Egg-derived anti-SARS-CoV-2 immunoglobulin Y (IgY) with broad variant activity as intranasal prophylaxis against COVID-19: preclinical studies and randomized controlled phase 1 clinical trial

COVID-19 emergency use authorizations and approvals for vaccines were achieved in record time. However, there remains a need to develop additional safe, effective, easy-to-produce, and inexpensive prevention to reduce the risk of acquiring SARS-CoV-2 infection. This need is due to difficulties in vaccine manufacturing and distribution, vaccine hesitancy, and, critically, the increased prevalence of SARS-CoV-2 variants with greater contagiousness or reduced sensitivity to immunity. Antibodies from eggs of hens (immunoglobulin Y; IgY) that were administered receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were developed as nasal drops to capture the virus on the nasal mucosa. Although initially raised against the 2019 novel coronavirus index strain (2019-nCoV), these anti-SARS-CoV-2 RBD IgY surprisingly had indistinguishable enzyme-linked immunosorbent assay binding against variants of concern that have emerged, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529). This is distinct for sera from immunized or convalescent patients. Culture neutralization titers against available Alpha, Beta, and Delta were also indistinguishable from the index SARS-CoV-2 strain. Efforts to develop these IgY for clinical use demonstrated that the intranasal anti-SARS-CoV-2 RBD IgY preparation showed no binding (cross-reactivity) to a variety of human tissues and had an excellent safety profile in rats following 28-day intranasal delivery of the formulated IgY. A double-blind, randomized, placebo-controlled phase 1 study evaluating single-ascending and multiple doses of anti-SARS-CoV-2 RBD IgY administered intranasally for 14 days in 48 healthy participants also demonstrated an excellent safety and tolerability profile, and no evidence of systemic absorption. As these antiviral IgY have broad selectivity against many variants of concern, are fast to produce, and are a low-cost product, their use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation.

Development of SarS-Cov-2 antigen Detection Kit Based on Immunoglobulin Y (Igy) Using Surface Plasmon resonance (SPr)

SARS disease reappeared at the end of 2019 with a new name as Coronavirus Disease 2019 (COVID-19) caused by a new virus called SARS-CoV-2. This virus has spread throughout the world until recently and caused massive deaths and losses. The nucleic acid test in the form of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is very important to diagnose COVID-19 in patients, but this method has several drawbacks such as operators who have to be trained, the diagnosis results appear in a relatively long time, and the examination price relatively expensive. This research was conducted to produce immunoglobulin Y (IgY) extracted from chicken egg yolk targeting the S-protein receptor-binding domain (RBD) on SARS-CoV-2 as a component of the surface plasmon resonance (SPR) SARS-CoV-2 antigen detection kit. This research was started by extracting IgY from hyperimmune chicken egg yolks with the polyethylene glycol (PEG) precipitation method and continued with dialysis. The extracted IgY was further purified using thiophilic adsorption chromatography and concentrated by using Amicon® Ultra-15 ultrafiltration. The IgY activity against SARS-CoV-2 RBD was tested qualitatively using the agar gel precipitation test (AGPT) technique and the total protein content was determined using the Lowry method. IgY was tested for its affinity against SARS-CoV-2 RBD using SPR. The IgY concentration obtained was 11 mg/mL. The AGPT result showed the presence of IgY activity against SARS-CoV-2 RBD isolated from egg yolk and chicken serum after 8 weeks after the first vaccination of chickens. The SDS-PAGE results showed a very clear band of IgY characters. The obtained IgY showed adequate interaction with commercial SARS-CoV-2 RBD on an SPR device. The purified IgY was able to bind with protein-S RBD and showed a fairly good affinity for the SARS-CoV-2 antigen sample. The results of these observations indicate that IgY anti-S-protein SARS-CoV-2 can be produced and purified from chicken egg yolk and can be used as a diagnostic component to detect SARS-CoV-2 antigen, especially on SPR.

Immunoglobulin Y for Potential Diagnostic and Therapeutic Applications in Infectious Diseases

Antiviral, antibacterial, and antiparasitic drugs and vaccines are essential to maintaining the health of humans and animals. Yet, their production can be slow and expensive, and efficacy lost once pathogens mount resistance. Chicken immunoglobulin Y (IgY) is a highly conserved homolog of human immunoglobulin G (IgG) that has shown benefits and a favorable safety profile, primarily in animal models of human infectious diseases. IgY is fast-acting, easy to produce, and low cost. IgY antibodies can readily be generated in large quantities with minimal environmental harm or infrastructure investment by using egg-laying hens. We summarize a variety of IgY uses, focusing on their potential for the detection, prevention, and treatment of human and animal infections.

Evaluation of Immunoglobulin-Y in Place of Tylosin Phosphate in the Diets Fed to Holstein Steers and Preliminary Analysis of Liver Abscess Duration on Animal Growth Performance

Despite the regular use of feed-grade macrolide-antibiotics, bovine liver abscesses persist, representing a financial burden to pre- and post-mortem sectors of the beef industry. An immunoglobulin-Y (IGY) additive developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses. Research is needed for the impact of liver abscess severity as well as abscess duration on steer performance and carcass characteristics. Holstein steers (n=64; initial body weight (BW)=372.5±2.41 kg) consuming a finishing diet for 188 d were used in a completely randomized design where treatments included: TYL (tylosin phosphate 90 mg/day; n=32) or IGY (2.5 g/day; n=32) and steer was the experimental unit. Feed intake was recorded daily while BW and liver ultrasound outcome (normal or abnormal) was recorded every 28 d until slaughter to estimate duration of abscess presence (DUR). Continuous variables of animal growth performance and carcass characteristics were analyzed using the MIXED procedure of SAS. Categorical quality grade and liver data were analyzed using the GLIMMIX procedure of SAS. Treatment did not affect live or carcass-adjusted growth performance (P≥0.131). However, steers fed TYL had greater (P=0.042) empty body fat (EBF) % and a greater proportion of carcasses grading premium choice than steers fed IGY (P=0.030). Treatment did not affect prevalence of abscessed livers, abscess severity or estimated abscess duration (P≥0.213) but datasets with greater experimental units are needed to substantiate this outcome. Increasing abscess severity tended (P≤0.10) to linearly reduce carcass-adjusted gain to feed (G:F), fat thickness, and EBF. Carcass dressing % was only affected by severe (A+ and A+AD) abscess scores (P=0.010). Carcass-adjusted final BW, average daily gain, G:F, and hot carcass weight was decreased only when the estimated DUR was ≥140 d (P≤0.05). Carcass dressing %, however, was linearly affected by estimated liver abscess DUR (P≤ 0.005), regardless of abscess severity. Preliminary evidence suggests that measuring the duration of liver abscess affliction during the feeding period may also give insight to the degree of performance reduction.

83 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses & reduction of macrolide-resistant bacteria dissemination

Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384± 13.5kg) consuming a finishing diet (90% concentrate) for 188 days (Final BW= 660± 42 kg) were used in a completely randomized design where steer was experimental unit and treatments included: TYL (Tylosin phosphate fed at 90mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n= 32). Feed intake was recorded daily and BW was recorded every 28 days until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data were analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P= 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P < 0.02) and consequently greater numerical yield grade (P < 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL=63% vs. IGY=52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P < 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in feedlot cattle diets.

81 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses & reduction of macrolide-resistant bacteria dissemination

Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum andTrueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384±13.5kg) consuming a finishing diet (90% concentrate) for 188 d (Final BW= 660±42 kg) were used in a completely randomized design where treatments included: TYL (Tylosin phosphate fed at 90 mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n = 32). Feed intake was recorded daily and BW was recorded every 28 d until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data was analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P = 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P < 0.02) and consequently greater numerical yield grade (P < 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL= 63% vs. IGY= 52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P < 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in the diets of feedlot cattle.