Three mono or bi-cistronic plasmid constructs, pGO-1001, pGO-1002, and pGO-1003, were designed encoding sequence gene cassettes for SARS-CoV-2 Spike, Spike plus ORF3a, and ORF3a, respectively ( Figure 1A ). The consensus sequences for Spike and ORF3a were generated from publicly available SARS-CoV-2 sequences through mid-February 2020 that were subsequently codon-optimized. For the bi-cistronic construct pGO-1002, a furin cleavage site and a self-cleaving T2A peptide sequence were added between the Spike and ORF3a coding sequences to ensure both proteins were generated after expression and translation of the antigen cassette. An IgE leader sequence was added upstream of the antigen cassettes in each construct. All constructs were made in the pGLS-101 expression vector, including an upstream human cytomegalovirus enhancer/promoter (6).

To confirm that each antigen was adequately expressed, lysates collected from 293T cells transfected with each of the three plasmids were subjected to Western blot and immunofluorescent analysis (IFA) using antibodies specific to SARS-CoV-2 Spike and ORF3a. Western blot analysis showed that lysates from cells transfected with either mono-cistronic plasmid (pGO-1001 or pGO-1003) contained bands of the expected size for Spike (~140 kDa) or ORF3a (~31 kDa). Lysates from cells transfected with the bi-cistronic pGO-1002 construct had bands of the anticipated sizes of both Spike and ORF3a ( Figure 1B ). In addition, IFA of 293T cells detected both Spike and ORF3a proteins in pGO-1002 transfected cells ( Figure 1C ). These data indicate that the production of SARS-CoV-2 Spike and/or ORF3a occurred as expected from each vaccine construct.

Safety and immunogenicity of the three candidate vaccines were first evaluated in mice. Groups of five mice (7 weeks of age) were immunized intramuscularly (IM) in the anterior tibialis muscle with 50µg of one of the three vaccine plasmids or empty pGLS-101 ( Supplementary Figure S1A ). Electroporation (6 pulses at 200V/cm, 50ms, 1Hz) was applied to the injection site using the BTX ECM 830 device. Animals were immunized twice, 14 days apart, and bled at days 0, 14, and 28 to collect sera for immunoassays. Animals were sacrificed on day 28 to obtain spleens for evaluation of cellular responses to the vaccine antigens. All animals remained healthy, without signs of illness and had similar weight gains through the study ( Supplementary Figure S1B ).

The presence of antibodies to the Spike S1 subunit and to ORF3a in sera were determined by ELISA. A single immunization with pGO-1001 or pGO-1002 induced full seroconversion to Spike in both groups, and equivalent endpoint titers (~10⁴) were seen in both groups by 2 weeks post second immunization ( Figure 2A ). Two immunizations with either pGO-1003 or pGO-1002 were required for full seroconversion to ORF3a and yielded endpoint titers after the 2^nd immunization of 3x10³ and ~10³, respectively ( Figure 2B ).

To assess the humoral responses’ functionality, we performed surrogate neutralization assays and/or traditional plaque reduction neutralization titer (PRNT₅₀) assays using sera collected on day 28 (2 weeks post-second injection) from immunized mice. The surrogate neutralization assay measures the ability of sera to inhibit binding between a recombinant SARS-CoV-2 Spike RBD protein and plate-bound ACE2 cellular receptor protein. In this assay, sera from mice receiving two immunizations of either pGO-1001 or pGO-1002 inhibited RBD-ACE2 binding by over 80% ( Figure 2C ). PRNT₅₀ assays were performed under BSL3 conditions by the Korean Center for Disease Control (KCDC). They measured the ability of each serum to block in vitro infection of Vero cells by the SARS-CoV-2 clinical isolate β-CoV/Korea/KCKC03/2020. In this assay, sera from mice immunized twice with either pGO-1001 or pGO-1002 neutralized infection to an equivalent level (PRNT₅₀ titer ~100) while sera from mice immunized with pGO-1003 were unable to neutralize infection ( Figure 2D ). These results show that immunization with pGO-1002 induces humoral responses to both encoded SARS-CoV-2 antigens, Spike and ORF3a, and the induced responses to Spike are functional toward blocking binding between the Spike RBD and ACE2 as well as neutralizing infection by a clinical SARS-CoV-2 isolate.

Induction of cellular responses to Spike and/or ORF3a was evaluated by performing an IFN-γ ELISpot assay on splenocytes isolated from spleens collected from immunized mice on day 28. Splenocytes were stimulated in vitro with each of four linear pools of peptides spanning the full length of Spike and a pool of linear peptides spanning the full length of ORF3a. Mice immunized with pGO-1001 generated strong cellular responses to each Spike peptide pool, while mice receiving pGO-1003 developed cellular responses to the ORF3a peptide pool. Mice immunized with bi-cistronic construct pGO-1002 generated T cell responses against both the Spike peptide pools and ORF3a peptide pool that mirrored those seen against each peptide pool in mice immunized with pGO-1001 or pGO-1003 ( Figure 2E ). These results show that the bi-cistronic construct pGO-1002 induced equivalent humoral and cellular responses to Spike and ORF3a as induced by mono-cistronic constructs of each antigen.

These results establish that pGO-1002 induced Spike- and ORF3a-specific humoral and cellular immune responses with similar kinetics and magnitude as induced by monocistronic constructs of each antigen. Based on these results we selected to advance pGO-1002 for further evaluation.

In anticipation of future clinical investigation of pGO-1002, we moved to investigate pGO-1002 immunogenicity after intradermal (ID) vaccination as this route is more accessible and more tolerable than intramuscular injection. In a recent study, we developed a new ID delivery protocol for DNA plasmids that involves the application of suction to the ID injection site which they found led to more rapid and robust transgene expression in rat skin (32). Furthermore, they demonstrated that ID immunization with a DNA vaccine followed by suction applied by our GeneDerm device led to more rapid and stronger antibody responses in rats. Here we assessed the immunogenicity of pGO-1002 following GeneDerm-assisted ID delivery in Sprague Dawley (S.D.) rats. Groups of five rats were immunized twice, 2 weeks apart, with either 30µg or 300µg of pGO-1002 or 300µg of empty pGLS-101 by ID injection followed by GeneDerm-applied suction (30 seconds, 65 kPa) at the injection site ( Figure 3A ). Blood was collected from all animals at days 0, 14 (week 2), and 28 (week 4) for sera to evaluate humoral responses, and animals were sacrificed at day 28 to obtain spleens for evaluation of cellular immune responses.

Rats with antibodies to SARS-CoV-2 Spike as measured by S1 ELISA were seen after a single immunization of 30μg or 300μg pGO-1002 and the S1 titers were boosted following a second injection of either dose ( Figure 3B ). All five animals that received two immunizations of 300μg pGO-1002 seroconverted to Spike and had a Spike S1 endpoint titer of ~1.31x10⁴ while four of five animals that received two immunizations of 30μg pGO-1002 seroconverted with a log lower Spike S1 endpoint titer than seen in the 300μg group. The kinetics and Spike antibody endpoint titers in rats receiving pGO-1002 by GeneDerm-assisted ID delivery matched those seen when 300μg pGO-1002 was administered using PharmaJet or EP-enhanced ID delivery ( Supplementary Figure S2A ). Antibody responses to ORF3a were detected in some animals only in the group receiving two immunizations with 300μg pGO-1002 (data not shown). Sera collected on week 4 from pGO-1002 immunized rats were next evaluated in surrogate neutralization and PRNT₅₀ assays. Sera from rats that received two 30μg doses showed low activity in both the surrogate neutralization and PRNT₅₀ assays ( Figures 3C, D ). By contrast, sera from rats receiving two 300μg doses of pGO-1002 inhibited RBD-ACE2 binding by 70% in the surrogate neutralization assay, and neutralized in vitro infection by the SARS-CoV-2 clinical isolate β-CoV/Korea/KCKC03/2020 with an average PRNT₅₀ titer ~220 ( Figures 3C, D ). Surrogate neutralization and PRNT₅₀ titers observed in rats immunized by GeneDerm-assisted ID delivery matched those seen in rats that received pGO-1002 by PharmJet or electroporation enhanced ID delivery ( Supplementary Figures S2B , S2C ).

An IFN-γ ELISpot was next performed on splenocytes collected from pGO-1002 immunized rats to evaluate the induction of cellular responses to Spike and ORF3a ( Figure 3E ). Splenocytes isolated on week 4 were stimulated in vitro with each of four linear pools of peptides spanning the full length of Spike and a pool of linear peptides spanning the full length of ORF3a. GeneDerm-assisted ID delivery of pGO-1002 to rats induced robust total cellular responses to S and ORF3a (~650 SFU/10^6 splenocytes). Total cellular responses induced by GeneDerm-assisted ID delivery were higher than cellular responses seen when pGO-1002 was administered ID by Pharmajet or by EP-enhanced ID injection ( Supplementary Figure S2D ). Notably, T cell responses for animals immunized with 30µg of pGO-1002 by ID+GeneDerm induced an equivalent cellular response as seen in the group receiving 300µg by ID+GeneDerm. Broad reactivity to multiple regions of Spike and ORF3a were seen in pGO-1002 immunized rats. Isotyping of Spike-specific antibodies in rats immunized with pGO-1002 by ID+GeneDerm showed that they were predominantly of the IgG2a/2b subtype consistent with an induction of a Th1 profile of T cell responses by pGO-1002 ( Supplementary Figure S3 ).

These results confirm that GeneDerm-assisted ID delivery of pGO-1002 induces strong cellular responses to Spike and ORF3a as well as humoral responses capable of blocking Spike RBD-ACE2 binding and neutralizing SARS-CoV-2 infection.

Based on the positive immunogenicity results in mice and rats, pGO-1002 was reformulated with 1X SSC buffer and designated as GLS-5310. The ability of GLS-5310 to provide protection against SARS-CoV-2 was first evaluated in a Syrian hamster challenge model. Groups of six hamsters were immunized intradermally (ID) twice with 150μg of GLS-5310 or empty pGLS-101 vector on days 0 and 21 (week 3) followed by GeneDerm-applied suction (30 seconds, 65 kPa). Sera were collected from immunized animals on days 0, 21 (week 3), and 41 (week 6). All animals were challenged on day 42 with either a wild type (WT;2019-nCoV/USA-WA1/2020) SARS-CoV-2 strain or the B.1.351 (Beta; 2019-nCoV/South Africa/KRISP-K005325/2020) SARS-CoV-2 variant strain. Challenged animals were sacrificed on day 47 (5 days post-challenge) to collect blood, lung, and nasal turbinate tissues.

Sera collected from immunized animals were evaluated in a modified high-throughput surrogate neutralization assay that uses homogenous time resolved fluorescence (HTRF) technology, where SARS-CoV-2 spike RBD from WT (USA-WA1/2020), Beta (B.1.351), or Delta (B.1.617.2) variants were assessed for ability to interact with ACE2 protein. In this assay, initial experiments showed that 50% human control plasma (i.e., from individuals without SARS-CoV-2 exposure or COVID-19) had no effect on the HTRF signal, while the control inhibitory antibody REGN10933 (casirivimab) blocked HTRF signal due to WT, Beta, or Delta variant RBDs in the presence of human plasma with half-maximal inhibitory concentrations (IC50s) of 47.3, 937.5, and 35.4 ng/mL, consistent with previous reports (33, 34). Serial dilutions of day 0 and 41 sera from hamsters were then evaluated for their ability to inhibit binding between ACE2 and the RBD variants of SARS-CoV-2. Humoral responses in day 41 sera collected from GLS-5310 immunized hamsters were able to inhibit binding between ACE2 and the RBDs of WT or Delta variant SARS-CoV-2 but were unable to block binding between the ACE2 and the RBD from the Beta SARS-CoV-2 variant ( Figure 4A ). Hamsters immunized with GLS-5310 completely cleared the infectious virus in the lungs and substantially reduced virus levels in the nares 5 days post-challenge with the wt. SARS-CoV-2 strain ( Figure 4B ). Hamsters immunized with GLS-5310 and challenged with the Beta SARS-CoV-2 variant also had no infectious virus in their lungs 5 days post-challenge ( Figure 4C ).

A separate group of hamsters were immunized twice, 21 days apart with 150μg of GLS-5310 by ID+GeneDerm and 150μg GLS-5310 by intranasal administration and then challenged 3 weeks later (day 42) with wt. SARS-CoV-2. Sera collected on day 41, one day prior to challenge, was able to inhibit binding between ACE2 and the RBDs of wt. or Delta variant SARS-CoV-2, but not between ACE2 and the RBD of the Beta SARS-CoV-2 similar to what was observed in animals immunized with GLS-5310 by ID+GeneDerm alone ( Figure 5A ). In addition, of five hamsters immunized with GLS-5310 by this ID+GeneDerm/IN route, one demonstrated breakthrough infection in the lungs 5 days post-challenge with wt. SARS-CoV-2, whereas TCID₅₀ for the other four animals were below the limit of detection ( Figure 5B ).

Immune serum was then further assessed for live-virus neutralization and inhibition of cytopathic effect (CPE). Sera collected from a hamster immunized with pGLS-101 and a hamster immunized with GLS-5310 on day 41 were evaluated for their ability to block SARS-CoV-2 replication and cytopathic effect (CPE) in Vero-E6 cells. Dilutions of heat-inactivated immune sera collected from each animal were pre-incubated with wt SARS-CoV-2 and then added to Vero-E6 cell cultures. In these studies, sera from the pGLS-101 injected hamster was unable to block virus-induced CPE and loss of viability, while day 41 immune sera from a hamster immunized with GLS-5310 reduced virus-induced CPE with dose-dependence, as confirmed by direct visual imaging, and maintained or improved infected cell viability, as monitored by resazurin viability dye ( Figures 6A, B ). These results support that GLS-5310 immunization induces immune responses capable of blocking SARS-CoV-2 infection in hamsters.

Human embryonic kidney cells 293T (HEK 293T; ATCC, cat#CRL-3216) were obtained from ATCC and cultured in Dulbecco’s Modified Eagle Media (DMEM; Welgene, cat#LM001-05) supplemented with 10% fetal bovine serum (FBS; Welgene, cat#S101-07). Antibody to SARS-CoV-2 Spike was obtained from Sino Biological (cat#40591-MM42). Human hepatoma cell line Huh-7 (ATCC, CVCL-0336) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at a 37°C incubator, respectively (6). Antibody to SARS-CoV-2 ORF3a obtained from immunized mice serum. Antibody to β-actin was obtained from Abcam (cat#ab8226).

Male, 7-week-old BALB/c mice were purchased from RaonBio (Seoul, Korea). Female, 6- to 8-week-old Sprague Dawley rats were purchased from OrientBio (Seoul Korea). Male and female Syrian golden hamsters (6– to 8-week-old) were acquired from Charles River Laboratories and housed at Bioqual, Inc., for the duration of the study. All animal testing and research complied with relevant ethical regulations and studies received approval from each institution’s IACUC. For mouse studies, 50µg empty vector or vaccine plasmids were administered intramuscularly twice, 2 weeks apart, by needle into the tibialis anterior (T.A.) muscle and then in vivo electroporation (EP) was applied to the injection site using BTX ECM 830 series device BTX, MA, USA. The machine was set to deliver six pulses at 200V/cm, 50ms, 1Hz. Mice were weighed starting 1 week prior to first immunization and then every 3 to 4 days until mice were sacrificed on day 28 post injection to obtain spleens for analysis of cellular immune responses. On days 0, 14, and 28, blood was collected from mice by tail vein sampling.

For intradermal (ID) immunizations, rat skin was first prepared by gentle clipping hair in a spot on the backs of animals and then a depilatory cream was applied to spot to remove remaining hair. Control (pGLS-101; 300µg) or pGO-1002 (30 or 300µg) plasmid was administered using one of three methods; (1) plasmid (100µl) was injected to the shallow intradermal space using a tuberculin syringe (BD, Cat.328820) followed by application of 65 kPa suction for 30 sec using GeneDerm device such that the bleb induced by injection was contained within the opening of the cap of the device (32); (2) plasmid (100µl) was injected using PharmaJet Tropis^® needle-free device following manufacturer’s protocol; or (3) plasmid (100µl) was injected to the shallow intradermal space using a tuberculin syringe and electroporation 200V/cm, 50ms, 1 Hz, 6 pulses, 1s interval (BTX) was applied with a EP device.

Hamsters were vaccinated in a manner similar to rats with either pGLS-101 or GLS-5310. GLS-5310 is a formulated drug product of pGO-1002 at a concentration of 6 mg/mL in 1X saline sodium citrate (SSC) buffer for human use. Prior to use, vaccine was diluted with 1X SSC. For animals vaccinated ID only, 150 µg of pGLS-101 or GLS-5310 vaccine in a 50µl volume was administered whereas for animals vaccinated ID and IN 150 µg of vaccine in a 50 µl volume was administered to both sites for a total dose of 300µg.

Cultures of HEK293T cells were transfected using PEI MAX transfection reagent (Polysciences, Inc., cat# 24765-1, following manufacturer’s protocol) with 100µg empty pGLS-101 vector or one of the three vaccine candidate plasmids (pGO-1001, pGO-1002, pGO-1003). Each cell culture was lysed 48 hours later in 1% Triton X-100 buffer. Proteins in lysates were separated by SDS-PAGE (Bio-Rad, Mini-PROTEAN Tetra System) and then transferred to PVDF membrane. Membrane was blotted with antibodies to SARS-CoV-2 Spike (1:5000 dilution); ORF3a (1:200 dilution), and β-actin (1:1000 Dilution) followed by blotting with horseradish peroxidase (HRP)-conjugated anti-mouse IgG. Blots were developed using ECL system (Biomax, cat# BWD0100).

Cultures of Huh7 cells were transfected with pGLS-101 or pGO-1002 plasmids as described above. Forty-eight hours post transfection cells were washed twice in 1×PBS and fixed in 4% v/v formaldehyde (Fisher) supplemented with 0.0075% v/v glutaraldehyde (Sigma) in PBS for 30 minutes at room temperature. Cell membranes were permeabilized in 0.1% Triton X-100 in PBS for 30 minutes. Autofluorescence was quenched using 0.1 M glycine in PBS for 15 minutes Blocking was performed with 3% w/v bovine serum albumin (BSA) for at least 1h at room temperature, were incubated with primary antibodies for 90 minutes at room temperature or overnight at 4°C, Immunofluorescence labeling was performed by incubation with the mouse anti-SARS-CoV-2 Spike antibody or anti-SARS-CoV-2-ORF3a antibody followed incubation with goat anti-rabbit IgG-Alexa 488 and goat anti-mouse IgG-Alexa 568 antibodies. DAPI panels show control staining of cell nuclei (6).

Corning ELISA plates (cat #3366) were coated with recombinant protein antigens in phosphate-buffered saline (1x PBS) overnight at 4°C. Plates were blocked with blocking buffer (Seracare, Cat.5140-0011) for 2 hours at 37°C. Plates were then washed and incubated for 2 hours at 37°C with 50µl of 4-fold serial dilutions of mouse or rat sera made in blocking buffer. Plates were again washed and then incubated for 1 hour at room temperature (R.T.) with horse radish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rat IgG secondary antibody diluted in blocking buffer. After final washes, plates were developed using ABTS^® Peroxidase Substrate System (KPL, cat. 5120-0032) and the reaction stopped with ABTS^® Peroxidase Stop Solution (KPL, cat. 5150-0017). Plates were read at 405 nm wavelength within 30 minutes using a Synergy HTX (Bio Tek Instruments, Highland Park, VT). Calculation of cutoffs for endpoint titer determined using formula: Day 0 of each group; (each value - blank) Avg +(1.923 x SD) as previously described (61).

For mouse sera ELISA, wash buffer was 1x PBS with 0.05% Tween 20 (diluted Phosphate-Buffered Saline (10X) 1:10 (v/v) and Tween 20 1:20 (v/v) with deionized water), binding antigens tested included recombinant S1 subunit of SARS-CoV-2 Spike (1µg/ml; Sino Biological, 40591-V02H) or internally purified recombinant SARS-CoV-2 ORF3a protein (1µg/ml), and secondary antibody was HRP-goat anti-mouse IgG (H+L) (diluted 1:2500; Invitrogen, Cat# 31430). For rat sera ELISA, wash buffer was 1x PBS with 5% Tween 20, binding antigens tested included recombinant S1 subunit of SARS-CoV-2 Spike (4µg/ml) or recombinant SARS-CoV-2 ORF3 protein (5µg/ml; Bioworld technology, NCP0026P), and secondary antibody was HRP-goat anti-rat IgG (H+L) (diluted 1:2500 for S1 ELISA and 1:1000 for ORF3 ELISA; Sigma, Cat. A9037).

Spleens collected from mice and rats were collected individually and processed into single cell suspensions in R10 media: RPMI1640 media (Gibco, Cat 11875135) supplemented with 10% FBS (Welgene, Cat S101-07) and (1%) penicillin/streptomycin (Gibco, cat 15140-122). Cell pellets were re-suspended in 5 mL of ACK lysis buffer for 8 minutes at R.T. and then R10 was added to stop the reaction. The samples were again centrifuged at 1400 rpm for 5 minutes and cell pellets were re-suspended in R10 and counted. ELISpot assays for mice and rat splenocytes were performed using Mouse IFN-γ or Rat IFN-γ ELISpotPLUS plates (MABTECH, cat. 3221-4APW-10 and 3220-4APW-10respectively) following manufacturer’s protocol. Basically, 96-well ELISpot plates were precoated with INF-α capture antibody and blocked with R10 medium for 1hr at room temperature. 200,000 mouse or rat splenocytes were plated into each well and stimulated for 14 hours with: (1) one of 5 pools of 15-mer peptides overlapping by ten amino acids spanning the SARS CoV-2 Spike (Genscript); or (2) ORF3a (Mimotopes). Cells were stimulated with a final concentration of 1µg/ml of each peptide per well in R10 media. The spots were developed based on manufacturer’s instructions. R10+5% DMSO (Sigma, # D2650) and Concanavalin A (Con-A; Sigma, cat#: C5275) were used for negative and positive controls, respectively. Spots were scanned and quantified by ImmunoSpot CTL reader. Spot-forming unit (SFU) per million cells was calculated by subtracting the R10+5% DMSO control wells. The cutoff values were determined as previously described using the following equation: Cutoff = Mean of Spot numbers in overlapping peptides (OLPs) + (Standard deviation of spot numbers in OLPs * 2 with the sample used for cut off coming from splenocytes of unvaccinated mice or rats (6, 61).

SARS-CoV-2/South Korea/2020 isolate neutralization assays were performed at Korea Centers for Disease Control and Prevention. Neutralizing virus titers were measured for mice or rat sera that had been heat-inactivated at 56°C for 30 minutes SARS-CoV-2 (βCoV/Korea/KCDC03/2020) was diluted to a concentration of 60 pfu/ml and mixed 50:50 in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100μg/ml streptomycin, with serial serum dilutions from 1:4 to 1:2040 in a 96-well U-bottomed plate. The plate was incubated at 37°C in a humidified box for 1 hour before the virus was transferred into the wells of 12-well plate that had been seeded the previous day at 2.5 × 10^⁵ Vero cells (ATCC, CCL-81) per well in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100μg/ml streptomycin. Virus was allowed to adsorb at 37°C for 1 hour and then plaque assay overlay media (2× MEM/1.5% Agar/4% FBS final) was added to cells. After 3 days incubation at 37°C in a humidified box, the plates were fixed, stained and plaques counted. Cytopathic effect (CPE) of each well was recorded under microscopes and the neutralizing titer was calculated by the dilution number of 50% protective condition (inverse of the dilution of sera where plaques first appeared). PRNT₅₀ values were determined by the following equation: PRNT₅₀=DL + ((PRNT₅₀-PL)(DH-DL)/(PH-PL))-DL: plaque is the reciprocal of the lower dilution bracketing the 50% endpoint.-DH: is the reciprocal of the higher dilution bracketing the 50% endpoint; -P50: is the number of plaques at the 50% endpoint; PL.: is the number of plaques at the lower dilution bracketing the 50% endpoint; PH.: is the number of plaques at the higher dilution bracketing the 50% endpoint.

A surrogate neutralization assay was performed on immune sera from mice and rats to detect whether circulating antibodies could block the interaction between the receptor binding domain (Genscript, Cat #: L00847) of the SARS-CoV-2 Spike glycoprotein with the ACE2 cell surface receptor (Genscript, cat#: L00847 or Ray Biotech, Cat# CoV-ACE2S2). In separate tubes, diluted positive control (Genscript, Cat#L00847), diluted negative control (Genscript, Cat#L00847), and the sera samples were mixed with diluted HRP-RBD solution (concentration 1:999) at a volume ratio of 1:1. Mixtures were incubated at 37°C for 30 minutes, and then 100µl each of the positive control mixture, the negative control mixture, and the sera sample mixture was added to the corresponding wells that were pre-coated with recombinant ACE2 protein. Plate was sealed and incubated at 37°C for 15 minutes. Plate seal was removed, and plate washed four times with 260µl of 1x Wash solution and then blotted dry. Then 100µl of TMB Solution (Genscript, Cat#L00847) was added to each well of plate which was then incubated in the dark at 20-25°C for 15 minutes before 50µl of Stop solution added to wells to quench the reaction. Absorbance at 450nm for wells of plate read immediately using the microtiter plate reader (Biotek, Synergy^™ HTX). Cutoff values were determined by the following equation: Inhibition = (1-O.D. Value of sample/OD value of Negative Control) * 100%. The cut off value is based on validation with Genscript panel of confirmed COVID-19 patient sera and healthy control sera. Values over 20% constituted a positive result meaning SARS-CoV-2 neutralizing antibody was present in the sera sample while vales under 20% constituted a negative result meaning no detectable SARS-CoV-2 neutralizing antibody was present.

Plasma samples were serially diluted 1:2 in assay buffer (25 mM Tris, pH 7.4, 150 mM KCl, 0.05% CHAPS, 0.1% BSA), and dilutions were pre-incubated with 2 nM HIS-CoV-Spike RBD (either wild-type, Beta, or Delta variants, Sino Biological) prebound to 600 ng/mL anti-HIS-d2 HTRF acceptor (PerkinElmer) in a total volume of 10μL of assay buffer in white, low-volume 384 well plates. After 1 hour, assays were initiated by adding 5μL of 6 nM biotin-ACE2 (2 nM final concentration, Acros BioSciences) prebound to 50 ng/mL streptavidin-terbium HTRF donor (PerkinElmer). After an additional 2 hours incubation, HTRF signals were measured using a ClarioStar plate reader (BMG LabTech) at 320 nm excitation and 620/665 nm emission with a 50μs delay and window time of 200μs. The raw data at each wavelength were then converted to the HTRF ratio by RFU 665/RFU 620 x 10000. Ratio values were then converted to percent inhibition, where 0% was equal to the HTRF value in the absence of plasma and 100% was equal to the HTRF value in the absence of HIS-CoV-Spike RBD. To calculate IC50s, percent inhibition values were then fit to a 4-parameter dose-response curves using the dilution factor as the X coordinate, the top parameter fixed to 100% and the slope constrained between 1-2. REGN10933 was obtained from excess aliquot volumes at the Perelman School of Medicine, University of Pennsylvania, PA, USA which could not be used for patients (a gift from Dr. Pablo Tebas).

Day 41 sera from a pGLS-101 and GLS-5310 immunized hamster were heat-inactivated at 56°C for 30 minutes, serially diluted 2-fold from 1:20-1:1280 in DMEM+2% FBS, and then incubated with 100x TCID₅₀ of USA-WA1/2020 SARS-CoV-2 for 1 hour before addition to cultures of 20,000 Vero-E cells plated 24 hours previously in 96 well plates. For each experiment, all culture conditions were performed in duplicate. After 72 hours, cell cultures were imaged at 20X magnification and then treated with resazurin (Sigma-Aldrich) to a final concentration of 20μg/ml. Cells were incubated for an additional 4 hours before fluorescence intensity was measured using a ClarioStar plate reader (BMG Labtech). Background fluorescence was subtracted from wells containing resazurin and media but no cells.

Syrian hamsters were immunized on study days 0 and 21 via ID/suction route (or IN depending on the groups being used) and challenged on Study Day 42, via intranasal (IN) route, with 6x10³ PFU of SARS-CoV-2 (2019-nCoV/USA-WA1/2020, LOT #12152020-1235, derived from BEI seed stock Cat # NR-52281 Lot # 70036318) or 3.67x10² PFU of SARS-CoV-2-RSA (2019-nCoV/South Africa/KRISP-K005325/2020 in Calu-3, LOT 030621-750, derived from BEI seed stock cat # NR-54974, Lot # 70041987) challenge dose per hamster. Prior to challenge procedure, the animals were anesthetized by injecting 80 mg/kg ketamine and 5 mg/kg xylazine via the intramuscular (IM) route. The animals were injected with an antisedan, at 1 mg/kg via IM route, 20 minutes post challenge. All animals were monitored until completely recovered. Post-challenge, animals were observed for weight loss and clinical symptoms of disease until the terminal day, study day 5 post-challenge, when hamsters were euthanized, and tissues collected for viral RNA quantification and histopathology analysis. Differences viral load between experimental groups of animals were analyzed statistically using t-test.

Statistical analyses were conducted using Student’s t test in Prism (version 9) software (GraphPad). Adjusted probability p values (p) of smaller than 0.05 was considered statistically significant.