Chimerism of avian IgY-scFv and truncated IgG-Fc: A novel strategy in cross-species antibody generation and enhancement.

Chicken single-chain fragment variable (IgY-scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half-life due to the absence of antibody Fc region compared with the full-length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti-canine parvovirus-like particles avian IgY-scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv-CH2, scFv-CH3, and scFv-Fc can bind with antigen specifically and dose-dependently. Surface plasmon resonance investigation confirmed that scFv-CH2, scFv-CH3, and scFv-Fc had different degrees of binding to FcRn, with scFv-Fc showing the highest affinity. scFv-Fc had a significantly longer half-life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc-based therapeutic antibodies and proteins with longer half-lives. The avian IgY-scFv-mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine.

Passive protection of chicken egg yolk immunoglobulin (IgY) against Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus).

IgY is an immunoglobulin primarily found in the serum and egg yolk of birds, amphibians, and reptiles. Recent years, IgY is considered to have a good application prospect in the immunodiagnostics and passive immunotherapy of aquatic diseases. In this study, we prepared a specific IgY against Streptococcus agalactiae in tilapia after immunizing the hens for 4 times. The result of ELISA detection showed that the IgY titers in water-soluble fraction (WSF) after 6 weeks of immunization reached 1:51200 and last for 4 weeks. Western blot (WB) analysis data showed that the specific IgY could recognize the target band, the specific IgY showed a concentration-dependent inhibitory effect on the growth of S. agalactiae, altered cell wall structure and aggluted of S. agalactiae. The quantitative reverse transcription PCR (qRT-PCR) analysis data suggested that the specific IgY downregulated the expression of pro-inflammatory factors (IL-8, TNF-α), upregulated the anti-inflammatory factors (IL-10, TGF-ß). In addition, the histopathological results showed that the specific IgY significantly decreased the pathological manifestations, dramatically improved the survival rates of tilapia in injection, feeding, and immersion experiments. Collectively, our findings demonstrated that the broad potential of specific IgY for the prevention and treatment of S. agalactiae infection in tilapia.

Real-world evidence of a novel tetravalent immunoglobulin Y effectiveness and safety in patients with the refractory Helicobacter pylori infection.

BACKGROUND: Refractory Helicobacter pylori (H. pylori) infection inevitably increase the difficulty of drug selection. Here, we described our experience with the use of a novel tetravalent IgY against H. pylori for the treatment of patients with refractory H. pylori infection. METHODS: Patients were randomly assigned to receive the standard quadruple therapy (amoxicillin, clarithromycin, omeprazole and bismuth potassium citrate ) for 2 weeks or 250 mg of avian polyclonal IgY orally twice a day for 4 weeks. The binding efficacy of IgY to H. pylori antigens was detected by western blotting13. C-urea breath test was performed to evaluate the eradication therap's efficacy. The side effects of IgY were evaluated via various routine tests. The questionnaire was used to gather clinical symptoms and adverse reactions. RESULTS: Western blot analysis showed that tetravalent IgY simultaneously bind to VacA, HpaA, CagA and UreB of H. pylori. Tetravalent IgY had an eradication rate of 50.74% in patients with refractory H. pylori and an inhibition rate of 50.04% against DOB (delta over baseline) of 13C-urea. The symptom relief rate was 61.76% in thirty-four patients with clinical symptoms, and no adverse reactions were observed during tetravalent IgY treatment period. CONCLUSIONS: Polyclonal avian tetravalent IgY reduced H. pylori infection, and showed good efficacy and safety in the treatment of refractory H. pylori infection patients, which represented an effective therapeutic option of choice for patients with refractory H. pylori infection.

Rapid development and mass production of SARS-CoV-2 neutralizing chicken egg yolk antibodies with protective efficacy in hamsters.

Despite the record speed of developing vaccines and therapeutics against the SARS-CoV-2 virus, it is not a given that such success can be secured in future pandemics. In addition, COVID-19 vaccination and application of therapeutics remain low in developing countries. Rapid and low cost mass production of antiviral IgY antibodies could be an attractive alternative or complementary option for vaccine and therapeutic development. In this article, we rapidly produced SARS-CoV-2 antigens, immunized hens and purified IgY antibodies in 2 months after the SARS-CoV-2 gene sequence became public. We further demonstrated that the IgY antibodies competitively block RBD binding to ACE2, neutralize authentic SARS-CoV-2 virus and effectively protect hamsters from SARS-CoV-2 challenge by preventing weight loss and lung pathology, representing the first comprehensive study with IgY antibodies. The process of mass production can be easily implemented in most developing countries and hence could become a new vital option in our toolbox for combating viral pandemics. This study could stimulate further studies, optimization and potential applications of IgY antibodies as therapeutics and prophylactics for human and animals.

Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

Production and purification of Clostridium perfringens type C beta-toxin and IgG and IgY antitoxins.

OBJECTIVES: This study aimed to produce and purify Clostridium perfringens type C beta-toxin, sheep anti-beta toxin immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY). METHODS: Two methods were used for beta-toxin purification: single-step metal affinity chromatography (MAC) using zinc as a chelator and ion exchange chromatography (IEX). The purified and inactivated beta-toxoids were then administered to sheep and chickens in order to produce IgG and IgY. RESULTS: All assays using the IEX failed. In contrast, MAC purified more than 21 mg of toxin per run in a single-step protocol. The purified and inactivated beta-toxoids were then administered to sheep and chickens, and IgG and IgY were purified with a high yield, medium antibody titer of 50 IU/mL, and high avidity (73.2 %). CONCLUSIONS: C. perfringens type C beta-toxin and sheep or chicken anti-beta toxin IgG and IgY antibodies were successfully produced and purified using a simple protocol. This protocol can be used for the production of components used in the diagnosis and research of necrotic enteritis caused by C. perfringens type C, as well as for the evaluation of existing vaccines and the development of new preventive methods against this disease.

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 µg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

H9N2 Avian Influenza Virus Downregulates FcRY Expression in Chicken Macrophage Cell Line HD11 by Activating the JNK MAPK Pathway.

The H9N2 avian influenza virus causes reduced production performance and immunosuppression in chickens. The chicken yolk sac immunoglobulins (IgY) receptor (FcRY) transports from the yolk into the embryo, providing offspring with passive immunity to infection against common poultry pathogens. FcRY is expressed in many tissues/organs of the chicken; however, there are no reports investigating FcRY expression in chicken macrophage cells, and how H9N2-infected HD11 cells (a chicken macrophage-like cell line) regulate FcRY expression remains uninvestigated. This study used the H9N2 virus as a model pathogen to explore the regulation of FcRY expression in avian macrophages. FcRY was highly expressed in HD11 cells, as shown by reverse transcription polymerase chain reactions, and indirect immunofluorescence indicated that FcRY was widely expressed in HD11 cells. HD11 cells infected with live H9N2 virus exhibited downregulated FcRY expression. Transfection of eukaryotic expression plasmids encoding each viral protein of H9N2 into HD11 cells revealed that nonstructural protein (NS1) and matrix protein (M1) downregulated FcRY expression. In addition, the use of a c-jun N-terminal kinase (JNK) activator inhibited the expression of FcRY, while a JNK inhibitor antagonized the downregulation of FcRY expression by live H9N2 virus, NS1 and M1 proteins. Finally, a dual luciferase reporter system showed that both the M1 protein and the transcription factor c-jun inhibited FcRY expression at the transcriptional level. Taken together, the transcription factor c-jun was a negative regulator of FcRY, while the live H9N2 virus, NS1, and M1 proteins downregulated the FcRY expression through activating the JNK signaling pathway. This provides an experimental basis for a novel mechanism of immunosuppression in the H9N2 avian influenza virus.

Production of IgY against iron permease Ftr1 from Candida albicans and evaluation of its antifungal activity using Galleria mellonella as a model of systemic infection.

Candida albicans is one of the leading pathological agents of mucosal and deep tissue infections. Considering that the variety of antifungals is restricted and that toxicity limits their use, immunotherapies against pathogenic fungi have been viewed as alternatives with reduced adverse effects. In this context, C. albicans has a protein used to capture iron from the environment and the host, known as the high-affinity iron permease Ftr1. This protein may be a new target of action for novel antifungal therapies, as it influences the virulence of this yeast. Thus, the aim of the present study was to produce and conduct the biological characterization of IgY antibodies against C. albicans Ftr1. Immunization of laying hens with an Ftr1-derived peptide resulted in IgY antibodies extracted from egg yolks capable of binding to the antigen with high affinity (avidity index = 66.6 ± 0.3%). These antibodies reduced the growth and even eliminated C. albicans under iron restriction, a favorable condition for the expression of Ftr1. This also occurred with a mutant strain that does not produce Ftr1 in the presence of iron, a circumstance in which the protein analog of iron permease, Ftr2, is expressed. Furthermore, the survival of G. mellonella larvae infected with C. albicans and treated with the antibodies was 90% higher than the control group, which did not receive treatment (p < 0.0001). Therefore, our data suggest that IgY antibodies against Ftr1 from C. albicans can inhibit yeast propagation by blocking iron uptake.

Generation and characterization of avian IgY antibodies for detecting beta-casein A1 in bovine milk.

Beta-casein is a primary milk protein that constitutes approximately 30% of the casein in bovine milk, with the two most common types in cattle being A1 and A2. The A2 protein differs from the A1 version due to a mutation in the codon at position 67, resulting in a histidine to proline substitution. However, the bioactive peptide, beta-casomorphine-7 (BCM7), which originates from partial proteolysis of the A1 variant, has been linked to several gastrointestinal disorders in humans. Production of A1 beta casein-free products is increasing demand in the milk market, worldwide. This study generated and characterized a polyclonal IgY antibody that specifically recognizes the A1 beta-casein protein present in cow's milk. A commercially available IgY anti-A1 antibody was used as a positive control, and the sensitivity and specificity of both the commercial and produced anti-A1 antibodies were evaluated. The results showed 100% sensitivity and specificity of 100% of the commercial IgY anti-A1. The in-house produced anti-A1 antibody demonstrated a sensitivity of 95.2% and a specificity of 100%, indicating its potential as a reliable and cost effective tool for detecting A1 beta-casein protein in milk samples.

DNA starvation/stationary phase protection protein of Helicobacter pylori as a potential immunodominant antigen for infection detection.

BACKGROUND: Application of chicken egg yolk immunoglobulin Y (IgY) for Helicobacter pylori (H. pylori, HP) has gained much interest in recent years. Comparing with for treatment, IgY may be more advantageous when used for H. pylori detection. METHODS: Nine strains of H. pylori with different genetic backgrounds were inactivated and used to immunize hens, respectively, for the preparation of polyclonal anti-H. pylori immunoglobulin Y (anti-HP IgY). The proteins of H. pylori with reactivity to anti-HP IgY were detected by Western Blot. The five protein bands that can be well recognized by anti-HP IgY of each group, and were prevalent in all nine strains were excised from SDS-PAGE gel, digested and identified by Nano-HPLC-MS/MS analysis. The potential of these identified proteins as antigen detection targets was then assessed by sequence analysis. RESULTS: Anti-HP IgY derived from each group of specific strain immunized hens can recognize self-strain and non-self-strain antigens well. Five immunodominant antigens were identified as chaperonin GroEL, flagellin A, urease subunit alpha, peroxiredoxin and DNA starvation/stationary phase protection protein. Sequences analysis showed that both peroxiredoxin and DNA starvation/stationary phase protection protein were present in all 1000 strains of H. pylori queried, and the amino acid sequences were highly conserved. The highest sequence consistency between the DNA starvation/stationary phase protection protein of H. pylori and non-Helicobacter organisms was 52.59%, and the consistent sites were scattered and there was no continuous long fragment consensus sequence. CONCLUSION: DNA starvation/stationary phase protection protein was identified as an immunodominant antigen of H. pylori and sequence analysis indicated that it could serve as a potential antigen target for the diagnosis of H. pylori infection.

Polyvalent passive vaccine candidates from egg yolk antibodies (IgY) of important outer membrane proteins (PF1380 and ExbB) of Pseudomonas fluorescens in fish.

Polyvalent antibodies can resist multiple bacterial species, and immunoglobulin Y (IgY) antibody can be economically prepared in large quantities from egg yolk; further, IgY polyvalent antibodies have application value in aquaculture. The outer membrane proteins (OMPs) PF1380 and ExbB of Pseudomonas fluorescens were expressed and purified, and the corresponding IgY antibodies were prepared. PF1380, ExbB, and the corresponding IgY antibodies could activate the innate immune responses of chicken and Carassius auratus. The passive immunization to C. auratus showed that the IgY antibodies of PF1380 and ExbB had an immune protection rate, down-regulated the expression of antioxidant-related factors (MDA, SOD, GSH-Px, and CAT) to reduce the antioxidant reaction, down-regulated the expression of inflammation-related genes (IL-6, IL-8, TNF-α, and IL-1ß) to reduce the inflammatory reaction, maintained the integrity of visceral tissue structure, and reduced apoptosis and damage of tissue cells in relation to P. fluorescens and Aeromonas hydrophila infections. Thus, the IgY antibodies of PF1380 and ExbB could be considered as passive polyvalent vaccine candidates in aquaculture.

Protective effects of anti-CfaB-EtpA-LTB IgY antibody against adherence and toxicity of enterotoxigenic Escherichia coli (ETEC).

AIM: Production of IgY antibodies against CfaB-EtpA-LTB (CEL) chimeric protein and evaluation of its protective effects against enterotoxigenic Escherichia coli (ETEC) by in vivo and in vitro investigation. METHODS AND RESULTS: Indirect ELISA and immunoblotting methods were applied to assess the immunogenicity and specificity of IgYs and also to evaluate the efficacy of IgYs in binding prevention and neutralizing the heat-labile (LT) toxin of ETEC bacteria. The results indicated that the anti-CEL IgY at a concentration of 2 mg ml-1 could decrease the bacterial adhesion to HT-29 cells by 74% compared to the control group.At a concentration of 750 µg ml-1, the IgY antibody managed to neutralize the disruptive LT toxin effect on the Y1 cell line. At a concentration of 2 mg ml-1, 81% reduction was observed in the fluid accumulation in the ileal loop assay. CONCLUSION: According to our findings, passive immunotherapy with anti-CEL IgY can prevent bacterial colonization and toxicity, thus facilitating in controlling the enteric diseases caused by ETEC infection.

Use of adjuvant ISA VG 71 to produce neutralizing egg yolk antibodies against bothropic venom.

The use of egg yolk antibodies-IgY technology-represents an alternative to the production of mammalian immunoglobulins and has several advantages regarding animal welfare and lower costs of production. The use of adjuvants to achieve the hyperimmunization of laying hens plays a key role in the success of the production of high levels of the antibodies. In the present work, two different adjuvant systems (Freund's adjuvants and MontanideTM ISA 71 VG) were compared to produce IgY anti-Bothrops alternatus. For the first immunization, formalin-inactivated Salmonella was added to MontanideTM ISA 71 VG to emulate Freund's complete adjuvant which includes a mycobacteria antigen. After eight immunizations, IgY produced by using either adjuvant was able to neutralize the lethal activity of the venom in a mouse model, but differences were found regarding the recognition of components of the venom between the two adjuvants tested. Overall, MontanideTM adjuvant used in this work could be a good alternative choice to produce antibodies capable of neutralizing the lethality of complex antigens. This adjuvant is commercially available and used in the formulation of several poultry vaccines and could be used for the IgY technology instead of traditional immunomodulators such as Freund's adjuvants. Key points • IgY extracts recognized major components of the venom.• Avidity indexes of the IgY extracts increased after the successive immunizations.• IgY obtained by two adjuvant systems neutralized the lethal activity of the venom.

Production of anti-tetanus toxin IgY and study of its protective effects in a mouse model.

Tetanus is an acute and often fatal infectious disease caused by Clostridium tetani. Tetanus toxin (TT) is responsible for spastic paralysis observed in tetanus. Anti-tetanus antibodies obtained from horses and humans are the most antitoxins used for tetanus treatment, although some clinical side effects and disadvantages have been reported in their application. The aim of this study is the production of anti-TT IgY and evaluation of its protective effects in a mouse model. Anti-TT IgY was purified from the egg yolk using PEG6000 precipitation and water dilution methods, and its purity was verified by SDS-PAGE. Finally, the potency of purified anti-TT IgY in neutralizing the lethal effects of TT was studied in vivo using a mouse model. PEG6000 precipitation method had better results. Animal studies showed that the purified IgY neutralized the toxic effects of 100 MLD of TT and multiple intravenous-dose injections of anti-TT IgY also had a continuous effect of TT neutralization. The purified anti-TT IgY was effective in neutralizing the lethal activity of TT in a mouse model. Our results suggested that IgY could be an alternative therapeutic source for the management of tetanus in the future.Abbreviations Anti-TT, Anti-tetanus toxin; ELISA, Enzyme-linked immunosorbent assay; IgY, Immunoglobulin Y; MLD, Minimum lethal dose; PBS, Phosphate buffer solution; PEG, Polyethylene glycol; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TIG, Tetanus immune globulin; TT, Tetanus toxin; WD, Water dilution; RT, Room temperature.

A review on immunoglobulin Y (IgY) conjugated with metal nanoparticles and biomedical uses.

Today, the use of nanoparticles has attracted considerable attention in biomedical investigations and applications. Antibody-nanoparticle conjugates have proven to be useful tools for raising accuracy and sensitivity in in vitro diagnostics. IgY antibodies have benefits over different antibodies in terms of minimizing animal harm, reducing reactivity with mammalian factors, and cost-effective extraction. Metal nanoparticles are widely used for various medical and biological applications and are potential candidates for identifying pathogens and treating them, which can be mostly related to their special properties, including their shape and size. Avian IgY antibodies conjugated with nanoparticles have been widely used for the detection of parasitic, viral, and bacterial infections as well as allergens and toxicological and pharmaceutical molecules. This review aimed to investigate avian antibodies conjugated with metal nanoparticles and their biological applications.

Effects of different protectants on the IgY content and physico-chemical properties of spray-dried egg yolk powder.

BACKGROUND: Egg yolk powder (EYP) with high immunoglobulin of yolk (IgY) content and good solubility is in great demand in the market of functional foods. In this article, the properties of spray-dried EYP with the addition of five protectants (maltodextrin, trehalose, mannitol, maltitol and sucrose) were investigated. RESULTS: All the protectants increased IgY activity and solubility of EYP. Among them, EYP with maltodextrin displayed the highest activity of IgY (27.11 mg/g), the highest solubility (66.39%) and the lowest surface hydrophobicity. Moreover, the average particle size of EYP with maltodextrin was the smallest (9.78 µm). The egg yolk particles obtained by adding the protectants are more uniformly distributed and have smaller particle size. Fourier-transform infrared spectroscopy confirmed the structural integrity of the proteins, indicating that the protectants addition enhanced the hydrogen bonding forces between the EYP protein molecules. CONCLUSION: The addition of protectants can significantly improve the IgY content, solubility and structural stability of EYP. © 2023 Society of Chemical Industry.

Assessment of maternal immunity against Salmonella enterica serovar Heidelberg in progeny of broiler breeders vaccinated with different formulations of bacterins.

RESEARCH HIGHLIGHTSProgeny from vaccinated 30-week-old breeders presented less SH in caecal content compared to the control.High titres of maternal anti-Salmonella IgY could be associated with lower SH faecal shedding.

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.