Affordable IgY-based antiviral prophylaxis for resource-limited settings to address epidemic and pandemic risks

Abstract

Background: The COVID-19 pandemic caused by SARS-CoV-2 exposed a global problem, as highly effective vaccines are challenging to produce and distribute, particularly in regions with limited resources and funding. As an alternative, immunoglobulins produced in eggs of immunized hens (IgY) can be a simple and inexpensive source for a topical and temporary prophylaxis. Here, we developed a method to extract and purify IgY antibodies from egg yolks of hens immunized against viral pathogen-derived proteins using low-cost, readily available materials, for use in resource-limited settings.

Methods: Existing protocols for IgY purification and equipment were modified, including extraction from yolks and separation of water-soluble IgY using common household reagents and tools. A replacement for a commercial centrifuge was developed, using a home food processor equipped with a 3D printed adapter to enable IgY precipitation. IgY purification was verified using standard gel electrophoresis and Western blot analyses.

Results: We developed a step-by-step protocol for IgY purification for two settings in low- and middle-income countries (LMIC): a local laboratory, where commercial centrifuges are available, or a more rural setting, where an alternative for expensive centrifuges can be used. Gel electrophoresis and Western blot analyses confirmed that the method produced highly enriched IgY preparation; each commercial egg produced ~ 90 mg of IgY. We also designed a kit for IgY production in these two settings and provided a cost estimate of the kit.

Conclusion: IgY purified from eggs of immunized local hens can offer a fast and affordable prophylaxis, provided that purification can be performed in a resource-limited setting. Here, we created a low-cost method that can be used anywhere where electricity is available using inexpensive, readily available materials in place of costly, specialized laboratory equipment and chemicals. This procedure can readily be used now to make an anti-SARS-CoV-2 prophylaxis in areas where vaccines are unavailable, and can be modified to combat future threats from viral epidemics and pandemics.

Figure 1

Summary of the IgY purification procedure. Panel A. Summary of steps in the IgY purification process. Panel B. Image of yolk separation from albumen, using an egg separator; leftover albumen are pulled off from under the separator using gloved hands. Panel C. 125 mL bottle filled with final solution from B. Panel D. Image of removal of yolk aggregates by filtration, after acidification and freezing of the mixture; yellow residues remained in the Whatman filter paper and the filtered solution in the bottle was clear. Panel E. Image of the makeshift centrifuge (left) and the final precipitated pellet in the 15 mL tubes.

Figure 2

Experimental confirmation of IgY purification. Panel A. Stain-free SDS-PAGE of lane 1-4: preparations of purified IgY using the new protocol. Lane 5; purified ovalbumin from the albumen. Each at 0.5 ug protein/lane. The upper and lower proteins in lanes 1-4 are the heavy and the light chains of IgY, respectively. Panel B. Western Blot analysis with anti-IgY and anti-ovalbumin identifying the heavy chain of IgY in lanes 1-4 (upper band) and ovalbumin (lower band), in lane 5. The levels of ovalbumin in the IgY preparations (lanes 1-4) were almost undetected (compared to lane 5; purified albumin) especially in the later preparations (lanes 3,4).

Figure 3

Components of the IgY kit. All kit materials listed in Table 1 are shown not to scale. All kit materials except the food processor easily fit into a small (15 × 15 × 15 inch) box.

Figure 4

High-level implementation of the new IgY protocol and required facilities. A scheme illustrating the implementation of the IgY purification process in two LMIC settings: Laboratory equipped with centrifuges; and a rural facility with electricity, a freezer and running water. The chosen recombinant protein (immunogen) can be produced in a specialized academic or industry laboratory and shipped for immunization as dry material (highly stable and requires no special refrigeration). Hens can then be centrally immunized by a vet, or by rural hen growers, and the resultant IgY-containing eggs can be purified either in a central facility or at a rural setting. The centrifugation step can be performed either at the central laboratory facility, equipped with a centrifuge, or locally, using our repurposed centrifugation device and printed adapter. Finally, the purified IgY is formulated into dropper bottles and can be used intranasally.