT. Membranes were incubated with the appropriate IgY dilutions (1:1,000 anti-S1; 1:2,500 anti-VLP, and Unimmunized IgY) in blocking buffer at RT. After 1 hr, the primary antibody dilutions were aspirated, and each blot was washed three times (5 min) with TBST. Blots were then incubated for 1 hr with a 1:1,000 dilution of Goat anti-chicken HRP-conjugated IgG (ImmunoReagents, Inc., Raleigh, NC, USA) in blocking buffer at RT. Secondary antibody solution was then aspirated, and blots were washed three times (10 min followed by two 5 min washes) with TBST and washed a final time (5 min) with TBS. Color change was observed using TMB chemical substrate (VWR International, Radnor, PA, USA), per manufacturer’s directions. The reaction was quenched using TBS, and images were captured using a standard camera.

(TIF)

Click here for additional data file. (2.3MB, tif)
S3 Fig. IgY production over time.

ELISA titers (log2) of IgY targeting norovirus virus-like particles and bacterial multiepitope fusion antigen over 24 weeks are shown as line plots. Viral and bacterial immunizations were carried out intramuscularly, as mentioned in the “Materials and methods” section, at weeks 0, 2, and 4.

(TIF)

Click here for additional data file. (641.3KB, tif)
S1 File. SARS-CoV-2 Inhibitor Screening Kit method verification.

SARS-CoV-2 inhibitor screen kit method verification was performed following the manufacturer’s specifications [18].

(DOCX)

Click here for additional data file. (14.3KB, docx)
S2 File. Raw data file for plaque reduction assay.

(XLSX)

Click here for additional data file. (19.1KB, xlsx)
S1 Raw image. SDS-PAGE analysis of SARS-CoV-2 S1 spike-specific IgY carried out under reducing conditions–raw image.

(PDF)

Click here for additional data file. (1.3MB, pdf)

Acknowledgments

The authors recognize the efforts of our Laboratory Director, Mohamed Ait Ichou, PhD, Laboratory Technicians Nnebuefe Idegwu and Drupad Patel, and David Myers, who provided animal husbandry of laying hens and who carried out yolk harvests.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was funded by Scaled Microbiomics, LLC. The funder provided support in the form of salaries for authors CA and JG, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “author contributions” section.

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Decision Letter 0

Paulo Lee Ho

19 Oct 2020

PONE-D-20-16731

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

PLOS ONE

Dear Dr. Goepp,

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Paulo Lee Ho, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer Comments: The following corrections have to be done by the authors for the manuscript approval.

1. Line no.94 – mcg unit has to be corrected as “µg”

2. Line no.101: vaccine preparation – what is the adjuvant used for immunization during sensitization dose and Line no.104: booster immunizations - for booster dose on 14th day of immunizations?

- Clearly mention the antigen-adjuvant ratio preparations for immunization (v/v).

3. Line no.102: each breast of each of one hen pairing – Route of immunization? Intramuscular or subcutaneous has to be mentioned.

4. Line no. 105, 106: Protocols for hen maintenance, immunization, and phlebotomy were approved by the Scaled Microbiomics, LLC Animal Use and Care Committee – Ethical approval reference number need to be mentioned.

5. line no.107: Serum IgY antiviral reactivity:

Serum antibody titre representation using simple bar graphs with clear explanations to show the significant increase in antibody titre levels has to be shown.

6. Line no.124: Total protein concentration of egg yolk and serum:

Standard curve and dynamics of total protein concentration from both egg yolk and serum has to be represented graphically. Even simple graphs are sufficient but it should be clearly self explanatory as per the results obtained.

7. Line no.195: Avian IgY was successfully isolated from yolks, yielding up to 16.8l mg/ml

At which titre day egg this antibody yield was calculated?

8. Line no.196: from serum, yielding up to 23.1mg/ml

Similarly at which titre day of the serum collected post immunization, this antibody yield was calculated?

9. And from both the Line no.195, 196; are antigen-specific IgY antibodies titre has raised as per represented within a couple of week and only with one booster immunization? - JUSTIFY

10. Line no. 177: Plaque reduction assay – Show the results of cell lines and viral plaque titration evaluations.

11. Line no. 211: IgY titers in serum, with titers of 1:10,000 at weeks 3 and 4 (data not shown). – The data has to be represented in graphs showing the dynamics of significant increase in the antibody titration both in serum and the yolk.

12. Line no.166: Binding Inhibition of S1 to ACE2 and Line no. 216: Inhibition of S1 spike binding to ACE2 protein by S1-specific IgY – Show the resulting proof for inhibition. And Western blotting results are must to prove the binding efficacy and specificity of antibodies but it has not been submitted here. Show western blotting results also.

13. Figure 2: Titre of antibodies increased in serum and yolk at the same time period.? How is that possible? Because, it takes a week time for transfer of antibodies from serum to yolk. – Justify with proper results.

Reviewer #2: This manuscript presents excellent evidence for moving forward with an avian antibody approach to the treatment and/or prevention of COVID19. The authors demonstrated a robust and specific IG-Y immune response against SARS-CoV2 S1 spike protein. The approach described in the manuscript is well-conceived and the evidence is compelling. The data presented therefore support the author's conclusions. The statistical analysis is a standard one and it is appropriate for this experimental approach. The underlying PAGE gel data for the statistical analysis is a part of the manuscript.

I do think that discussion should be tweaked a bit: this evidence for this approach as compelling for the treatment of COVID19 based on the use of polyclonal antibodies in other clinical contexts is not germane to a PLOS ONE manuscript. So the discussion should be less speculative.

**********

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Reviewer #2: No

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PLoS One. 2021 May 28;16(5):e0252399. doi: 10.1371/journal.pone.0252399.r002

Author response to Decision Letter 0

2 Mar 2021

Responses to reviewers are contained in the Revised Cover Letter/Rebuttal Letter in tabular form. They are reproduced here but Table formatting is not preserved. Please advise if we should submit this information in a different format.

Original Line Number Reviewer #1 Comments New Line Number Author Response

26-28 (Abstract) Author-initiated change 26-28 Modified language about vaccine availability to reflect recent developments

Introduction, first paragraph Author-initiated change 54-62 Modified language to reflect recent developments on the vaccine front.

94 mcg unit has to be corrected as “µg” 115 Change has been made

101 vaccine preparation – what is the adjuvant used for immunization during sensitization dose 123 Adjuvant is Montanide ISA 70 VG, specified in new lines 113-114; the phrase “vaccine/adjuvant” has been added for clarity

N/A Clearly mention the antigen-adjuvant ratio preparations for immunization (v/v) 114 This ratio now appears immediately after naming the adjuvant (30% antigen in PBS, 70% adjuvant, v/v). The phrase “30%/70% v/v” has been added to line 125 to reinforce this.

104 booster immunizations - for booster dose on 14th day of immunizations? 126-127 Added this statement: “booster immunizations were performed identically to immunization day 1 for each hen pair.” The awkward word “pairing” was replaced with “pair.”

102 each breast of each of one hen pairing – Route of immunization? Intramuscular or subcutaneous has to be mentioned 124 The word “intramuscularly” has been inserted.

105, 106 Protocols for hen maintenance, immunization, and phlebotomy were approved by the Scaled Microbiomics, LLC Animal Use and Care Committee – Ethical approval reference number need to be mentioned 129 This number now appears in the text.

107 Serum IgY antiviral reactivity:

Serum antibody titre representation using simple bar graphs with clear explanations to show the significant increase in antibody titre levels has to be shown. 130-139 Please See Notes Regarding Serum IgY Titers Below at ***

124 Total protein concentration of egg yolk and serum:

Standard curve and dynamics of total protein concentration from both egg yolk and serum has to be represented graphically. Even simple graphs are sufficient, but it should be clearly self-explanatory as per the results obtained. 168 The standard curve for total protein concentration in yolk-derived IgY is now provided as new S1 Fig., and is so indicated at Line 168. As noted above, serum collection was discontinued after samples were obtained at Weeks 3 and 4, hence, no intelligible graph of these results is possible. We stress again that this study was intended to determine parameters of yolk-derived IgY, and we do not believe that this missing data impairs that original intent.

195 Avian IgY was successfully isolated from yolks, yielding up to 16.8l mg/ml

At which titre day egg this antibody yield was calculated? 246 ff Four weeks (28 days) after initial immunization, now noted in text at line 244.

196 from serum, yielding up to 23.1mg/ml

Similarly at which titre day of the serum collected post immunization, this antibody yield was calculated? 248 Three weeks (21 days) following the initial immunization, now noted in text at line 246.

195, 196 are antigen-specific IgY antibodies titre has raised as per represented within a couple of week and only with one booster immunization? – JUSTIFY 259 ff The section cited by the reviewer here pertains to antibody concentration, not titer. Under the assumption that the reviewer intended reference to [new] lines 259 ff, where titers are reported, we note that this titer rise is consistent with those seen in our other work on IgY raised against both viral and bacterial antigens; language to this effect is now at Lines 261-264. We routinely provide a single primary and one booster injection spaced 14 days apart. Only if Day 14 titers remain below 1:210 (1:2024) do we re-boost, which was not done in this case given the high initial titers, short duration of study, and high cost of antigen. We provide a graph in a new Supplement (S6 Fig.) at Line 262, similar to Figure 1, showing the rapid rise of egg-derived IgY titers against two other antigens, one bacterial and one viral, to illustrate that the results reported here are typical for our work. In all cases, the IgY titer reported is defined as the maximal dilution returning a signal > 2.1x background. This definition is clarified in Line 204, which now includes the inequality > 2.1 times control.

177 Plaque reduction assay – Show the results of cell lines and viral plaque titration evaluations. 222-241 The reviewer is asking to show the results of the plaque assay in the cell lines. We are providing the neutralizing ab titer dataset in a new Supplementary File (S5 File), indicated in the MS. at Line 242.

211 IgY titers in serum, with titers of 1:10,000 at weeks 3 and 4 (data not shown). – The data has to be represented in graphs showing the dynamics of significant increase in the antibody titration both in serum and the yolk. 260-263 As noted, serum collection was done only at weeks 3 and 4, at both of which time points yolk anti-S1 IgY titers were detectable. Also as noted, this study focused on characteristics of yolk-derived, not serum, IgY.

166; 216 Show the resulting proof for inhibition…. And Western blotting results are must to prove the binding efficacy and specificity of antibodies, but it has not been submitted here. Show western blotting results also. 213-215; 222; 182-183 At 214 ff, we have added information about the S1/ACE2 inhibition kit from Acro Biosystems, and the Method Verification dataset generated prior to the test. The kit is internally validated by the manufacturer; the manufacturer’s Protocol for use in this test is at the URL provided in reference #18, with attention directed to page 9, “Method Verification,” which shows binding of biotinylated human ACE2 protein to immobilized SARS-CoV-2 S protein receptor binding domain, along with a curve showing inhibition of human ACE2/SARS-CoV-2 S protein binding by a standardized inhibitor (anti-SARS-CoV-2 IgG antibody). We believe that this information, along with our existing Figures 3 and 4, address the request for “proof for inhibition.”

Regarding Western Blotting: We used a purified Spike S1 glycoprotein (The Native Antigen Company), rather than an intact viral particle, as the immunogen, for reasons related to biosafety. We therefore believe that Western Blotting would add little useful information beyond the ELISA results in terms of proving the binding efficacy and specificity of antibodies. We have provided a Dot Blot image as a Supplementary Figure (S3 Fig.; line 181/2), which demonstrates that our anti-S1 IgY binds specifically to the purified S1 antigen, with no non-specific binding. We believe that this should be sufficiently confirmatory that the antibodies are specific for their intended targets.

Figure 2 Titre of antibodies increased in serum and yolk at the same time period.? How is that possible? Because it takes a week time for transfer of antibodies from serum to yolk. – Justify with proper results. Figure 2 As noted, serum levels were evaluated only at Weeks 3 and 4 after immunization, by which time specific reactivity was apparent in the yolk-derived IgY samples. We believe this is consistent with the reviewer’s observation, and further, that this study’s focus on characteristics of egg-derived IgY as the therapeutic/prophylactic entity make serum levels less relevant.

Reviewer #2 Comments

Discussion I do think that discussion should be tweaked a bit: this evidence for this approach as compelling for the treatment of COVID19 based on the use of polyclonal antibodies in other clinical contexts is not germane to a PLOS ONE manuscript. So the discussion should be less speculative. Text at original lines 284-290, 302-311, and 316-320 has been deleted We believe the reviewer’s concerns center around three paragraphs in Discussion: 284-290, regarding prior use of polyclonal antibodies, have been deleted; 302-311, in which we speculated about economic and other factors that may have prevented uptake of IgY technology, and 316-320, a discussion of use of human polyclonal IgA in intranasal use against upper respiratory infections, have all been deleted. Minor edits to remaining text have been made to assure continuity: deleted “also” in new line 284, Deleted “previous positive” at beginning of new line 295 and inserted “These,” deleted from that line “on intranasal prophylaxis of influenza.”

Acknowledgements From Authors: We inadvertently neglected to acknowledge Dr. Mohamed Ait Ichou, who directs our laboratory. Added Dr. Ait Ichou’s name in Acknowledgements

Decision Letter 1

Paulo Lee Ho

17 May 2021

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

PONE-D-20-16731R1

Dear Dr. Goepp,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Paulo Lee Ho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have satisfied my concerns and I believe that this manuscript is now ready for publication. The design and execution of the experiments was well conceived. The results are interesting and will help advance our fight against the current pandemic.

Reviewer #3: The authors of this study have evaluated IgY antibodies as potential tool for passive immunotherapy against SARS-COV-2 through blocking the interaction between the virus and ACE2 receptor. There is one concern:

The main point in the manuscript is inhibition of adhesion of viral spike glycoproteins to human ACE2 protein receptors, so, evaluation of binding Inhibition of S1 to ACE2 in methodology section need further details

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Acceptance letter

Paulo Lee Ho

20 May 2021

PONE-D-20-16731R1

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

Dear Dr. Goepp:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Paulo Lee Ho

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Standard curve for bicinchoninic acid (BCA).

    Total protein concentration was determined by BCA method targeting SARS-CoV-2 S1. Curve shows linearity over eight serial dilutions (R2 = 0.99).

    (TIF)

    Click here for additional data file. (537.7KB, tif)
    S2 Fig. Dot-blot image showing IgY binding specificity against purified S1 glycoprotein.

    Hens were immunized using norovirus virus-like particles (NVLP), and IgY was prepared as mentioned in the “Materials and methods” section. IgY solutions (NVLP, S1, and unimmunized) were spotted onto a nitrocellulose membrane (Azure Biosystems, Dublin, CA, USA) and allowed to dry at room temperature (RT) for 30 minutes. Each membrane was blocked in tris-buffered saline and Tween 20 (TBST) supplemented with 5% (v/v) skim milk for 1 hr at RT. Membranes were incubated with the appropriate IgY dilutions (1:1,000 anti-S1; 1:2,500 anti-VLP, and Unimmunized IgY) in blocking buffer at RT. After 1 hr, the primary antibody dilutions were aspirated, and each blot was washed three times (5 min) with TBST. Blots were then incubated for 1 hr with a 1:1,000 dilution of Goat anti-chicken HRP-conjugated IgG (ImmunoReagents, Inc., Raleigh, NC, USA) in blocking buffer at RT. Secondary antibody solution was then aspirated, and blots were washed three times (10 min followed by two 5 min washes) with TBST and washed a final time (5 min) with TBS. Color change was observed using TMB chemical substrate (VWR International, Radnor, PA, USA), per manufacturer’s directions. The reaction was quenched using TBS, and images were captured using a standard camera.

    (TIF)

    Click here for additional data file. (2.3MB, tif)
    S3 Fig. IgY production over time.

    ELISA titers (log2) of IgY targeting norovirus virus-like particles and bacterial multiepitope fusion antigen over 24 weeks are shown as line plots. Viral and bacterial immunizations were carried out intramuscularly, as mentioned in the “Materials and methods” section, at weeks 0, 2, and 4.

    (TIF)

    Click here for additional data file. (641.3KB, tif)
    S1 File. SARS-CoV-2 Inhibitor Screening Kit method verification.

    SARS-CoV-2 inhibitor screen kit method verification was performed following the manufacturer’s specifications [18].

    (DOCX)

    Click here for additional data file. (14.3KB, docx)
    S2 File. Raw data file for plaque reduction assay.

    (XLSX)

    Click here for additional data file. (19.1KB, xlsx)
    S1 Raw image. SDS-PAGE analysis of SARS-CoV-2 S1 spike-specific IgY carried out under reducing conditions–raw image.

    (PDF)

    Click here for additional data file. (1.3MB, pdf)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

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