1. Introduction
More than two years after the outbreak of the coronavirus disease 2019 (COVID-19), the World Health Organization (WHO) has documented approximately 500 million confirmed cases and more than 6 million deaths. This pandemic is characterized by the high infectivity of the virus and associated mortality and has greatly impacted the global economy and society [
1]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic virus of the coronavirus family that causes COVID-19. This pathogen is widely accepted to predominantly infect the respiratory system, resulting in symptoms ranging from respiratory tract infection to severe pneumonia and acute respiratory distress syndrome [
2]. However, several reports revealed that SARS-CoV-2 also infects extrapulmonary organs [
3,
4], including the heart [
5], brain [
6], and liver [
7]. In line with these observations, the primary SARS-CoV-2 receptor, angiotensin-converting enzyme-II (ACE2), and the transmembrane protease serine type 2 (TMPRSS2) receptor have been detected in various extrapulmonary organs [
3,
4,
5,
6,
7]. The entry of SARS-CoV-2 into the host cells via the cell membrane is mediated through the binding of the viral homotrimeric spike (S) protein to ACE2 and subsequent priming by TMPRSS2 [
8].
Scientists have employed different innovative approaches and technologies to develop novel therapeutics against SARS-CoV-2 infection. Nevertheless, despite the immense global effort to combat SARS-CoV-2 infection, the pandemic remains poorly controlled and several SARS-CoV-2 variants have been consecutively verified. These variants include the Wuhan variant, Alpha (α) variant, Delta (δ) variant, and Omicron (ο) variant [
9]. These strains have replaced the Wuhan strain, which was the first to be identified but has gradually disappeared. Among these variants, the Omicron (ο) variant is the most recently identified SARS-CoV-2 variant and has become the dominant strain globally [
10]. Unexpectedly, the development of vaccines and therapeutics remains challenging and could not completely prevent SARS-CoV-2 from spreading.
To respond to the current challenge of effectively controlling and managing the pandemic, several innovative approaches have been designed and tested. Passive immunization [
11] utilizes antibodies from the serum of immunized animals, immunized humans [
12], and convalescent patients [
13]. The generation of monoclonal antibodies was considered to be the best choice due to their high specificity. However, their high production costs may limit their development and availability [
14], and this production cost may be even higher for respiratory infections due to the demand for additional antibodies [
15]. The use of polyclonal antibodies has multiple hurdles including the safety of patients and standardization. In recent years, immunoglobulin Y (IgY) has drawn remarkable attention from the field and has become an interesting approach to produce inexpensive and bioavailable antibodies in large quantities [
16]. IgY has similar functions to mammalian IgGs and serves as the primary circulating antibody isotype in birds, reptiles, and amphibians [
17]. In addition, IgYs are transferred to egg yolks in high concentrations and are eventually passed to developing embryos to provide passive immunization [
18,
19]. High-yield production of IgY has shown promising potential for both prophylaxis and the treatment of bacterial and viral infections [
20,
21,
22,
23,
24]. Collectively, these findings demonstrate that IgY-based technologies hold great promise in biomedical research, diagnostics, and therapeutics. Since IgYs can be obtained easily and reliably in large amounts from the egg yolks of immunized chickens in a non-invasive and cost-effective way, it remains an interesting question whether IgY may represent an alternative and optional antibody with efficacy equivalent to IgGs in clinical uses.
Considering the advantages of IgYs in biomedical applications, scientists have proposed a new hypothesis that passive immunization with IgYs may represent a potential therapeutic approach for treating viral respiratory infections [
25]. Recently, Artman et al. produced IgYs targeting the SARS-CoV-2 S protein and demonstrated the ability of IgYs to lower the binding affinity of SARS-CoV-2 S proteins for ACE2 and subsequently reduce viral replication in host cells [
26]. In the present study, we immunized laying hens with antigens for various subunits of the SARS-CoV-2 S protein and then extracted and purified IgYs specific for the corresponding antigens from egg yolks. We evaluated whether these IgYs were able to neutralize SARS-CoV-2 infectivity in Vero E6 cells in vitro. In addition, we infected Syrian hamsters with SARS-CoV-2 and subsequently examined whether IgY administration showed efficacy in prophylaxis and treatment against SARS-CoV-2 infection in vivo. In the present study, our findings have provided evidence demonstrating the prophylactic and therapeutic utilities of IgY antibodies against SARS-CoV-2 infection in vivo.
2. Materials and Methods
2.1. Immunization of Laying Hens
Eighty micrograms of each purified SARS-CoV-2 spike protein subunit (Spike S1-His Recombinant Protein, Cat: 40591-V08B1, Spike RBD-His Recombinant Protein, Cat: 40592-V08B, or Spike S2 ECD-His Recombinant Protein, Cat: 40590-V08B (Sino Biological Inc., Beijing, China) was dissolved in 0.5 mL of normal saline solution then emulsified with 0.5 mL of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). The resultant emulsified product was then used for immunizing the laying hens via intramuscular injection into 5 different sites on the muscles of the thorax. Blood samples and the laying eggs were collected once per two weeks for the purification of IgY antibodies which were used for the detection of the plateau of passive immunization. Four weeks after the immunization of laying hens by the 1st injection, the antigen proteins and incomplete Freund’s adjuvant were mixed and emulsified, followed by the 2nd immunization of these hens using the same procedures. Two weeks after the final injection, eggs were harvested and stored at 4 °C. Mock IgY was produced using the same adjuvant without antigens.
2.2. IgY Extraction
The extraction of IgYs was conducted using our patented method for extracting IgY from egg yolks (Wang et al., United States Patent; Patent No.: US0096.05052B2 Date of Patent: 28 March 2017). The method for extracting IgY from egg yolks is conducted as follows. Overall, a buffer solution, a yolk sample, and an inorganic salt solution were used for the extraction. Briefly, the yolk sample with the buffer solution was diluted with the acetate-based buffer solution with a pH ranging from 4.6 to 5.4. The mixture was then stirred for at least 30 min at 4 °C, followed by centrifugation. After centrifugation of the mixture at 14,000× g for 30 min, the supernatant was collected. Thereafter, the inorganic ammonium sulfate salt solution with a concentration ranging from 0.05 M to 0.15 M and a saturation degree from 30% to 60% was added into the supernatant to salt out the IgYs. After another stirring for 30 min, the mixture was centrifuged again at 14,000× g for 30 min to precipitate the IgYs. The whole extraction process requires approximately 2 h.
2.3. Specific IgY Enzyme-Linked Immunosorbent Assay (ELISA)
To quantitatively detect IgY antibodies against the SARS-CoV-2 spike protein subunits, we used an indirect ELISA. Ninety-six-well ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated for 16 h at 4 °C with purified SARS-CoV-2 spike protein subunit (Spike S1-His Recombinant Protein, Cat: 40591-V08B1, Spike RBD-His Recombinant Protein, Cat: 40592-V08B, or Spike S2 ECD-His Recombinant Protein, Cat: 40590-V08B (Sino Biological Inc., Beijing, China), diluted in carbonatebicarbonate buffer to a concentration of 1 μg/mL. The plates were washed with 0.05% PBST, incubated with a blocking buffer consisting of 3% non-fat dried milk in PBS for 1 h at room temperature, and then washed. Chicken anti-sera and purified IgY of egg yolk (10 µg/mL) after immunization of laying hens were prepared by diluting in blocking buffer, followed by 10-fold serial dilutions. These were added in triplicate to the wells and incubated for 1.5 h at 37 °C. Samples were added to corresponding wells and incubated for 1 h at room temperature. The plates were washed and incubated with 100 μL per well of HRP-conjugated goat anti-chicken IgY (1:3000, Rabbit anti-Chicken IgY, Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C. After washing, 100 μL of SureBlue™ TMB 1-Component Microwell Peroxidase Substrate (SeraCare 5120-0076, LGC Clinical Diagnostics, Milford, MA, USA) was added to the wells and incubated for 5 min at room temperature. The reaction was terminated with 100 μL of TMB BlueSTOP™ Solution (SeraCare 5150-0024, LGC Clinical Diagnostics, Milford, MA, USA). Optical density (O.D.) was measured at 450 nm with subtraction of the O.D. on an ELISA reader (Molecular Devices). The endpoint titer was defined as the highest IgY dilution at which the OD450 value was ≥0.2. All experiments were performed in triplicates.
2.4. Cell Lines and Viruses
All the SARS-CoV-2 strains used in this study, including the SARS-CoV-2 WA strain (hCoV-19/Taiwan/4/2020), and the three variants of concern (VOCs), i.e., B.1.1.7 (hCoV-19/Taiwan/792/2020, α variant), B.1.617.2 (hCoV-19/Taiwan/1144/2021, δ variant), and Omicron BA.1 (hCoV-19/Taiwan/IPM-63/2022, ο variant) were provided by the Taiwan Centers for Disease of the Ministry of Health and Welfare. The Vero E6 cells (ATCC CRL-1586) were generally maintained in high-glucose DMEM (HyClone) which was supplemented with an antibiotic–antimycotic (Gibco) and 10% fetal bovine serum (Hyclone). For virus propagation, SARS-CoV-2 strains were propagated in Vero E6 cells cultured in high-glucose DMEM containing 2% FBS. The 2nd passage of viruses was subjected to all the experiments in this study. Viral titers were examined using the plaque assay and the viral stocks were stored as aliquots at −80 °C. All the SARS-CoV-2 experiments, excluding those using inactivated SARS-CoV-2, were performed in a biosafety level 3 (BSL-3) or BSL-4 laboratory.
2.5. Western Blot
The protein lysates of SARS-CoV-2-infected cells were dissolved with RIPA buffer. The protein expression was analyzed by Western blot analysis in which the TGN Gel 4–15% (QP5510, SMOBIO) with an SE260 Mighty Small II Deluxe Mini Vertical Protein Electrophoresis Unit (Hoefer, Inc., Holliston, MA, USA) was used. A YesBlot™ Western Marker I (10–200 kDa) (WM1000 Smobio, Taiwan) was used as a molecular weight marker. Antibodies including the mouse monoclonal anti-spike (1:5000; GeneTex Cat #GTX632604) and mouse monoclonal anti-nucleocapsid (1:2000; GeneTex Cat# GTX632269) antibodies were used to detect the SARS-CoV-2 spike and nucleocapsid proteins, respectively. The mouse monoclonal GAPDH antibody (GTX627408, GeneTex, 1:5000) was used as the loading control. The anti-chicken IgY (IgG) (whole molecule)–peroxidase antibody produced in rabbits (Sigma-Aldrich A9046) and the anti-rabbit IgG (whole molecule)–peroxidase antibody produced in goats (Sigma-Aldrich A0545) were used as the secondary antibodies as required. The luminol-based detection of Western blot signals was performed using the Amersham ECL Western Blotting Detection Kit (Amersham Biosciences, Buckinghamshire, UK). The images were captured and analyzed using Image Quant™ LAS 4000 (GE Healthcare, Chicago, IL, USA).
2.6. The Plaque Reduction Neutralization Test (PRNT) with Infectious SARS-CoV-2
To determine the titers of neutralizing antibodies (nAbs) from anti-S protein IgYs, IgYs were first diluted ten-fold (starting at a concentration of 10,000 μg) in the DMEM. Following the heat inactivation of the complement, 0.1 mL of the serial dilutions was incubated with a virus solution containing ~100 pfu of SARS-CoV-2 Wuhan or α strain. The mixture of IgY and SARS-CoV-2 solution was added to the culture of Vero E6 cells in 24-well plates incubated at 37 °C. The Vero E6 cells were then cultured in DMEM supplemented with 2% FBS and 0.3% agarose for 3 days. Crystal violet solution was then used for the staining of the plaques. After the staining, the plaques were counted. The titers for IgY neutralizing antibody were calculated as the reciprocal of the maximal dilution of the 50% virus titer reduction compared to the negative control mock IgY produced using the same adjuvant without antigens.
2.7. Determination of IgY In Vitro Specificity
The SARS-CoV-2-infected Vero E6 cells were lysed using the RIPA buffer supplemented with protease inhibitor (Merck Millipore, Billerica, MA, USA) and incubated at 100 °C for 5 min. A total of 15 μL of each sample was loaded into wells with the A YesBlot™ Western Marker I (10–200kDa) (WM1000 Smobio, Taiwan). Membranes were immunoreacted with IgYs against SARS-CoV-2 S protein subunits or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (GeneTex, Cat# GTX627408, 1:5000). Reactive bands were visualized by Amersham ECL Western Blotting Detection Kit (Amersham)
2.8. SARS-CoV-2 Infection in Syrian Hamsters and IgY Treatment
Owing to the expression of endogenous ACE2 expression, Syrian golden hamsters were used as the in vivo model for SARS-CoV-2 infection. The in vivo experiments were conducted as described previously [
27] and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Defense Medical Center Laboratory Animal Center (AN-110-15 and AN-110-16). Eight-week-old male Syrian hamsters were purchased from the National Laboratory Animal Center (Taipei, Taiwan). To infect the Syrian hamsters, 1000 pfu of SARS-CoV-2 (Wuhan strain) were delivered intranasally. IgYs specific for S1-RBD and S2 subunits were mixed and given simultaneously via the intranasal and intraperitoneal routes to neutralize SARS-CoV-2 infectivity in the nasal cavity and in the circulation. Syrian hamsters were treated with IgY antibodies 1.5 h before or after SARS-CoV-2 infection. Syrian hamsters were randomly separated into 6 groups: (A) IgY Mock; (B) IgY Mock + CoV2 (IgYs were given 1.5 h before viral infection); (C) CoV2 + IgY Mock (IgYs were given 1.5 h after viral infection); (D) IgY S1-RBD/S2; (E) IgY S1-RBD/S2 + CoV2 (IgYs were given 1.5 h before viral infection); (F) CoV2 + IgY S1-RBD/S2 (IgYs were given 1.5 h after viral infection). Each group contained at least three hamsters. After infection and the administration of IgYs, the body weight of Syrian hamsters was monitored for 7 days. On day 4 post-infection, lung sections were subjected to H&E staining and immunohistochemistry. Since the SARS-CoV-2 spike (S) protein tightly interacts with ACE2 after viral infection, this marker is seldom assessed in SARS-CoV-2-infected host cells. Syrian hamsters were challenged by SARS-CoV-2 in animal biosafety level 3 (ABSL-3) laboratories. Considering the high infectivity and biohazards of SARS-CoV-2, we only examined the changes in body weight and the expression of SARS-CoV-2 nucleocapsid, a viral protein implicated in SARS-CoV-2 replication protein after viral infection. The Syrian golden hamster model for in vivo SARS-CoV-2 infection is highly reproducible and routinely conducted in our laboratory. All animal procedures complied with the ARRIVE guidelines (
https://arriveguidelines.org/ (31 December 2020) and were reviewed and approved by the Institute of Preventive Medicine, National Defense Medical Center Animal Care and Use Committee (approval numbers: AN-110-15 and AN-110-16)
2.9. Immunohistochemistry
After the SARS-CoV-2 infection, lung tissue from uninfected control hamsters or infected hamsters was autopsied and fixed in 4% paraformaldehyde for 48 hours. Subsequently, paraffin sections (4 μm in thickness) were made and H&E staining was used to identify the histopathological changes. In addition, immunohistochemistry staining (IHC) was used for the detection of the SARS-CoV-2 nucleocapsid protein with SARS-CoV-2 (COVID-19) nucleocapsid antibody (GeneTex, Cat #GTX135357; 1:1000). The lobe sections were obtained over the whole lung and all of the slides were subjected to the automatic sample preparation system BenchMark XT and the overview scan was performed using the VENTANA DP 200 slide scanner (Hong Jing Co., Ltd., New Taipei, Taiwan).
2.10. Statistical Analysis
Data were expressed as mean ± SD and analyzed using GraphPad Prism v. 6.0.1. (GraphPad Software, La Jolla, CA, USA). Statistically significant differences among groups were detected by one-way ANOVA. Once a significantly difference was detected, post hoc Tukey tests were conducted. Differences in the values between the two groups were analyzed with the unpaired Student’s two-tailed t-test. The criterion for significance was set at p < 0.05.
4. Discussion
The COVID-19 pandemic represents a great threat to public health and the global economy. Accordingly, numerous efforts in the development of innovative strategies to overcome this challenge have been made. Passive immunization is one of these novel therapeutic options that may hold promise in pandemic control [
11]. Although monoclonal antibodies are accepted to be the best type of antibodies generated for passive immunization, they are also time-consuming and expensive to make [
14]. In contrast, IgYs have drawn substantial attention due to their high functional similarities to mammalian IgG, ideal bioavailability, ease of high-yield production, and low production cost [
16]. Numerous studies have demonstrated the treatment efficacy of IgY against bacterial and viral infections [
20,
21,
22,
23,
24]. The SARS-CoV-2 S protein consists of a signal peptide located on the N-terminus, an S1 subunit, and an S2 subunit [
31]. The S1 subunit consists of an N-terminal domain and an RBD, and the S2 subunit comprises a fusion peptide, heptapeptide repeat sequence 1, heptapeptide repeat sequence 2, a transmembrane domain, and a cytoplasmic domain. The RBD of the S1 subunit is responsible for the recognition and binding to the host ACE2 receptor and the S2 subunit governs membrane fusion [
31]. In the present study, we immunized laying hens and generated egg yolk IgYs specific for the S1, S1-RBD, and S2 subunits of the SARS-CoV-2 S protein. Egg yolk IgY titers increased in a time-dependent manner in parallel with the elevation of serum IgY titers in immunized hens (
Figure 1).
In addition to the characterization of IgY features, we further evaluated the in vitro specificity of IgYs and their ability to neutralize viral infectivity. IgYs specific for each S protein subunit showed excellent in vitro specificity for full-length spike protein derived from SARS-CoV-2 Wuhan, α, δ, or ο strains (
Figure 3). To examine the neutralization potential of these IgYs in vitro, we pre-mixed IgYs with different SARS-CoV-2 strains and assessed viral titers using plaque assays. Notably, IgY antibodies specific for S1 and S1-RBD subunits and those specific for the S2 subunit were able to potently neutralize viral infectivity (
Figure 2). A bioinformatic study using phylogenetic analysis and 3D structural modeling revealed that the SARS-CoV-2 S protein is highly conserved across variants. From the NCBI database, the identity of the overall percent protein sequence of the SARS-CoV-2 spike protein sequences was 99.68. The S1 subunit was more conserved than the S2 subunit (99.70% versus 99.66%). Based on the NCBI database of the infected countries, the 319-541 residues of amino acids within the S1 domain were 100% similar among the collected SARS-CoV-2 S protein sequences. Therefore, the SARS-CoV-2 S1-RBD subunit has been widely used as a target region for the development of vaccines [
32]. Interestingly, IgY targeting the S2 subunit, a component that mediates membrane fusion, also fully neutralized viral infectivity at 100 μg. These data revealed that, similar to IgYs specific for S1 or S1-RBD subunits, IgYs that specifically target the spike protein S2 subunit may also possess the potential to neutralize the SARS-CoV-2 viral infectivity.
IgYs specific for S1, S1-RBD, and S2 subunits all exhibited the ability to neutralize SARS-CoV-2 infectivity in vitro. We examined the combined treatment efficacy of IgY specific for the S1-RBD subunit and IgY specific for the S2 subunit before or after SARS-CoV-2 infection in Syrian hamsters, an established in vivo experimental model for SARS-CoV-2 infection. Antibodies were delivered via both intranasal and intraperitoneal routes. Intranasal IgYs were administered to neutralize intranasally delivered SARS-CoV-2 and intraperitoneal injection of IgYs was employed to suppress systemic viremia induced by SARS-CoV-2 infection. The combined treatment with IgYs specific for S1-RBD and S2 subunits rescued the weight loss phenotype induced by SARS-CoV-2 infection and reduced the size of lung lesions and areas positive for SARS-CoV-2 N protein staining (
Figure 4,
Figure 5 and
Figure 6). Pre-treatment with IgYs targeting S protein subunits restored body weight to a greater extent than treatment with these IgYs after SARS-CoV-2 infection. In addition, pre-treatment with these IgYs was more effective than post-infection treatment at reducing lung lesions and areas positive for SARS-CoV-2 nucleocapsid staining. It is encouraging that the IgY treatment after viral infection remains efficacious to rescue body weight loss and intrapulmonary lesions. In infected animals, SARS-CoV-2 may lead to secondary viral infection after viral replication and subsequently damage host cells. It is possible that post-infection treatment with IgYs specific for different SARS-CoV-2 subunits ameliorated the disease phenotype by suppressing secondary viral infections. Alternatively, pre-treatment with IgYs specific for different SARS-CoV-2 subunits may neutralize viral infectivity before cells are infected by SARS-CoV-2, leading to better outcomes.