key: cord-0260720-cx6td7uz authors: Frumkin, L.; Lucas, M.; Scribner, C.; Ortega-Heinly, N.; Rogers, J.; Yin, G.; Hallam, T. J.; Yam, A.; Bedard, K.; Begley, R.; Cohen, C. A.; Badger, C. V.; Abbasi, S. A.; Dye, J. M.; McMillan, B.; Wallach, M.; Bricker, T. L.; Joshi, A.; Boon, A. C. M.; Pokhrel, S.; Kraemer, B. R.; Lee, L.; Kargotich, S.; Agogiya, M.; St. John, T.; Mochly-Rosen, D. title: 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 date: 2022-01-10 journal: nan DOI: 10.1101/2022.01.07.22268914 sha: 406970b0fa70cf929d53c9ce31eeae937a8e4934 doc_id: 260720 cord_uid: cx6td7uz 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. selectivity against many variants of concern, are fast to produce, and are a low-cost product, their 63 use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation. 64 Introduction 65 As of January 4, 2022, over 290 million persons with coronavirus disease 2019 (COVID-19) 66 had been identified in 222 countries and territories with an estimated 5.4 million deaths [1] . It 67 is estimated that only 48% of the world population has been fully vaccinated [2], a figure 68 dramatically lower than the 70%-80% believed needed to reach herd immunity to stop the The main entry route for SARS-CoV-2 is the nasal mucosa, which has high levels of the 85 human angiotensin-converting enzyme 2 (hACE2) receptor that is used by the virus to gain 86 cellular entry [14] . Viral binding to the hACE2 receptor is mediated by the spike (S) protein on 87 the surface of the viral envelope [15] for all SARS-CoV-2 variants identified so far; even the 88 highly mutated Omicron variant, with 15 mutations in the receptor-binding domain (RBD) of 89 the S protein, is still dependent on hACE2 for its infectivity [16] . Therefore, the nasal mucosa 90 is an excellent site as a critical barrier to reduce SARS-CoV-2 entry; antibodies against the 91 SARS-CoV-2 RBD can compete with viral binding to the hACE2 receptor. In addition, 92 antibodies on epithelial surfaces can greatly inhibit lateral viral motility, agglutinate viral 93 particles, and anchor the virus to the extracellular matrix [17, 18] , thus making intranasally 94 administered antibodies a potentially important antiviral strategy. Indeed, intranasal antibody 95 prophylaxis has been an effective means against multiple viral pathogens in humans and 96 veterinary applications, including respiratory tract viruses [18] . Thus, covering the nasal 97 mucosa with anti-SARS-CoV-2 antibodies could prevent SARS-CoV-2 infection in naïve 98 individuals and may also reduce viral transmission from an infected individual by reducing 99 levels of active virus. 100 Because the RBD remains essential for SARS-CoV-2 infection, even for variants of 101 concern, we chose recombinant RBD of the S protein (amino acids 328-533) as the 7 Briefly, cell-free reactions were prepared by the addition of 37.5% v/v iodoacetamide-132 treated S30 extract, 5 µg/mL plasmid, and a supermix containing amino acids, nano- 133 microspheres, and small molecules for energy generation [20] . T7 RNA polymerase was over-134 expressed in Escherichia coli and added to the cell-free reaction as a reagent lysate at <1% v/v. 135 Reactions were carried out in a DASbox stirred tank (Eppendorf) at 250 mL volume with pH, 136 dissolved oxygen, and temperature control. Reactions were run for 16 hours at a temperature of 137 25°C, pH was controlled at 7.0 using 1 M citrate and 1 M potassium hydroxide, and dissolved 138 oxygen was maintained at 20%. 139 The XpressCF reaction of SARS-CoV-2-RBD 328-533 was clarified by centrifugation at 140 10,000 rpm for 20 minutes (Beckman, JLA-10.500 rotor) and filtered through a 0.22-μm 141 membrane filter. The clarified material was loaded onto a 5-mL his-Trap Excel affinity column 142 equilibrated with binding buffer (15 mM Tris-HCl, 500 mM NaCl, pH 8.0). After 20 column 143 volumes were applied to wash unbound impurities, the bound proteins were eluted with 20 mM 144 Tris-HCl, 300 mM imidazole, pH 8.0. The eluted fractions were then analyzed by 4-12% sodium 145 dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentrations 146 were determined by measured absorbance at 280 nm by NanoDrop (Thermo Fisher Scientific). 147 The his-SUMO tag was removed by Ulp1 protease digestion for 1 hour at room temperature. The 148 digested reaction was analyzed by 4-12% SDS-PAGE to verify full cleavage of the his-SUMO 149 tag before flow-through mode purification by Capto Q (Cytiva). Twenty mM Tris-HCl, pH 8.0, 150 was used to equilibrate the column, and the flow-through containing the cleaved protein was 151 collected. To further purify the CoV-2-RBD, the Capto Q flow-through fraction was bound to a 152 Capto SP ImpRes cation column equilibrated with 20 mM Tris-HCl, pH 8.0. The bound protein 153 was eluted with a 30-column volume linear gradient using elution buffer (20 mM Tris-HCl, 300 154 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint mM NaCl, pH 8.0). An Amicon Ultra-15, 3kD centrifugal filter was used to concentrate and 155 buffer exchange the target peak fraction into 6% sucrose in PBS at pH 7.2. The final purity of the 156 product was demonstrated by high-performance liquid chromatography (HPLC) analysis. 157 Characterization of the cell-free expressed recombinant RBD using surface 158 plasmon resonance 159 Binding kinetic of cell-free expressed SARS-CoV-2 RBD construct or mammalian expressed 160 his-tagged RBD control (ACROBiosystems SPD-C52H1) were then measured on a Biacore 161 T200 instrument, using Fc-tagged hACE2 receptor protein (ACROBiosystems AC2-H5257). To 162 this end, an anti-human Fc antibody (Jackson ImmunoResearch Labs) was immobilized on all 163 flow cells of a CM5 chip (GE Healthcare). Fc-tagged human ACE2 receptor protein 164 (ACROBiosystems AC2-H5257) was captured at ~100 replication units (RU). Binding of cell- 165 free expressed SARS-CoV-2 S protein RBD construct or mammalian expressed RBD control 166 (ACROBiosystems SPD-C52H1) was measured at concentrations up to 100 nM. Kinetic 167 experiments were performed at 25°C at a flow rate of 50 μL/min. RBD samples were diluted in 168 HBS-EP buffer (Teknova) and injected over the chip for 180 seconds followed by a 420-second 169 dissociation. The chip was regenerated with 10 mM glycine pH 1.5 after each injection. 170 Affinities were calculated using Biacore T200 Evaluation Software. 171 Hen immunization and IgY purification and characterization 172 Nine SPF hens were obtained from Charles River Laboratories and housed in a filtered air, 173 positive pressure barrier room (3 more hens from the same lot were added to the study after 5 174 months) at Avian Vaccine Services, Charles River Laboratories (Storrs, CT). Each hen was 175 caged individually with access to feed and water. Upon receipt, hens were immunized with an 176 9 inoculum containing 50 µg of recombinant cell-free expressed RBD fragment derived from S1 177 spike protein and water-in-oil adjuvant. Test bleeds were taken before the first immunization and 178 every 2 weeks after the first immunization throughout the project. Serum samples from each hen 179 were tested using an indirect enzyme-linked immunoassay (ELISA) and Western blot. Hens 180 received a boost 14 days after immunization and every 4 weeks after the previous immunization, 181 unless otherwise indicated. 182 Eggs were collected weekly. Yolks in batches up to 100 eggs/batch were separated and 183 IgY was purified. ELISA titration of IgY binding to full-length S1 (ACROBiosystems, S1N-184 C5255) expressed in human 293 cells and the cell-free expressed RBD of the SARS-CoV-2 spike 185 protein was performed in 96-well plates. IgY was then purified from yolks. Eggs were collected 186 weekly and egg white was separated from the egg yolk and discarded. Yolks were stored frozen 187 at -20˚C. For purification of IgY, yolks were thawed and diluted 1:10 in sterile PBS. The pH was 188 reduced to 5.0 and the yolk solution was incubated at 4˚C overnight. Yolk solution was then 189 centrifuged for 10 minutes at 9000 rpm. Supernatant was removed, pooled, pH was increased to 190 7.0, and final filtration (0.45 µm) was performed. 191 Hen sera and purified IgY were tested by ELISA and Western blot analysis using both 192 the immunogen (RBD) and full length glycosylated S1 protein (ACROBiosystems, cat# S1N-193 C5255). Lot Y0120 of purified IgY was derived from 50 yolks, starting 2 weeks after the 194 primary immunization. Lot Y0130 was derived from the next 50 yolks, starting 3 weeks after the 195 primary immunization. Lot Y0140 and above were derived from the next 100 yolks collected 196 over the preceding 2 weeks. Lots of purified IgY were tested by indirect ELISA and Western 197 blot to determine antibody response levels within the yolk after purification as well as affinity to 198 the cell-free expressed RBD fragment of S1 spike protein and the entire glycosylated S1 protein 199 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. Luminescence was read on a plate reader and analyzed with SoftMaxPro 6.0 (Molecular 213 Devices). 214 Hen sera were diluted with 1% BSA at various dilutions and tested in ELISA. One 215 hundred microliters were added to wells and the plate was incubated at room temperature for 1 216 hour. Plate wells were then rinsed with 1% Tween 20 wash solution, as indicated above. Anti-217 chicken HRP conjugate (Invitrogen, cat# SA-19509) diluted 1:5000 was added to each well. The 218 plate was incubated at room temperature for 1 hour, followed by another 1% Tween 20 wash. 219 3,3′,5,5′-tetramethylbenzidine substrate (KPL, cat# 5120-0050) was added to each well and the 220 plate was incubated at room temperature for 15 minutes. Optical density was measured using an 221 ELISA reader (Molecular Devices) at 650 nm. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint Western blot analysis was performed with the cell-free expressed RBD fragment of the 223 index S1 protein or the entire mammalian-expressed S1 protein (ACROBiosystems). One 224 microgram of denatured protein was added to Mini-PROTEAN TGX 4-20% SDS-PAGE. Gel 225 was run at 200V for 45 minutes in Tris-glycine buffer and stained using Simply Blue Safestain 226 (Invitrogen) at room temperature for 1 hour. Gels were destained twice using deionized water. 227 An unstained SDS-PAGE gel was transferred to the polyvinylidene difluoride membrane 228 using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) and cut into strips. Strips were 229 then stored at -20˚C until ready to use. Five percent nonfat dry milk was added to strips and 230 incubated at room temperature for 30-60 minutes. Test bleed sera and IgY were diluted 1:2000 231 and purified IgY were diluted 1:500 in 0.2 µm-filtered PBS and added to strips. Strips were 232 incubated at room temperature for 2 hours followed by 3 washes in filtered PBS. Rabbit anti-233 chicken IgY (H+L) secondary antibody, HRP (Invitrogen) diluted 1:3000 was added to each strip 234 and incubated at room temperature for 1-2 hours. Strips were washed once again in filtered PBS. 235 Opti-4CN substrate (Bio-Rad Laboratories) was added to each strip and incubated at room 236 temperature for up to 30 minutes in a rocking incubator. Strips were washed twice in deionized 237 water and images were taken using Bio-Rad EZ Gel Imager. 239 ELISA titer of the final IgY preparation used in the clinical studies against the Alpha, Beta, 240 Delta, and Omicron-derived RBD [amino acids 319-537; ACROBiosystems SPD-C52H1(index), 241 SPD-C52Hn (Alpha), SPD-C52Hp (Beta), SPD-C525e (Delta), and SPD-C522e (Omicron)]) was 242 carried out as described above. In culture viral neutralization studies using pseudovirus 244 12 The neutralization assays using pseudovirus were performed at RetroVirox (San Diego, 245 CA Quality controls for the pseudovirus neutralization assays were performed to determine: 265 1) signal-to-background values; 2) variation of the assay, estimated as the average of the 266 coefficient of variation for data points for which 50% or greater infection (RLUs) was observed 267 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. for 1 hour at 37°C with serially diluted antibodies. Vero-E6 monolayers were exposed to the 281 antibody-virus mixture at 37°C for 1 hour. Following incubation, viral inoculum was removed 282 and fresh cell culture media was added for an additional 23 hours at 37°C. Cells were washed 283 with PBS, fixed in 10% formalin, and permeabilized with 0.2% Triton-X for 10 minutes. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. Using Good Manufacturing Practice (GMP), anti-S1 RBD IgY was formulated for use as 328 intranasal drops as 0 (placebo control), 5, 10, and 20 mg/mL anti-S1 RBD IgY preparations in 329 sterile 2% microcrystalline cellulose and carboxymethylcellulose sodium at Bravado 330 Pharmaceuticals (Lutz, FL). The suspension was packed in a 1.5-mL dropper bottle and tests for 331 GMP drug product release complied with the relevant standards and methods, including 332 microbiological examination of nonsterile products (USP <1111>, <61> and <62>). All 333 formulated products were 100% stable as measured by physical and analytical properties, 334 including HPLC, when stored for at least 6 months at 2-8 o C and about 1 month when stored at 335 room temperature. Ten and 20 mg/mL solutions stored at room temperature showed 100% 336 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; Following 1-hour incubation at room temperature with a biotinylated detection antibody, 383 streptavidin-PE was added for 30 minutes while shaking. Plates were washed as described above 384 and PBS was added to wells for reading in the Luminex FLEXMAP 3D Instrument with a lower 385 bound of 50 beads per sample per cytokine. Each sample was measured in duplicate. Custom 386 Assay Chex control beads were purchased from Radix BioSolutions and added to all wells. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; weighting equation of 1/y2. The concentrations of anti-SARS-CoV-2 IgY in the samples were 405 determined from the calibration curve. After 4 analytical method validations, lower and upper 406 limit of detection, intra-and inter-assay precision and accuracy, dilution integrity, and short-term 407 stability, IgY levels in the blood samples of rats before and after 28 days of treatment were 408 below the limit of detection. Human tissue cross-reactivity study 410 A GLP study examining human tissue reactivity of the anti-SARS-CoV-2 RBD IgY was 411 conducted at Charles River Laboratories (Frederick, MD) using at least 3 tissues from at least 3 412 donors (S4 Table) . Ten mL of 10 mg/mL or 20 mg/mL IgY control (negative control) and anti- CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; Syrian hamsters develop mild-to-moderate disease with progressive weight loss that starts 428 several days after SARS-CoV-2 infection by intranasal inoculation [22, 23] . SARS-CoV-2 (strain 429 2019-nCoV/USA-WA1/2020) was propagated on Vero-TMPRSS2 cells, and the virus titer was 430 determined by plaque assays on Vero-hACE2 and Vero-hACE2-TMPRSS2 cells. Briefly, cells 431 were seeded in 24-well plates, and the next day, virus stocks were serially diluted 10-fold, CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; CoV-2 RBD IgY with 1 x 10 4 PFU (5 animals). Note that the viral titer in Vero-hACE2-452 hTMPRSS2 cells, which express the essential protease to liberate the RBD from the S protein on 453 the surface of the virus [24] , was subsequently found to be almost 100-fold higher for Groups 1 454 and 3 (4 x 10 6 PFU) and Groups 2 and 4 (0.8 x 10 6 PFU). 455 Animal weight was recorded daily. Three days after challenge, the animals were are available as supporting information (S1 Protocol and S1 CONSORT checklist). 492 Figure 1 . CONSORT flow diagram of phase 1 single-ascending and multiple-dose study 493 The primary objective of the study was to assess the safety and tolerability of anti-SARS- 494 CoV-2 IgY. A secondary objective was to assess the PK of anti-SARS-CoV-2 IgY. Evaluation of 495 immunoglobulin E (IgE) and anti-IgE antibodies was an exploratory objective. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; Healthy male and female participants ≥18 and ≤ 45 years old with a body weight ≥ 50 kg 497 and a body mass index ≥ 18.0 and ≤ 32.0 kg/m 2 were eligible for this study. Females of 498 childbearing potential who were pregnant or lactating or planning to become pregnant during the 499 study and participants with a history of alcohol and drug abuse, current smoking, clinically 500 significant laboratory abnormalities, history of nasal surgical procedures, frequent or recurrent 501 nasal conditions, current use of any nasal preparations, evidence of or history of clinically 502 significant conditions, or positive test for hepatitis B, hepatitis C, human immunodeficiency 503 virus, or SARS-CoV-2 nucleic acid or serology were excluded from participation. Full eligibility 504 criteria are summarized in S1 Protocol. 505 The master randomization schedule and the associated code break envelope files were 506 produced by an unblinded statistician using a computer-generated (SAS® v9.4 PLAN procedure) 507 pseudo-random permutation procedure. For Part 1, the first two randomization numbers for each 508 cohort were randomly assigned in a 1:1 ratio (anti-SARS-CoV-2 IgY: Placebo) to allow for 509 sentinel dosing, and the remainder of the numbers for each cohort was generated in a 5:1 (anti-510 SARS-CoV-2 IgY: Placebo) ratio using a permuted blocked randomization with a block size of 511 six. For Part 2, 24 numbers were generated in a 1:1:1:1 ratio (6 mg anti-SARS-CoV-2 IgY: 12 512 mg anti-SARS-CoV-2 IgY: 24 mg anti-SARS-CoV-2 IgY: Placebo) using a permuted blocked 513 randomization with a block size of six. The block sizes were kept confidential during the study. 514 The site personnel randomized eligible participants on Day 1 by assigning the next 515 available randomization number for the specific study part to the participant and reporting the 516 randomization number on the case report form. Study drug was prepared by an unblinded 517 pharmacist based on the treatment corresponding to the assigned randomization number on the 518 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; randomization schedule that was only available to the pharmacist. In the event of an emergency, 519 authorized personnel were able to unblind a participant through the code break envelope 520 associated with the randomization number assigned to the participant. 521 In Part 1, participants were randomly assigned to receive a single dose of anti-SARS- 522 CoV-2 RBD IgY antibodies or placebo in a sequential escalating manner. Three groups were 523 sequentially dosed with 8 healthy participants per group (6 active and 2 placebo in each group). CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; participants who received study drug. All variables were summarized by descriptive statistics for 608 each treatment group. The statistics for continuous variables included mean, median, standard 609 deviation, and number of observations. Categorical variables were tabulated using frequencies 610 and percentages. The incidence of all reported adverse events and treatment-related adverse 611 events was tabulated by treatment group. Adverse events were also classified by system organ 612 class and preferred term using the Medical Dictionary for Regulatory Activities. Adverse events 613 were to be listed and summarized by treatment group, preferred term, severity, seriousness, and 614 relationship to study drug. In the event of multiple occurrences of the same adverse events with 615 the same preferred term in one participant, the adverse event was counted once as the worst 625 The presence of anti-SARS-CoV-2 IgY in sera of study participants was evaluated using GLP 626 standards in a qualified ELISA assay at Charles River Laboratories (Reno, NV). ELISA 96-well 627 plates were coated with goat anti-chicken IgY (Thermo Fisher; A16056) and, after blocking, 628 were incubated with the samples containing anti-SARS-CoV-2 IgY at various concentrations for 629 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; 1 hour at room temperature. After washing the microplate, rabbit anti-chicken IgY (Thermo CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. (Fig. 2C) . The purified SARS-CoV-2 RBD was eluted as a 671 single peak by analytical size-exclusion chromatography with >95% monomer content (Fig. 2C) . 672 Bacterial endotoxin contamination was determined to be <0.1 EU/mg by Charles River Endosafe 673 LAL cartridge system. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint Integrity of the cell-free (non-glycosylated) SARS-CoV-2 RBD was then verified by 675 kinetic binding to the hACE2 receptor. Binding kinetics and affinity were similar to a 676 mammalian expressed and glycosylated S1 fragment (Fig. 2D ) and were consistent with 677 previously described binding affinities, suggesting the RBD expressed cell-free was properly 678 folded and bioactive. (amino acids 328-533) and mammalian-expressed full-length S1 to the hACE2 using Biacore. 686 Cell-free expressed RBD ( Fig. 2B ; 50 µg in simple oil emulsion) was injected into 9 SPF hens 687 (46-weeks old) and IgY was extracted from egg yolks using a water-based method 2 weeks after 688 the second immunization and thereafter. The IgY preparation was subjected to protein and 689 Western blot analyses (Fig. 3A) . The IgY preparations were >95% pure; a quantitative Western 690 blot analysis demonstrated that this preparation contained less than 2% ovalbumin by weight 691 (Fig. 3A) . Chromatography of the IgY preparations on size-exclusion HPLC identified 5 peaks 692 (Fig. 3B) ; SDS-PAGE and Western blot analysis of the peaks collected between 17 and 30 693 minutes confirmed that these peaks all contain IgY. The anti-SARS-CoV-2 RBD IgY antibodies 694 recognized both the immunogen, cell-free expressed RBD, and the mammalian-expressed full-695 length and glycosylated S1 protein (Fig. 3C) . One egg yolk of the SPF hens provided about 500 696 mg of purified IgY and each of >10 independent batches of IgY, purified from 100 eggs, each 697 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; yielded an average of 47 ± 13 g (SD) of purified IgY (Fig. 3D) . There was limited variability in 698 the affinity of the various IgY batches for the S glycosylated protein as judged by ELISA (Fig. 699 3D; average titer against full-length S1 was 1:18,000). Furthermore, there was almost no 700 difference in titer of individual hens towards the full-length glycosylated S protein, suggesting 701 minimal variability between hens (Fig. 3E, F) . Over 11 months, IgY was collected in batches of 702 100 eggs per preparation with a similar yield of IgY per preparation and a similar response; 703 interruption of immunization for 3 months did not result in a drop in titer (Fig. 3F, right panel) . 704 Approximately 300 µg/mL IgY provided 50% neutralization of the index virus in culture ( is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint IgY preparations were tested. The neutralization activity of the purified IgY, defined as the 728 concentration inhibiting 50% of the viruses (IC50) was ~170 µg/mL (Fig. 3G) . Importantly, ~10-729 fold higher neutralization activity towards the index SARS-CoV-2 virus was observed when 730 using a live index virus (Fig. 3H) . 731 Because at least 13 common variants of SARS-CoV-2 with amino acid mutations in the 732 RBD had emerged since December 2020 [27-29] (Fig. 4A) , we tested the activity of the anti-733 SARS-CoV-2 IgY against several variants (including Beta, Delta, and Omicron; Fig. 4A , B) and 734 D614G, an amino acid substitution outside the RBD that is now found in most variants. Beta, 735 Delta, and Omicron were classified as variants of concern, associated with increased is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint uses the hACE2 receptor to infect human cells [10, 30] , the RBD contains a total of 15 mutations 743 compared to the index virus, 11 of which were not found in the previous variants (Fig. 4A, B) . 744 Yet, the ELISA titer of IgY against the Omicron RBD was also equivalent or slightly better than 745 that towards the RBD of the index virus (Fig. 4D) . 746 Next, we tested the neutralization titer of anti-SARS-CoV-2 RBD IgY (lot Y0180) 747 against the RBD of the index, Alpha, and Beta variants, thus including all the amino acid is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint virus-induced CPE in infected cell monolayers (Fig. 4H ). The human serum had a similar degree 766 of neutralization against the index pseudovirus and Delta (Fig. 4H ) when tested at a dose that is 767 three times higher than that required for 50% neutralization (1:640 in Fig. 4H , middle panels, vs. 768 1:2,000 in Figure 4F ). In contrast, anti-SARS-CoV-2 IgY tested at 400 µg/mL (Fig. 4H, right 769 panels), a dose below that required for 50% neutralization (~650 µg/mL; Fig. 4G is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; (Fig. 5A) survived to scheduled euthanasia with no mortality, test article-related organ weight 811 changes, or gross or microscopic findings (see Fig. 5B ). There were also no differences between 812 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; female and male groups in each treatment arm. The GLP-qualified assay detected no anti-SARS- 813 CoV-2 RBD IgY in the sera of animals after 28 days of daily treatment with 4 mg anti-SARS- 814 CoV-2 RBD IgY (lower limit of detection of 30 ng/mL). 815 There was no evidence of significant systemic immune activation in the rats (20/group) CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; 6 hours apart, at 24 hours after the first treatment (D1H24); 28 days of twice-daily treatments 836 and 4 hours of the treatment of that day (D28H4); and 28 days of twice-daily treatments and 24 837 hours of the treatment of that day (D28H24). Red indicates a statistical difference with a false 838 discovery rate (FDR) significant p-value (p < 0.05). Human tissue cross-reactivity study 840 We determined potential cross-reactivity of the anti-SARS-CoV-2 RBD IgY protein to a full 841 panel of human tissues (at least 3 donors per tissue; see S4 Table for . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; Safety and tolerability 868 The overall incidence of treatment-emergent adverse events was 29% (7 of 24 participants; Table 869 3) in the single-dose part of the study and 58% (14 of 24 participants; Table 4 ) in the multiple-870 dose part of the study, with similar incidence rates between anti-SARS-CoV-2 RBD IgY (42%) 871 and placebo (50%) groups (Tables 3, 4) . The most frequent treatment-emergent adverse event 872 was headache, with similar rates between placebo (17%) and anti-SARS-CoV-2 RBD IgY (14%) 873 (Tables 3,4). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; tested before treatment (D1 pre-dose), 2 hours after dosing on day 1 (D1 H2); day 2, before 893 treatment (D2 pre-dose), and 2 hours after the first dosing on day 2 (D2 H2, Fig. S2 ). There were 894 slight but statistically significant decreases (red histograms) in 10 of the 80 cytokines (Fig. S2) . 895 These slight declines in CCL27, CXCL9, IL23, IL27, LIF, MIP5, RESISTIN, TNFα, TNFβ, and 896 TNFRSF6 were noted only in the 12 mg/day group compared to the pretreatment levels ( Fig. 897 S2 ). These declines also occurred only 2 hours after the first dose (D1 H2) and were not 898 sustained, except for MIP5 (Fig. S2) . There was also a small decrease in IL3, 2 hours after the 899 first dose of 6 mg/day group anti-SARS-CoV-2 RBD IgY, but there were no changes in this 900 cytokine at any other times or doses (Fig. S2) . These slight changes, which were also not CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; including in pediatric populations. Our decision to protect from viral entry at the nasal mucosa 961 stems from the observation that levels of lung hACE2 are much lower than in the nose; infection 962 of lung tissue is >5 orders of magnitude lower compared with nasal mucosa [49, 50] . Therefore, 963 inhibition of viral entry at the nose is likely the correct target site for optimal efficacy. 964 Our product is egg-derived immunoglobulins, which could contain potentially antigenic 965 residual chicken proteins. However, it is not indicated for those who are allergic to egg yolks. 966 Note also that most humans are exposed to egg-derived antigens through their diet and are not 967 allergic. Furthermore, anaphylaxis for those who consume eggs regularly is rare. However, the 968 safety and tolerability of hen-derived IgY as intranasal treatment in humans have not been variants. 995 Our work shows that, although the IgY was raised against the ancestral (index) strain 996 RBD, the repertoire of the antibodies raised in hens was diverse and polyclonal so that binding 997 affinities measured by ELISA for the single (Alpha), double (Delta), and triple amino acid (Beta) 998 substitutions, or the Omicron variant with 15 amino acid substitutions in the RBD, were not 999 different from the affinity for the index RBD or full-length S protein (Fig. 4C, D) . We then 1000 confirmed that there was no difference between Alpha, Beta, and Delta variants, and the index 1001 strain in a neutralization assay in culture using a VSV-S pseudovirus or live virus (Fig. 4E, G) , 1002 whereas a reduced neutralization activity of human serum was observed in side-by-side 1003 experiments (Fig. 4F, H) . The viral neutralization studies reported here were conducted by a 1004 commercial provider (RetroVirox) and by an established laboratory at the USAMRIID, both 1005 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; comparing the results with either convalescent sera or an immunized human, for relative titer glycosylation sites as that used as an immunogen in vaccinated individuals; many studies use the generation is inexpensive and fast; one egg of an SPF hen produces 20-80 daily doses (at 6 production of the cell-free expressed RBD, Roxanne Lopardo at Avian Vaccine Services, 1098 Charles River Laboratories for her work in IgY testing, and David Goodkin for helpful advice 1099 and critical review. We also thank the study staff at Linear Clinical Research Ltd and Resolutum is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. The information contained in this protocol is confidential and intended for the use of the study staff. The information in this document is the property of the sponsor and may not be disclosed unless federal or state law or regulations require such disclosure. Subject to the foregoing, this information may be disclosed only to those persons involved in the study who need to know, with the obligation not to further disseminate this information. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint Background and objectives 2a Scientific background and explanation of rationale 4-7 2b Specific objectives or hypotheses 6-7 Trial design 3a Description of trial design (such as parallel, factorial) including allocation ratio 11-18 3b Important changes to methods after trial commencement (such as eligibility criteria), with reasons NA Participants 4a Eligibility criteria for participants 11 4b Settings and locations where the data were collected 11 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 11-18 6b Any changes to trial outcomes after the trial commenced, with reasons NA Sample size 7a How sample size was determined 16 7b When applicable, explanation of any interim analyses and stopping guidelines 16-17 Randomisation: Sequence generation 8a Method used to generate the random allocation sequence 11-12 8b Type of randomisation; details of any restriction (such as blocking and block size) 11-12 Allocation concealment mechanism 9 Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned 11-12 Implementation 10 Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions Blinding 11a If done, who was blinded after assignment to interventions (for example, participants, care providers, those 11 S1 CONSORT Checklist . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. Sources of funding and other support (such as supply of drugs), role of funders 49 *We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials. Additional extensions are forthcoming: for those and for up to date references relevant to this checklist, see www.consort-statement.org. . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint A S2 Figure . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint S1 IgY SARS-CoV-2 RBD 100 20 3 a 3 a -a = These animals were for cytokine analysis only. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.07.22268914 doi: medRxiv preprint S4 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; University of Oxford. Vaccination by location What it will take to vaccinate the world against COVID-19 A framework 1112 for monitoring population immunity to SARS-CoV-2 Development and deployment of COVID-19 vaccines for those most vulnerable Development of vaccines and antivirals for combating viral 1117 pandemics 1.617.2 Delta variant replication and immune evasion Reduced 1121 neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum National Institutes of Health SARS-CoV-2 Variants & Therapeutics SARS-CoV-2 Omicron strain exhibits 1127 potent capabilities for immune evasion and viral entrance Broadly 1130 neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift Striking Antibody Evasion 1133 Manifested by the Omicron Variant of SARS-CoV-2 SARS-CoV-2 variants in analyzed sequences (by country) Characteristics of SARS-CoV-2 and COVID-19 Structure, function, 1141 and antigenicity of the SARS-CoV-2 spike glycoprotein International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity SARS-CoV-2 Omicron strain exhibits 1143 potent capabilities for immune evasion and viral entrance Antibody responses to viral infections: a structural 1146 perspective across three different enveloped viruses Intranasal antibody prophylaxis for protection against viral disease A simplified and 1150 robust protocol for immunoglobulin expression in Escherichia coli cell-free protein 1151 synthesis systems Microscale to 1153 manufacturing scale-up of cell-free cytokine production -a new approach for shortening 1154 protein production development timelines SAS Institute Inc. 2015. SAS/IML ® 14.1 User's Guide Simulation 1157 of the clinical and pathological manifestations of coronavirus disease Syrian hamster model: implications for disease pathogenesis and transmissibility A single 1161 intranasal or intramuscular immunization with chimpanzee CoV-2 vaccine protects against pneumonia in hamsters International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Establishment of a 1164 well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T 1165 cells with stable expression of ACE2 and TMPRSS2 Oral 1167 administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa 1168 infections in patients with cystic fibrosis: a phase I feasibility study An improved 1171 SUMO fusion protein system for effective production of native proteins Increased elastase 1174 sensitivity and decreased intramolecular interactions in the more transmissible 501Y.V1 1175 and 501Y.V2 SARS-CoV-2 variants' spike protein-an in silico analysis Natural variants in SARS-CoV Spike protein pinpoint structural and functional hotspots with implications for prophylaxis 1179 and therapeutic strategies Reduced 1183 neutralisation of SARS-CoV-2 omicron B.1.1.529 variant by post-immunisation serum 1184 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 31. Chemical Computing Group. Molecular Operating Environment (MOE) Centers for Disease Control and Prevention. Potential Rapid Increase of Omicron Variant 1191 Infections in the United States Chicken antibodies: a clinical chemistry perspective Immunoglobulin Y for 1197 potential diagnostic and therapeutic applications in infectious diseases Avian antibodies (IgY) targeting spike 1200 glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibit 1201 receptor binding and viral replication Anti-SARS-CoV-2 IgY isolated from 1203 egg yolks of hens immunized with inactivated SARS-CoV-2 for immunoprophylaxis of 1204 COVID-19 targeting SARS-CoV-2 S1 as potential virus entry blocker International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Chicken egg yolk antibodies (IgYs) 1209 block the binding of multiple SARS-CoV-2 spike protein variants to human ACE2 Generation of chicken IgY 1212 against SARS-COV-2 spike protein and epitope mapping The preparation of N-IgY targeting 1215 SARS-CoV-2 and its immunomodulation to IFN-γ production in vitro Nasal mucociliary clearance as a 1218 factor in nasal drug delivery Passive immunity in prevention and treatment of infectious 1220 diseases Nasal administration of immunoglobulin as effective 1222 prophylaxis against infections in elite cross-country skiers Effect of treatment with nasal IgA on the incidence of infectious 1225 disease in world-class canoeists Upper respiratory infections in 1227 children: Response to endonasal administration of IGA International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Intranasally administered immunoglobulin for the prevention of rhinitis in children. Pediatr Ragweed hay fever: treatment by local passive administration 1233 of IgG antibody CoV-2 Reverse genetics reveals a variable infection gradient in the respiratory tract Pathogenesis of COVID-19 from a cell biology perspective Egg yolk antibodies for passive immunity Development of Spike 1242 receptor-binding domain nanoparticles as a vaccine candidate against SARS-CoV-2 1243 infection in ferrets Phytopharmaceuticals mediated Furin and TMPRSS2 receptor blocking: can it be a 1246 potential therapeutic option for Covid-19? Use of antimicrobial mouthwashes (gargling) and nasal sprays by healthcare 1249 workers to protect them when treating patients with suspected or confirmed COVID-19 1250 infection International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Intranasal 1252 antiviral drug delivery and coronavirus disease 2019 (COVID-19): A state of the art 1253 review Nasal delivery of an IgM offers 1255 broad protection from SARS-CoV-2 variants Inhalable nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian 1258 hamsters at ultra-low doses Protective effects of sti-1260 2020 antibody delivered post-infection by the intranasal or intravenous route in a Syrian 1261 Vulnerabilities in coronavirus glycan shields despite extensive glycosylation 1268 Induction of hepatitis C virus E1 envelope protein-specific immune response can be 1269 enhanced by mutation of N-glycosylation sites Chicken egg yolk antibodies (IgY-technology): a review of progress in 1272 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) production and use in research and human and veterinary medicine Supporting information captions 1276 S1 Protocol. Phase 1 Clinical Trial Protocol Rat toxicity study protocol Bioanalytical sample collection from the rat toxicity study Cytokine sample collection for the rat toxicity study Human tissue cross-reactivity Efficacy study in hamsters. Placebo control and IgY-treated Syrian hamsters were 1283 challenged with 4 x 10 6 or 8 x 10 5 PFU SARS-CoV-2 Each symbol is a single animal. One animal in the placebo group challenged with a 1288 low dose of the virus did not have any detectable virus in the lung homogenate. This was ruled 1289 an outlier based on the viral RNA levels in other assays. Viral RNA levels were assessed by RT-1290 qPCR in lung homogenates of nasal swabs using 2 different primer-probe sets Blood cytokine levels before treatment (D1 pre-dose), 2 hours after the first 1294 treatment (D1H2), 24 hours after 3-times daily intranasal (D2 pre-dose), and 2 hours after the 1295 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) first dose on the second day (D2H2) of anti-SARS-CoV-2 Data are adjusted for nonspecific binding and plate artifacts plus the effects of individual 1297 persons. Red indicates a statistical difference with a false discovery rate (FDR) significant p-1298 value We are also grateful for 1302 the financial support from the Moonchu Foundation, the Human Immune Monitoring Center 1303 (HIMC) at Stanford University, and the generous monetary donations of many others. The 1304 funders had no role in study design, data collection and analysis John 1323 Investigation: Michaela Lucas, Nastassja Ortega-Heinly, Gang Yin Writing -original draft: Daria Mochly-Rosen, Lyn R. Frumkin 1332 Writing -review -all authors 1094 We thank our many SPARK at Stanford advisors for their support, both financially and through 1095 their invaluable advice. We wish to thank Miao Wen, Nina A. Carlos, Lawrence Huang, Cuong 1096 Tran, Stephanie Armstrong, and Daniel Calarese of Sutro Biopharma for their help in the evaluation. 1007 The culture neutralization titer of anti-SARS-CoV-2 RBD IgY is lower than the human 1008 anti-SARS-CoV-2 sera (e.g., Fig. 4F vs. E). However, this may reflect the need for protease- 1009 induced RBD exposure in the full-length S protein for binding by IgY, which might not occur 1010 effectively in the culture model. A comparison of titer values between our product and sera from 1011 an immunized person can also be calculated based on values of IgG levels in human sera (~15 1012 mg/mL); a titer of 1:2,000 ( Fig. 4F ; dashed line) is equivalent to ~7 ug/mL or 100-fold higher 1013 IgG titer than our IgY ( Fig. 4E ; ~600 ug/mL). However, as the dose of the IgY anti-SARS-CoV-1014 2 preparation in humans is planned to be 4 mg/dose, more important is the equal potency of the 1015 IgY towards the various variants when used even at ~1/10 of the intended IgY dose (Fig. 4H) . 1016 Another potentially important difference between our findings and the previously 1017 published study is the antigen used to raise the antibodies. When expressed in mammalian cells, 1018 the full-length S1 protein has at least 22 glycosylation sites per S monomer [59] . As glycosylated 1019 amino acids are more immunogenic, the affinity of the human antisera may reflect binding to the 1020 glycosylated determinants of the protein. However, as glycosylation sites in the S1 protein are 1021 heavily mutated and new sites may be formed in many of the variants [28], immune reactivity 1022 that is biased towards glycosylated sites may lead to loss of activity as the virus mutates. This original viral isolates rather than the common current variants. 1030 An important feature of our product is the ease of developing prophylaxis that can be 1031 quickly and inexpensively produced. We found that 24 mg total daily dose (divided into three 1032 equal doses) of intranasal anti-SARS-CoV-2 IgY for 14 days had an excellent safety profile in 1033 humans; this daily dose represents ~1/20 of one egg of immunized SPF hen and ~1/5 of an egg 1034 of commercial hen, underscoring that such an IgY dose is feasible for both production cost and 1035 effort. 1036 There are some limitations to our studies. Our phase 1 clinical trial in healthy volunteers 1037 was to assess initial safety, tolerability, and PK of anti-SARS-CoV-2 IgY and was not designed 1038 to evaluate efficacy. In addition, we were unable to obtain in vivo data showing viral 1039 neutralization (Fig. S1. ). This may reflect using too much virus in this animal model of COVID-1040 19; our study used 8 x 10 5 or 4 x 10 6 , vs. 1 or 5 x 10 4 TCID50 [55, 58] . In addition, our intranasal 1041 formulated IgY preparation was viscous to obtain better delivery in humans. As hamsters are 1042 obligatory nose-breathers, they may have blown out the formulated IgY. Finally, the virus that 1043 was delivered in 50 µL of liquid directly into each nare may have washed out some antibodies. 1044 Another limitation in our study is that the neutralization studies comparing the hen IgY vs. 1045 human sera were not comprehensive and included only 1 or 2 human samples. Nevertheless, our 1046 study is the first to demonstrate the broad selectivity of anti-SARS-CoV-2 IgY against all the 1047 current variants of concern and a favorable safety profile when used chronically as intranasal 1048 drops in rats and humans. 1049 There are also several advantages for the use of IgY as prophylaxis against other 1050 pathogens besides SARS-CoV-2 that cause disease in humans. As we noted above, IgY 1051 1052 mg/dose) within 3 weeks from the first injection (1 week after the first boost). We found a 1053 limited variability between individual immunized hens as determined by ELISA and Western 1054 blot analyses and a batch-to-batch consistency (Fig. 3D-G.) . IgY is also easy to distribute; 1055 besides the known long-term stability of purified IgY [61], we also found excellent stability of 1056 the formulated material at 2-8°C for at least 6 months (maximum time point measured so far) and 1057 greater than 2 weeks when stored at room temperature. This is in contrast to vaccines, some of The current COVID-19 pandemic illustrates the need for prophylactics that can be 1075 produced rapidly at low cost, are technically accessible anywhere in the world, and complement 1076 traditional vaccine development. The safety and benefit of IgY and the ease to produce it at low 1077 cost are well described for animal farms. In contrast, the clinical adaptation of IgY for human use 1078 has been slow, likely hampered by a lack of intellectual property that has hindered commercial 1079 development by industry. For that reason, we undertook the effort of establishing the ability of 1080 anti-SARS-CoV-2 IgY to neutralize variants of concern and the initial safety of the IgY 1081 preparation using industry GLP and GMP standards. We suggest that until vaccination that is 1082 highly effective against prevalent variants becomes available worldwide or herd immunity is 1083 achieved, intranasal delivery of anti-SARS-CoV-2 IgY may provide passive immunization, 1084 including for use as an add-on to personal protective equipment and other preventive measures 1085 for the general population. This IgY may also provide short-term protection in addition to 1086 vaccines in less well-ventilated environments, including in trains, airplanes, lecture halls, etc. We 1087 also suggest that this approach has the potential to provide a means to curb new threats of 1088 epidemics by airborne infectious agents; by providing the relevant immunogen for hen 1089 immunization at the geographical site where the threat was detected, an effective passive 1090 immunity can be initiated locally to stop the spread of the airborne infectious agent before it 1091 becomes an epidemic. We hope that this study will trigger further work to evaluate the safety and