The coronavirus disease 2019 (COVID-19) pandemic – caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – is still causing hundreds of thousands of new cases globally. Therapeutic antibodies have been used to attempt to counter and contain the virus. Convalescent plasma (CP) obtained from recovered COVID-19 patients is also in use in many regions, making use of passive immunity.
A new preprint on the bioRxiv* server extends this concept to antibodies raised in chickens against the receptor-binding domain (RBD) on the SARS-CoV-2 spike glycoprotein. The resulting immunoglobulin (Ig) Y was isolated and purified from egg yolk and assayed against the virus in vitro, showing high neutralizing capability, over a hundred-fold that of the widely used antiviral drug, remdesivir.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
The greatest need today is an effective means to arrest the spread of this virus, which threatens not only human health but the global economy. Some epidemiological research suggests that, based on the time of onset of the pandemic, the governmental policies taken to contain it, and preventive steps taken in various parts of the world, the pandemic may well last until 2024.
The viral spike protein
The virus has four major structural proteins, namely, the spike protein S, envelope E, membrane glycoprotein M and nucleocapsid N, all of which are essential for successful virus assembly and the release of new virions to infect neighboring cells. The greatest attention has been paid to the spike protein, a surface antigen that mediates virus attachment to and entry into the target host cell.
The spike protein exists in a trimeric form, each trimer comprising the S1 and the S2 subunit. These mediate spike attachment and viral fusion with the host cell, respectively. Structurally, the S1 subunit contains two independent domains, the N-terminal domain (NTD) and C-terminal domain (C-domain).
Depending on the type of coronavirus, the spike S1 subunit can attach to the host cell receptor via the NTD or the CTD. With SARS-CoV-2, the receptor-binding domain (RBD) is on the NTD, and attaches to the host cell receptor angiotensin-converting enzyme 2 (ACE2).
Drugs in use today
COVID-19 is primarily a lung pathogen, with the symptoms resulting from virus-induced inflammation. Beginning in most patients with fever, a dry cough, and fatigue, the condition progresses to interstitial pneumonia. This is seen as ground-glass opacities on a chest CT scan.
Risk factors for severe and critical COVID-19 include advancing age and the presence of chronic medical conditions. Dexamethasone, a potent glucocorticoid, has been found to significantly reduce 28-day mortality in patients who require supplemental oxygen or non-invasive mechanical ventilation.
The most widely used drug, however, possibly remains remdesivir, a nucleoside analog that was the first to be approved by the US Food and Drug Administration (FDA). Though it may shorten the recovery time in hospitalized adults with severe COVID-19, the World Health Organization (WHO) does not place this among the drugs of choice.
Passively administered antibodies
The way out for the world to recover from this pandemic is global vaccination against the virus. Vaccines have been rolled out in several parts of the world, and many more are nearing approval. The most effective of these vaccines claim a 95% protective efficacy, though the actual production of antibodies, and the duration of protective neutralizing humoral immunity, remains to be analyzed.
Highly potent neutralizing monoclonal antibodies against the viral RBD have been isolated from the B cells of COVID-19 patients. These appear to competitively inhibit viral attachment to ACE2. Recombinant human ACE2 has been found to neutralize the virus, reducing its ability to infect cells by up to 5,000-fold.
CP has been shown to produce good clinical and laboratory results in many patients with SARS-CoV-2 infection. However, the deficit of CP has led to its underuse in the clinical scenario. In this context, the current paper assumes new relevance.
IgY production
The researchers make use of egg yolk immunoglobulin Y (IgY), from the eggs of immunized poultry. This has been in use to prevent and treat infections in fowl and livestock. Its extension to human infectious disease management has been the focus of much recent research.
A few examples of this trend include the production of IgY against cholera, rotavirus diarrhea, influenza viruses H5N1 and H1N1, SARS-CoV and the hand-foot-and-mouth disease virus.
In the current study, the researchers expressed the SARS-CoV-2 RBD fusion protein with high purity in a fusion expression vector. The egg layers were then immunized with four doses of the RBD. The purified IgY was then confirmed to be specific for SARS-CoV-2 RBD and to have high binding affinity.
IgY inhibits viral entry and replication
Using the gold standard test, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the researchers found that the spike IgY was capable of efficient neutralization of viral entry into cells, with a low nanomolar 50% and 90% effective concentration (EC50 and EC90). Not only so, immunofluorescence showed that SARS-CoV-2 spike protein was not expressed within cells in culture exposed to the virus, indicating effective suppression of viral replication within the cells at a concentration of 110 nM.
![Vero E6 cells were incubated with the S-IgY for 2 h, followed by incubation with the secondary antibody (Alexa 488-labeled goat anti-mouse[1:500; Abcam]). The nuclei were stained with DAPI dye (Beyotime, China). The images were taken by fluorescence microscopy, Results were as follow: (A) NC:pre-immunized IgY: Full-field virus protein (B) SARS-CoV-2-IgY55nM:Viral protein decreased significantly in visual field,About 50% of the viruses were suppressed; (C) SARS-CoV-2-IgY110nM:Virus protein disappeared completely in visual field,The virus was completely suppressed; (D) Mock Cells without virus addition. It means that S-IgY can effectively inhibit the SARS-CoV-2 from entering cells and has a dose-dependent relationship.](https://d2jx2rerrg6sh3.cloudfront.net/image-handler/picture/2021/2/2021.02.16.430255v1.jpg "Vero E6 cells were incubated with the S-IgY for 2 h, followed by incubation with the secondary antibody (Alexa 488-labeled goat anti-mouse[1:500; Abcam]). The nuclei were stained with DAPI dye (Beyotime, China). The images were taken by fluorescence microscopy, Results were as follow: (A) NC:pre-immunized IgY: Full-field virus protein (B) SARS-CoV-2-IgY55nM:Viral protein decreased significantly in visual field,About 50% of the viruses were suppressed; (C) SARS-CoV-2-IgY110nM:Virus protein disappeared completely in visual field,The virus was completely suppressed; (D) Mock Cells without virus addition. It means that S-IgY can effectively inhibit the SARS-CoV-2 from entering cells and has a dose-dependent relationship.")
Vero E6 cells were incubated with the S-IgY for 2 h, followed by incubation with the secondary antibody (Alexa 488-labeled goat anti-mouse[1:500; Abcam]). The nuclei were stained with DAPI dye (Beyotime, China). The images were taken by fluorescence microscopy, Results were as follow: (A) NC:pre-immunized IgY: Full-field virus protein (B) SARS-CoV-2-IgY55nM:Viral protein decreased significantly in visual field,About 50% of the viruses were suppressed; (C) SARS-CoV-2-IgY110nM:Virus protein disappeared completely in visual field,The virus was completely suppressed; (D) Mock Cells without virus addition. It means that S-IgY can effectively inhibit the SARS-CoV-2 from entering cells and has a dose-dependent relationship.
In a head-to-head comparison with remdesivir, the researchers found that at different doses, the spike-IgY inhibited cellular viral replication. The viral copy number was significantly lower at an EC50 of 28 nM with anti-spike IgY, compared to ~3300 nM with remdesivir, indicating that the former has over a hundred-fold greater inhibitory capacity.
What are the implications?
COVID-19 vaccines are not universally administrable, and some immunosuppressed individuals are not eligible for certain types of vaccine. The immune response is not uniform in all vaccine recipients, and the durability and titer of neutralizing antibody response are yet to be determined.
In view of these facts, alternative methods of pandemic control are clearly necessary. The findings of this study indicate the efficacy of passive neutralizing IgY antibody therapy. This approach merits further attention, given the expense and cryopreservation requirements of human antibody use that restrict its large-scale use.
Not only do the preliminary results indicate a much higher neutralizing efficacy for IgY antibodies, relative to remdesivir. The findings indicate that it may be safer than other antibodies since it does not activate complement C5a. IgY is also more stable, physically than IgG, with greater thermostability.
It is also produced by a convenient and simple manufacturing process. IgY can be used orally, being resistant to both gastric acid and enzymes. This makes it particularly suitable for a situation like the present.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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