IntroductionThe influx and severity of COVID-19 infections has gone beyond our expectations. The unprecedented number of infections and extremely high rates of transmission have caused significant strain on hospitals and other healthcare facilities. Despite the use of available PPE (face masks or shields, gloves, disposable gowns), vigilant hand washing and disinfection, as well as vaccines, we have witnessed a large increase in the numbers of COVID-19 cases, particularly with the Omicron variants. Since the Omicron peak, there has been a sharp reduction in case numbers reaching a plateau, however, this does not reflect the true situation since many, or most COVID-19 cases today never get reported. This not only poses significant risk to the individual infected patient, but also the people around them, both within the healthcare facility (other workers and patients) and outside (general public and family members). Furthermore, new viral strains may suddenly appear that are highly virulent and transmissible. Thus, despite the success of the COVID-19 vaccines in helping to lower morbidity and mortality, there is a need for strategies that can strongly reduce disease transmission and spread.Over the past few years, we and others have published several articles demonstrating the use of chicken IgY in controlling infection and transmission by COVID-191,2,3,4 as well as other viruses such as SARS5,6 and Influenza7. This was demonstrated by immunizing laying hens with very small quantities of highly purified SARS-CoV-2 S1 glycoprotein8, leading to a strong antibody response in the sera of the chickens, which then appears in very large quantity of IgY in their egg yolk. Eggs were collected, the yolk separated and the IgY purified. Using this purified antibody, we demonstrated a very high titer against S1 antigen by both ELISA and Western blotting. It was also shown that it can neutralize the virus in vitro, as well as provide in vivo protection against infection in a hamster model9. Finally, a Phase 1 clinical trial was carried out demonstrating the safety of IgY as a nasal formulation in humans10.Based on the results obtained in animal model studies, it was concluded that chicken IgY raised against the SARS-CoV-2 S1 protein can act as a barrier against infection and last for at least 6–8 h. We hypothesized that the IgY can be formulated as either a nasal drop or an edible tablet, to be taken every 6–8 h when encountering potentially infected people. When administered as a tablet, the IgY antibodies will coat the mouth, oropharynx, larynx, and in particular the trachea13. As shown in previous studies, when the virus is inhaled, it will be sequestered and neutralized by the IgY, preventing passage of the virus to the lungs where it normally establishes an infection.In the current research presented here, we demonstrate the ability and feasibility to scale up and produce chicken IgY (in the form of dry egg yolk) in an edible tablet formulation produced on a large scale under food quality production standards. We also show how this can be accomplished and developed new methods for the scale up of immunization, quality control, epitope specificity and stability testing, demonstration of in vitro cross protection, and tablet formulation.Worldwide, there are approximately 7 billion laying hens11 which could be used in an emergency anywhere in the world to produce a vast quantity of protective IgY in as little as 30 days from first inoculation of the hens in a highly cost-effective manner. Our working hypothesis is that by producing large numbers of tablets (in the hundreds of millions or billions) commercially at a very cheap cost, they can be used to help control or slow down the transmission and spread of COVID-19 throughout a hospital, school or in the general public. Further, these tablets contain egg yolk powder and as such are amenable for use in children and the elderly. Although in its current egg-yolk powder form, the tablets are not safe for individuals with egg sensitivities, the IgY can be further purified from the egg yolk powder to allow for use by individuals with sensitivity to eggs12. This work is an important step in demonstrating the scalability and use of chicken IgY to control COVID-19 transmission, that can be marketed as a food product for human consumption. Therefore, the objective for this study was to show that these tablets can be manufactured on large scale in an economically feasible manner for the prevention and control of COVID-19 with the intent for the betterment of global health.Materials and methodsAntigensAntigens were sourced from ACROBiosystems Inc. The variant ID, lineage and origin are listed in Table 1 below.Table 1 ACROBiosystem Recombinant proteins used in this study. Abbreviations: wild-type (WT), receptor-binding domain (RBD), N-terminal domain (NTD).Full size tableVaccination protocol for small- and large-scale productionSeven young White Leghorn hens (38 weeks of age) were vaccinated with SARS-CoV-2 S1 antigen. The S1 glycosylated antigen used was from the Wuhan variant produced by ACROBiosystems. The antigen was produced in HEK cells labeled His and mixed with phosphate buffered saline (PBS) with Emulsigen-D (Phibro Animal Health Products, Teaneck, NJ, USA) following the manufacturer’s instructions at 50% (v/v). Each Hen received 5 µg of antigen diluted in Emulsigen-D twice, two weeks apart, to a total of 10 µg. Hens were vaccinated using a repeater injector and 1 mL was injected into the breast muscle of each bird. The first immunization was administered on one side of the breast and the second on the other side. Eggs were collected at different time points during the immunization schedule as shown in Table 2.Table 2 Timing of egg collection in relation to immunization.Full size tableThe same vaccination procedure was performed on the large-scale production of IgY in which 73,000 White Leghorn hens (44 weeks of age) were immunized. The sample size was chosen based on previous studies on other antigens where it was found a high level of consistency in the immune responsiveness of vaccinated laying hens against a variety of antigens13.Control IgY used in this study was prepared from eggs laid prior to immunization. The small-scale production included IgY pooled from 24 egg yolks, while the large-scale production was prepared from 96 egg yolks. All eggs used were randomly selected.All laying hens were raised following the National Chicken Council’s recommendations. Farm personnel followed strict environmental policies which included proper ventilation, indoor protection from predators and weather, and proper lighting which allowed for the ability to monitor disease. Flock health was inspected daily by trained personnel. On-staff veterinarians and poultry nutritionists designed diets to meet the unique physical needs of egg-laying hens which are customized to the age-specific needs of the flock. Feed and water quality were optimized for hen health. All hens were raised without the use of hormones.Only hens or eggs lost due to regular loss were excluded from the study. Hens that were not laying were removed during commercial production. No adverse events were observed throughout the duration of the study.This study is reported in accordance with the ARRIVE guidelines.IgY extraction and purification from hen egg yolkTwelve eggs from each vaccination time point (Table 2) were selected at random. The egg yolks were manually separated from the whites, the vitelline membranes pierced, and the egg yolk drained and pooled. The pooled yolk was gently mixed on a stir plate for 10 min at room temperature. 50 mL of homogenized yolks was combined with 150 mL of 4.67% (w/v) polyethylene glycol 8000 (PEG 8000) in PBS. The solution was centrifuged at 10,000 x g for 15 min at 4˚C. The resulting supernatant fluid was recovered through cheesecloth and the volume measured. A volume equivalent to one third the volume of the supernatant (~ 50 mL) of 37.5% (w/v) PEG 8000 in PBS was added to the supernatant and mixed gently on a stir plate for 10 min at room temperature. The solution was centrifuged at 10,000 x g for 15 min at 4˚C, supernatant was discarded, and the pellet was resuspended with 50 mL PBS. The solution was centrifuged again at 10,000 x g for 15 min at 4˚C to remove residual PEG 8000. The supernatant was discarded, and the pellet was resuspended in 50 mL PBS. The purified IgY was filter sterilized through a 0.2 μm PES membrane, aliquoted, and stored at either 4˚C or −20˚C depending on future use. The purified IgY preparations were analyzed using the A260/280 ratio, and concentrations approximated using the absorbance at 280 nm.ELISACorning Costar 96-well high-binding polystyrene were coated by applying 250 µL of ACROBiosystems recombinant protein (ACROBiosystems, Newark, DE, USA) at 1 µg ml−1 in carbonate bi-carbonate (CBC) buffer (pH 9.6) to test wells and CBC buffer only to control wells. Plates were incubated at 37˚C for 1 h. Plates were then aspirated, and each well blocked with 390 µL of 1% bovine serum albumin (BSA) for another hour at 37˚C. Wells were washed with Tris-buffered saline containing 0.05% (v/v) Tween 20 (WB) and air dried in a 25˚C incubator for 1 h. Plates were used immediately upon drying, or vacuum sealed and stored at 4˚C. Purified IgY samples to be tested were prepared by initially diluting the antibody 1:600 in BSA and serially diluting 1:2 in BSA. 125 µL of each dilution were transferred to ACROBiosystems recombinant protein-coated microwell plates. Plates were incubated at 25˚C for 1 h. The plates were washed three times with 400 µL WB per well. Plates were then conjugated with KPL goat anti-chicken IgY IgG HRP (Horseradish peroxidase; Thermo Fisher Scientific) at a concentration of 100 ng ml−1 for 1 h at 25˚C. Plates were then washed four times with 400 µL WB per well. The assay was developed in the dark using 100 µL per well of Tetramethylbenzidine (TMB) two component peroxidase substrate system (SeraCare Life Sciences, Inc., Milford, MA, USA) for 10 min. Developing was stopped using 100 µL of 1 M sulfuric acid. Absorbance of wells was measured at 450 nm and corrected using control well values.LDS-PAGE and western blottingWestern blotting was performed using the iBlot 2 and iBind systems (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sample preparation for gel electrophoresis was performed following the NuPAGE Bis-Tris Mini Gel protocol (Thermo Fisher Scientific). Every sample was comprised of NuPAGE LDS sample suffer and NuPAGE reducing agent at 1X concentration. The targeted protein amount for each sample was 2 µg/well. Deionized water was added to a final volume of 45 µL. Samples were heated at 100˚C for 3 min. Both chambers of a Life Technologies Mini Gel Tank (Thermo Fisher Scientific) were filled with 400 mL of 1X NuPAGE MES running buffer. NuPAGE 4–12% Bis-Tris mini gels were rinsed with 1X running buffer and placed in each chamber of the gel tank. A volume of 20 µL from each sample were loaded into both gels. Electrophoresis was performed at 200 V constant for 30 min. Upon completion, gels were washed in deionized water three times for 10 min each on a platform shaker. One gel was then stained with Imperial Protein Stain (Thermo Fisher Scientific) for final LDS-PAGE analysis, while the duplicate gel was placed on an iBlot 2 PVDF transfer stack.The transfer was performed on the iBlot 2 system at 20 V for 1 min, 23 V for 4 min, and then 25 V for 2 min per iBlot 2 protocol recommendation. The membrane was then washed in 1X iBind solution and placed in the iBind Western system. anti-SARS-CoV-2 S1 His-tag IgY was used as the primary antibody at a concentration of 15 µg ml−1. KPL goat anti-chicken IgY IgG HRP was applied as the secondary antibody at a concentration of 15 µg ml−1. After overnight incubation, the membranes were washed for 1 min in deionized water. Detection was performed using Novex HRP chromogenic substrate 3,3’,5,5’-Tetramethylbenzidine (TMB) for 5 min on a platform shaker. Membranes were allowed to dry in a dark drawer prior to image capture.Dot blotting2 µL of recombinant protein (S1, RBD, NTD) at a concentration of 0.5 mg ml−1 were dotted onto strips of nitrocellulose (Pall Corp., Port Washington, NY, USA). After drying, non-specific sites were blocked in 5% (v/v) BSA. Strips were incubated with anti-S1 IgY or anti-Normal IgY at 25 µg ml−1. Strips were washed in 0.05% PBS-Tween 20 (v/v). The strips were incubated in goat anti-chicken IgY IgG HRP-conjugated (Thermo Fischer Scientific) at 1 µg ml−1. After washing, the blots were developed with Novex HRP chromogenic TMB (Thermo Fischer Scientific).RBD binding assayTwo commercial ELISAs manufactured by ACROBiosystems catalog EP-107(WT) and EP-115(B.1.1.529) were used to look at the potential for the IgY to inhibit the RBD binding to the ACE2 receptor. The protocol was provided along with all the reagents by ACROBiosystems. The plate provided was precoated with Human ACE2 protein. Samples and controls were added to the wells along with the HRP labeled SARS-CoV 2 Spike RBD. The plates were incubated for 1 h at 37 °C, developed with TMB, and read at an absorbance of 450 nm.In vitro virus neutralizationThe chicken-derived IgY samples labelled Day 0, Day 14, Day 21, and Day 28 were assessed for their ability to neutralize SARS-CoV-2 virus using a PRNA assay. Briefly, the antibodies were diluted 1:10, 1:20,1:40, and 1:80 in culture medium and incubated with 200 plaque forming units (PFU) of the SARS-CoV-2 variant being tested. SARS-CoV-2 was diluted in supplemented (Dulbecco’s Modified Eagle’s Medium) DMEM to the appropriate concentration to achieve 200 PFU. The virus was then added to antibody samples and allowed to incubate for 1 h at 37 °C and 5% CO2. After incubation, a viral plaque assay was conducted to quantify viral titers using 12-well plates previously seeded with Vero cells (ATCC CCL-81) at a density of 2 × 105 cells per well. Media was aspirated from plates and virus-antibody samples were transferred to wells, one sample per well. Plates were inoculated for 1 h at 37 °C and 5% CO2. After infection, a 1:1 overlay consisting of 0.6% agarose and 2X Eagle’s Minimum Essential Medium without phenol red (Quality Biological, 115-073- 101), supplemented with 10% fetal bovine serum (FBS) (Gibco, 10,437,028), non-essential amino acids (Gibco, 11140-050), 1 mM sodium pyruvate (Corning, 25-000-Cl), 2 mM L-glutamine, 1% penicillin-streptomycin (P/S) was added to each well. Plates were incubated at 37 °C for 48 h. Cells were fixed with 10% formaldehyde for 1 h at room temperature. Formaldehyde was aspirated and the agarose overlay was removed. Cells were stained with crystal violet (1% w/v in a 20% ethanol solution). The viral titer of SARS-CoV-2 was determined by counting the number of plaques.Tablet formulationSeattle Gourmet Foods (Kent, WA, USA), a commercial candy manufacturer, was contracted for small pilot runs and a scale up to a production run. The edible tablets produced contained Dipack (sugar and maltodextrin; 49.5%), dextrose (26.5%), pasteurized egg yolk powder (15%), natural flavor (3.5%), and citric acid, malic acid, stearic acid, and magnesium stearate (≤ 2% each). The powders were mixed and made into tablets using a tablet press.Where tablets were tested via ELISA in this study, samples were prepared by dissolving three tablets (each containing 120 mg of egg yolk powder and approximately 5.4 mg of IgY) in 19 mL of Egg Extract Buffer (EEB).Statistical analysisThe statistical analyses were analyzed using the GraphPad Prism 8.0.1 software (GraphPad Software, Boston, Massachusetts USA). Statistical significance was performed using the T-test (2-tailed), where p