Study design and participants

Initial study of a phase 2 randomized, observer-blind, placebo-controlled study in healthy adults between 19 and 75 years old, who did not have known prior COVID-19 infection or vaccination (based on interviews, and not any specific screening for pre-existing SARS-CoV-2 antigens or antibodies), was carried out between July and October 2021 at 5 hospital sites in South Korea. Written informed consent was obtained from all participants. The trials were done according to the principles of the Declaration of Helsinki and Good Clinical Practice. This study was approved in South Korea by the Korea Ministry of Food and Drug Safety (approval #33,475) and Institutional Review Board at 5 sites; Catholic University of Korea, Eunpyeong St. Mary’s Hospital (approval #PC21BDDF0015), Soonchunhyang University Bucheon Hospital (approval #SCHBC 2021–03-017), Hanyang University College of Medicine (approval #GURI 2021–03-044), Catholic University of Korea, Bucheon St. Mary’s Hospital (approval #HC21BDDS0027), and Ewha Womans University Seoul Hospital (approval #SEUMC 2021–03-038). The study was registered with ClinicalTrials.gov (NCT04783311). The primary objective was to confirm the SARS-CoV-2 specific immune response when administering ECV19 to healthy adults. The secondary objective was to confirm the safety and tolerance to SARS-CoV-2. A total of 270 adults were screened, and 229 participants were enrolled. Twenty-four exclusion criteria for the trial were used and are listed in Additional file 1: Table S1.

Randomization and masking

This study used the Interactive Web Response System (IWRS) for randomization. Subjects were assigned to each treatment group in a ratio of 10:10:3 (low dose group to high dose group to placebo comparator group) and stratified by age (19 to 50 years of age versus 51 to 75 years of age). The randomization manager randomly chose a block size among the multiples of the number of treatment groups and generated randomization tables using SAS software (version 9.4 or above). Individuals who provided written informed consent to participation were given a screening number first. Afterwards, those who met the inclusion criteria were randomized by the central randomization plans. Allocation of unique codes by group was managed by IWRS at the central enrollment center. Pharmacists identified the subject’s randomization number via IWRS to release the appropriate investigational product.

Investigational product

The GMP-grade investigational product, ECV19, was manufactured by Eubiologics. ECV19 comprises the SARS-CoV-2 RBD antigen with a poly-histidine tag produced by a stable Chinese Hamster Ovary cell line. The sequence for the his-tagged RBD (Wuhan-Hu-1 strain, GenBank: MT380724.1) was cloned into the pcDNA3.4 expression plasmid to generate the stable cell line used to generate the purified RBD (Additional file 1: Figure S1). The RBD is displayed on nanoliposomes that include EcML and CoPoP, along with the inactive carrier lipids dioleoylphosphatidylcholine and cholesterol. The high-dose group received 20 μg RBD antigen, 20 μg EcML, and 40 μg CoPoP. The low-dose group received 10 μg RBD along with 10 μg EcML and 20 μg CoPoP. The placebo group received a saline injection (Daihan Isotonic Sodium Chloride).

Clinical procedures

The overall schedule for safety and immunogenicity assessment is shown in Additional file 1; Table S2. This study provides an interim analysis of safety and immunogenicity through 8 weekly visits during the study. Intramuscular vaccination with the investigational product occurred on Visit 2 (also referred to as day 0 herein) and Visit 5 (referred to as day 21), and serum was collected on visit 8 (referred to as day 42). No testing for COVID-19 was carried out during this period. Visit 2, 5, and 8 were conducted between July 27, 2021, and October 15, 2021. While the trial was designed as an observer-blind study, participants were not informed of which vaccine or placebo they received until 4 weeks after visit 8. All medical events occurring before the 1st dose of the investigational product were documented as medical history. Assessment of adverse events and severity of solicited or unsolicited adverse events in subjects enrolled in this study were classified based on “MFDS, Guidelines for the assessment of severity of adverse events in vaccine trials” [11]. Severities of unsolicited AEs including immediate adverse events (anaphylaxis related) observed within 30 min of dosing were classified based on the same guidance. Adverse events of special interest were considered according to the guidance document “MFDS, Considerations in COVID-19 vaccines development” [12]. The clinical protocol is included in Additional file 2.

Assessment of antibodies at the International Vaccine Institute (IVI)

The titer of the antibody binding to the spike protein (Acro Biosystems, cat# SPN-C52H9) (anti-S titer) was assessed by ELISA at IVI (Seoul, Korea) using qualified methods as previously described [13]. The antibody titer was expressed as binding antibody unit per mL (BAU/mL) based on a standard curve generated from dilutions of COVID-19 convalescent serum provided by Korea National Institutes of Health, which was calibrated to the unit of WHO international standard (NIBSC code 20/136). If at least two dilutions of each sample were not included within the standard curve due to low OD value, then the sample was considered as 0.5 BAU/mL. To measure the SARS-CoV-2-specific neutralizing antibody activity induced by the vaccine, a FRNT was performed using Wuhan strain of SARS-CoV-2 using qualified methods as described previously [14].

Assessment of virus neutralization by microneutralization assay (MNA)

A subset of samples was sent to Vismederi (Siena, Italy) for assessment of MNA of the Wuhan, Delta and Omicron strains. MNA was carried out as reported [15].

Assessment of RBD antibodies

SARS-CoV-2 RBD-specific antibody responses were determined by ELISA at Eubiologics. Briefly, ELISA plates (NUNC Maxisorp, Thermo Scientific) were coated with 100 ng/well of purified SARS-CoV-2 RBD protein in 100 μL of PBS overnight at 4 °C. Each antigen-coated well was blocked with PBS containing 2% non-fat milk for 1 h at 37 °C and washed with PBS containing 0.05% Tween-20 (PBST). Then, each serum was serially diluted and incubated for 1.5 h at 37 °C. The plates were washed, HRP Anti-human IgG (BD Pharmingen) was added, and the plates were incubated for 1 h at 37 °C. After incubation, the plates were washed six times with PBST and tetramethylbenzidine peroxidase substrate (TMB) was added to develop the color. The reaction was stopped by 0.5 M H3PO4, and the optical density (OD) was measured at a wavelength of 450 nm using ELISA plate reader. The RBD-ELISA titers were converted to binding antibody unit (BAU) using WHO international standard (NIBSC code: 20/136) and compared with the in-house standard serum (COVID-19 positive human convalescent serum panel obtained from Access biologicals).

Assessment of cellular response for a subset of samples from Eunpyeong St. Mary’s Hospital

Cellular response was assessed by analyzing the vaccine antigen-specific T cell count in peripheral blood mononuclear cells (PBMCs) (without proliferation by additional culture) in the blood of the participants treated with SARS-CoV-2 spike RBD vaccine antigens (low dose group, n = 17; high dose group, n = 21; placebo group, n = 7). After separation of PBMCs from blood samples of study subjects (treated with heparin), the PBMCs were slowly frozen at − 70 °C, and after 3 days, they were stored at <  − 190 °C in liquid nitrogen. After thawing, 2.5 × 105 cells were split into each well of a 96-well plate which was coated with anti-human IFNγ or anti IL-4 monoclonal antibody, and then, a SARS-CoV-2 S1 peptide pool (total 166 peptide) in RPMI (with 100 units/mL penicillin, 1 mg/mL streptomycin, 10% heat inactivated fetal calf serum) was added for 18–20 h. Then, detection monoclonal antibody and streptavidin alkaline phosphatase were cultured and treated with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate, and expression was measured. Spot forming cells (SFC)/105 cells were counted in each sample by an ELISpot counter.

Assessment of anti-S, anti-RBD, IgG subclass (anti-RBD) and anti-his-tag responses for a subset of samples from Eunpyeong St. Mary’s Hospital

A subset of samples was sent to the National Institutes of Health (Rockville, Maryland, USA) for exploratory analysis. The basic methodology for ELISA was carried out as described [16]. For anti-S and anti-RBD antibody measurement, the same spike and RBD proteins used at IVI and Eubiologics were utilized to coat ELISA plates, and ELISA units were determined for each test serum collected from this phase 2 study and post-immune Pfizer or Johnson & Johnson vaccination serum purchased from Access Biologicals (Vistas, USA). The WHO International Standards (National Institute for Biological Standards and Control, catalog #20/268, Hertfordshire, England) were also evaluated in the same assays, and the ELISA units of test sera were converted to BAU/mL concentrations.

For IgG subclass ELISA against RBD protein, all test samples were diluted to 2 anti-RBD BAU/mL total antibody level.

For anti-his-tag ELISA, serum samples were tested at 1:200 dilution using ELISA plates coated with 200 ng/well of 10 × His peptide (Abcam, Catalog # ab14943). As a standard, anti-His-Tag chimeric human monoclonal antibody (Sigma, Catalog # SAB5600096) was included at fourfold serial dilutions from 250 ng/mL.

Statistical analyses

Chi-square tests were utilized to compare demographic characteristics. For adverse event (AE), the difference in total number of AEs (including all grade 1, 2, and 3 AEs) among the placebo, low-dose, and high-dose group was compared by a chi-square test first. If significant, the difference between placebo and low-dose group and between placebo and high-dose group was evaluated by Fisher’s exact tests, and Bonferroni corrected p-values are shown. To compare ELISA and FRNT results among different groups at the same time, a Mann–Whitney test (to compare two groups) or a Kruskal–Wallis test followed by Dunn’s multiple comparison tests (to compare more than two groups) were used. The comparison among days 0, 21, and 42 within the group was done by a Friedman test followed by Dunn’s multiple comparison tests. Correlations between anti-RBD and anti-S titers and between anti-RBD titer and FRNT were determined by a Pearson test using log-transformed data. Statistical analysis was carried out using the GraphPad Prism software version 9.3.

Role of funding source

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HQ20C0076), and by Eubiologics. The Ministry of Health & Welfare had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. Staff at Eubiologics were involved in preparing and releasing the investigational product, coordinating contract research organizations, and assessing RBD antibody titers (Fig. 5).