Egg Yolk Antibody for Passive Immunization: Status, Challenges, and Prospects.

The immunoglobulin Y (IgY) derived from hyperimmune egg yolk is a promising passive immune agent to combat microbial infections in humans and livestock. Numerous studies have been performed to develop specific egg yolk IgY for pathogen control, but with limited success. To date, the efficacy of commercial IgY products, which are all delivered through an oral route, has not been approved or endorsed by any regulatory authorities. Several challenging issues of the IgY-based passive immunization, which were not fully recognized and holistically discussed in previous publications, have impeded the development of effective egg yolk IgY products for humans and animals. This review summarizes major challenges of this technology, including in vivo stability, purification, heterologous immunogenicity, and repertoire diversity of egg yolk IgY. To tackle these challenges, potential solutions, such as encapsulation technologies to stabilize IgY, are discussed. Exploration of this technology to combat the COVID-19 pandemic is also updated in this review.

Antiviral Immunoglobulins of Chicken Egg Yolk for Potential Prevention of SARS-CoV-2 Infection.

Background: Some viruses cause outbreaks, which require immediate attention. Neutralizing antibodies could be developed for viral outbreak management. However, the development of monoclonal antibodies is often long, laborious, and unprofitable. Here, we report the development of chicken polyclonal neutralizing antibodies against SARS-CoV-2 infection. Methods: Layers were immunized twice with 14-day intervals using the purified receptor-binding domain (RBD) of the S protein of SARS-CoV-2/Wuhan or SARS-CoV-2/Omicron. Eggs were harvested 14 days after the second immunization. Polyclonal IgY antibodies were extracted. Binding of anti-RBD IgYs was analyzed by immunoblot and indirect ELISA. Furthermore, the neutralization capacity of anti-RBD IgYs was measured in Vero-E6 cells infected with SARS-CoV-2-mCherry/Wuhan and SARS-CoV-2/Omicron using fluorescence and/or cell viability assays. In addition, the effect of IgYs on the expression of SARS-CoV-2 and host cytokine genes in the lungs of Syrian Golden hamsters was examined using qRT-PCR. Results: Anti-RBD IgYs efficiently bound viral RBDs in situ, neutralized the virus variants in vitro, and lowered viral RNA amplification, with minimal alteration of virus-mediated immune gene expression in vivo. Conclusions: Altogether, our results indicate that chicken polyclonal IgYs can be attractive targets for further pre-clinical and clinical development for the rapid management of outbreaks of emerging and re-emerging viruses.

Hyperimmunized Chickens Produce Neutralizing Antibodies against SARS-CoV-2.

The novel severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV-2, is responsible for the global COVID-19 pandemic. Effective interventions are urgently needed to mitigate the effects of COVID-19 and likely require multiple strategies. Egg-extracted antibody therapies are a low-cost and scalable strategy to protect at-risk individuals from SARS-CoV-2 infection. Commercial laying hens were hyperimmunized against the SARS-CoV-2 S1 protein using three different S1 recombinant proteins and three different doses. Sera and egg yolk were collected at three and six weeks after the second immunization for enzyme-linked immunosorbent assay and plaque-reduction neutralization assay to determine antigen-specific antibody titers and neutralizing antibody titers, respectively. In this study we demonstrate that hens hyperimmunized against the SARS-CoV-2 recombinant S1 and receptor binding domain (RBD) proteins produced neutralizing antibodies against SARS-CoV-2. We further demonstrate that antibody production was dependent on the dose and type of antigen administered. Our data suggests that antibodies purified from the egg yolk of hyperimmunized hens can be used as immunoprophylaxis in humans at risk of exposure to SARS-CoV-2.

Egg yolk immunoglobulin (IgY) targeting SARS-CoV-2 S1 as potential virus entry blocker.

AIMS: COVID-19 pandemic caused by SARS-CoV-2 has become a public health crisis worldwide. In this study, we aimed at demonstrating the neutralizing potential of the IgY produced after immunizing chicken with a recombinant SARS-CoV-2 spike protein S1 subunit. METHODS AND RESULTS: E. coli BL21 carrying plasmid pET28a-S1 was induced with IPTG for the expression of SARS-CoV-2 S1 protein. The recombinant His-tagged S1 was purified and verified by SDS-PAGE, Western blot and biolayer interferometry (BLI) assay. Then S1 protein emulsified with Freund's adjuvant was used to immunize layer chickens. Specific IgY against S1 (S1-IgY) produced from egg yolks of these chickens exhibited a high titer (1:25,600) and a strong binding affinity to S1 (KD  = 318 nmol L-1 ). The neutralizing ability of S1-IgY was quantified by a SARS-CoV-2 pseudotyped virus-based neutralization assay with an IC50  value of 0.99 mg ml-1 . In addition, S1-IgY exhibited a strong ability in blocking the binding of SARS-CoV-2 S1 to hACE2, and it could partially compete with hACE2 for the binding sites on S1 by BLI assays. CONCLUSIONS: We demonstrated here that after immunization of chickens with our recombinant S1 protein, IgY neutralizing antibodies were generated against the SARS-CoV-2 spike protein S1 subunit; therefore, showing the potential use of IgY to block the entry of this virus. SIGNIFICANCE AND IMPACT OF THE STUDY: IgY targeting S1 subunit of SARS-CoV-2 could be a promising candidate for pre- and post-exposure prophylaxis or treatment of COVID-19. Administration of IgY-based oral preparation, oral or nasal spray may have profound implications for blocking SARS-CoV-2.

The preparation of N-IgY targeting SARS-CoV-2 and its immunomodulation to IFN-γ production in vitro.

Specific antibodies against SARS-CoV-2 structural protein have a wide range of effects in the diagnose, prevention and treatment of the COVID-19 epidemic. Among them, egg yolk immunoglobulin Y (IgY), which has high safety, high yield, and without inducing antibody-dependent enhancement, is an important biological candidate. In this study, specific IgY against the conservative nucleocapsid protein (NP) of SARS-CoV-2 was obtained by immunizing hens. Through a series of optimized precipitation and ultrafiltration extraction schemes, its purity was increased to 98%. The hyperimmune IgY against NP (N-IgY) at a titer of 1:50,000 showed strong NP binding ability, which laid the foundation of N-IgY's application targeting NP. In an in vitro immunoregulatory study, N-IgY (1 mg/mL) modulated NP-induced immune response by alleviating type II interferon secretion stimulated by NP (20 µg/mL). In summary, N-IgY can be mass produced by achievable method, which endows it with potential value against the current COVID-19 pandemic.

Chicken IgY Antibodies Provide Mucosal Barrier against SARS-CoV-2 Virus and Other Pathogens.

BACKGROUND: This mini review includes two case descriptions. It introduces the use of chicken egg yolk antibody (IgY) solutions in the prevention and cure of viral and bacterial infections. Application for the protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rotavirus, and influenza viruses, as well as for the eradication of Pseudomonas aeruginosa, caries, various enteric bacteria and other pathogens, and toxins have been developed. This approach is a fast, reliable, safe, and tested method for producing molecular shield and protection against emerging pathogens and epidemics. In the current pandemic situation caused by coronavirus disease-2019 (COVID-19), this method of passive immunization could be applied for rapid protection against modifiable agents. The specific IgY antibodies start to accumulate into egg yolks about 3 weeks after the immunization of the chicken. The product can be collected safely, as the antigen is not found in the eggs. This method for microbial safety uses natural means and commonly used food substances, which have been tested and could be produced for both blocking epidemics and applying personalized medicine.

Chicken Egg Yolk Antibodies (IgYs) block the binding of multiple SARS-CoV-2 spike protein variants to human ACE2.

The SARS-CoV-2 virus is still spreading worldwide, and there is an urgent need to effectively prevent and control this pandemic. This study evaluated the potential efficacy of Egg Yolk Antibodies (IgY) as a neutralizing agent against the SARS-CoV-2. We investigated the neutralizing effect of anti-spike-S1 IgYs on the SARS-CoV-2 pseudovirus, as well as its inhibitory effect on the binding of the coronavirus spike protein mutants to human ACE2. Our results show that the anti-Spike-S1 IgYs showed significant neutralizing potency against SARS-CoV-2 pseudovirus, various spike protein mutants, and even SARS-CoV in vitro. It might be a feasible tool for the prevention and control of ongoing COVID-19.

An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review.

The present state of diagnostic and therapeutic developmental race for vaccines against the SARS CoV-2 (nCOVID-19) focuses on prevention and control of this global pandemic which also represents a critical challenge to the global health community. Although development of novel vaccines can prevent the SARS CoV-2 infections, it is still impeded by several other factors and therefore novel approaches towards treatment and management of this disease is the urgent need. Passive immunotherapy plays a vital role as a possible alternative to meet this challenge and among various antibody sources, chicken egg yolk antibodies (IgY) can be used as an alternative to mammalian antibodies which have been previously studied against SARS CoV outbreak in China. In this review, we discuss the strategies for the use of chicken egg yolk (IgY) antibodies in the development of rapid diagnosis and immunotherapy against SARS CoV-2. Also, IgY antibodies have previously been used against various respiratory bacterial and viral infections in humans and animals. Compared to mammalian antibodies (IgG), chicken egg yolk antibodies (IgY) have greater binding affinity to specific antigens, ease of extraction and lower production costs, hence possessing remarkable pathogen-neutralizing activity of pathogens in respiratory and lungs. We provide an overall importance for the use of monoclonal chicken egg yolk antibodies (IgY) using phage display method describing their potential passive immunotherapeutic application for the treatment and prevention of SARS CoV-2 infection which is simple, fast and safe way of approach for treating patients effectively.