Mass production of IgY-containing tablets for COVID-19 transmission control
Abstract: Despite the overall positive outcomes in hospitalization and mortality rates from the COVID-19 vaccines, COVID-19 infections remained prevalent around the world highlighting the need for alternative control strategies. Passive immunization with chicken IgY has long served as a feasible countermeasure, which gained further popularity in the research community during the recent pandemic. Here we demonstrate for the first time the scalability of anti-COVID-19 IgY production for effective distribution and potential use in large populations. Over 70,000 chickens were immunized against the SARS-CoV-2 S1 antigen to produce eggs containing anti-S1 IgY. The resulting egg yolk powder was formulated into commercially acceptable tablets for human consumption. QC and stability testing showed that the purified IgY and tablets maintained activity and stability for over a year. The resulting large batch of IgY tablets demonstrated equal immunoreactivity and virus neutralization potential against all leading COVID-19 variants. Our results demonstrate the feasibility of manufacturing egg yolk powder into edible tablets, and that can now be employed to block viral infectivity and transmission against all major COVID-19 variants affordably and effectively in both developed and developing countries.
Keywords: COVID-19, Chicken IgY, Egg yolk powder, Tablets, Passive immunization
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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.
Results
Immunoreactivity of IgY against SARS-CoV-2 S1 protein tested on a small scale
The SARS-CoV-2 S1 protein or RBD peptide reactivity of both purified IgY and egg yolk was examined in eggs collected from 7 hens at two weeks post second immunization (D21) with Fc tagged S1 glycoprotein using ELISA. The ELISA assay (Fig. 1) showed strong positive binding of both egg yolk-IgY and purified IgY to the S1 protein demonstrating that the S1 protein is highly immunogenic in hens. These antibodies also reacted very strongly with the RBD peptide by ELISA. Finally, there was only a small decrease in binding affinity of the purified anti-S1 IgY compared to that of the IgY in whole egg yolk.
Immunoreactivity of purified IgY from individual eggs
Individual eggs may demonstrate different IgY content leading to variability during production and manufacturing. Therefore, to determine the extent of variability of the IgY titer in individual hens, 12 eggs from different immunized hens were selected at random and their IgY isolated and tested by ELISA. This was compared to 8 eggs collected at random from non-immunized hens. As shown in Fig. 1B, we found that the IgY titer was high in all the eggs from immunized hens. In contrast, the negative controls (IgY from non-immunized hens), only gave consistent background levels of reactivity.
Immunoreactivity of purified IgY over time
To determine the rate at which anti-S1 antibody activity appears in the egg yolk, an ELISA was performed on purified egg yolk IgY taken at each stage of egg collection. As shown in Fig. 1C, no increase in IgY titer is seen at the first egg collection after the first immunization (D0). However, a significant increase in IgY titer is seen at the third egg collection (D21). The high IgY titer is maintained for the remaining samples collection over a period of 3 weeks and further work showed that the titer remains high for up to 3 months (Fig. 1D).
Western blot analysis of IgY
LDS gel and Western blot analysis was conducted to confirm both the purity of the IgY and its reactivity against the S1 antigen (reduced and non-reduced; ACROBiosystems). HRP conjugated goat anti-chicken IgY reacted strongly with a 180 kDa protein band of IgY under non-reducing conditions (Fig. 2, lane 1; Original full-length images can be seen in Figure S1). The anti-S1 IgY reacted strongly with both reduced and non-reduced forms of the SARS-CoV-2 S1 antigen, suggesting the anti-S1 IgY antigen recognition site is not affected in reduced conditions.
Virus neutralization study
IgY from hens immunized with the S1 antigen were tested using an in vitro plaque assay for virus neutralization. This assay was completed under blinded conditions. The Alpha variant of SARS-CoV-2 was used to infect Vero cells in culture. There was a significant reduction in plaque forming units reaching a maximum at 3 weeks post second immunization (D21; Fig. 3); a similar response was seen on D28. Interestingly, there was a significant reduction seen on D14 with a titer of 1:10.
Upscaling of IgY production
We next carried out large-scale production of the SARS-CoV-2 S1-specific IgY by vaccinating 73,000 hens with 5 µg of purified his tagged S1 glycoprotein (Wuhan variant produced by ACROBiosystems). An ELISA was performed to examine the IgY titer in both the purified IgY fractions and in the egg yolks sampled post immunization (Fig. 4). Consistent with the small-scale experiments, a significant increase in the SARS-CoV-2 S1 IgY in both purified IgY samples and egg samples was seen post immunization with the SARS-CoV-2 S1 protein antigen.
Following confirmation of the high titer of IgY in the egg yolks of the immunized hens, eggs were collected and sent to a commercial egg processing facility where the egg yolks were separated, pasteurized, and spray dried. After 12 weeks of collection and pooling into batches we produced a total of 63,000 kg of high titer, spray dried egg yolk powder.
To ensure that the spray dried egg yolk was free of contaminating pathogens, a quality control and batch safety test was carried out by the tablet manufacturer (Seattle Gourmet Foods, Washington, USA). The dried egg yolk was tested for the presence of common contaminant pathogens. No significant levels of contaminants were detected in the spray dried egg yolk which were therefore approved for food safety (Table 3).
Conclusion
In a pandemic characterized by a threat with a high reproduction rate like SARS-CoV-2, prompt implementation of solutions to curb transmission within a population may offer significant public health benefits. IgY technology presents a cost-effective and scalable solution, enabling the rapid production of effective antibodies within 30 days of antigen identification - much faster than traditional human vaccine development. This approach has demonstrated broad-spectrum applicability across various antigens (reviewed by Lee et al.13. Leveraging existing egg production infrastructure allows for efficient manufacturing and distribution, making IgY-based products particularly suitable for early response efforts. Potential applications include deployment in high-risk environments such as schools, hospitals, retirement villages and among immunocompromised populations, offering a valuable tool to reduce transmission while vaccines are developed and distributed.