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Safety and immunogenicity of a recombinant receptor‐binding domain‐based protein subunit vaccine (Noora vaccine™) against COVID‐19 in adults: A randomized, double‐blind, placebo‐controlled, Phase 1 trial

Abstract The development of a safe and effective vaccine is essential to protect populations against coronavirus disease 2019 (COVID‐19). There are several vaccine candidates under investigation with different mechanisms of action. In the present study, we have evaluated the safety and immunogenicity of a recombinant receptor‐binding domain (RBD)‐based protein subunit vaccine (Noora vaccine) against COVID‐19 in adults. This Phase 1 trial is a randomized, double‐blind, placebo‐controlled study to evaluate the safety and immunogenicity of the recombinant RBD‐based protein subunit vaccine (Noora vaccine) against COVID‐19 in healthy adults volunteers. Eligible participants were included in this study after evaluating their health status and considering the exclusion criteria. They were then randomized into three groups and received three doses of vaccine (80 µg, 120 µg, and placebo) on Days 0, 21, and 35. Primary outcomes including solicited, unsolicited, and medically attended adverse events were recorded during this study. Secondary outcomes including the humoral and cellular immunity (including anti‐RBD IgG antibody and neutralizing antibody) were measured on Days 0, 21, 28, 35, 42, and 49 by using the ELISA kit and the Virus Neutralization Test (VNT) was performed on day 49. Totally 70 cases were included in this Phase 1 trial and 60 of them completed the study. Safety assessments showed no severe adverse events. Local pain at the vaccine injection site occurred in 80% of the vaccinated volunteers. Induration and redness at the injection site were the other adverse reactions of this vaccine. There was no significant difference between the studied groups regarding adverse reactions. Anti‐RBD IgG antibody and neutralizing antibody assessment showed significant seroconversion in comparison to the placebo group (80%, and 100% respectively, p < 0.001). The cellular immunity panel also showed mild to moderate induction of TH1 responses and the VNT showed 78% of seroprotection. The results of this Phase 1 trial showed acceptable safety without serious adverse events and significant seroconversions in the humoral and cellular immunity panel. The dose of 80 µg is an appropriate dose for injection in the next phases of the trial.

Safety assessments showed no severe adverse events. Local pain at the vaccine injection site occurred in 80% of the vaccinated volunteers. Induration and redness at the injection site were the other adverse reactions of this vaccine. There was no significant difference between the studied groups regarding adverse reactions. Anti-RBD IgG antibody and neutralizing antibody assessment showed significant seroconversion in comparison to the placebo group (80%, and 100% respectively, p < 0.001). The cellular immunity panel also showed mild to moderate induction of TH1 responses and the VNT showed 78% of seroprotection. The results of this Phase 1 trial showed acceptable safety without serious adverse events and significant seroconversions in the humoral and cellular immunity panel. The dose of 80 μg is an appropriate dose for injection in the next phases of the trial.

| INTRODUCTION
The present pandemic of coronavirus disease 2019 (COVID- 19) which has been caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extremely impaired the lives of most people. 1 This pandemic has led to a significant public health crisis around the world. According to a report of the World Health Organization (WHO), up to July 2022, COVID-19 has affected 221 countries and territories with about 575 million confirmed cases and six million deaths. 2,3 COVID-19 is a severe infectious disease that mainly affects the respiratory system. 4,5 It is associated with lethal complications such as severe acute respiratory distress syndrome, pneumonia, cardiac problems, coagulation impairment, organ failure, and death. [6][7][8] Based on the abovementioned, it is necessary to design effective vaccines to protect populations against COVID-19. 9 An optimal vaccine against COVID-19 must have acceptable safety and protective efficacy to use in large populations. Hence, it is beneficial to develop COVID-19 vaccines with different platforms. 10  based, and protein subunit vaccines. 11 All platforms of the COVID- 19 vaccines have their distinct strengths and weaknesses. 12 Among the abovementioned platforms, the protein subunit platform is mostly used within 42% of the trials indicating that this platform is a feasible technology for the COVID-19 vaccine development.
Fifteen protein subunit-based vaccine candidates have entered Phase 3 or Phase 2/3 trials. In addition, there are 23 candidates in Phase 1 or Phase 1/2. They elicit the human immune system against spike (which is known as an RBD protein) to neutralize the virus. 13,14 The RBD engages the angiotensin-converting enzyme 2, a receptor for cell entry, and is an interesting vaccine platform to elicit immune responses to block the receptor binding. 15 The RBD has dominant epitopes in the S protein that effectively induces neutralizing antibodies. 16,17 During a previous preclinical study, an RBD-based protein recombinant vaccine candidate was developed which showed promising results regarding immunogenicity and safety in mice, rabbits, and primates. 18 This phase 1 trial aimed to evaluate the safety and immunogenicity of a recombinant RBD-based protein subunit vaccine (Noora vaccine) against COVID-19 in an Iranian adult population.

| Trial design and participants
This study was a randomized, double-blind, placebo-controlled, Phase 1 trial to assess the safety and immunogenicity of the recombinant RBD-based protein subunit vaccine (Noora vaccine) against COVID-19 in healthy Iranian adult volunteers. The trial was launched at the Baqiyatallah University of Medical Sciences (BUMS), Tehran, Iran.
Healthy volunteers were aged between 18 and 50 years. The health status of volunteers was evaluated during the screening period at the beginning of the study, by taking the medical history, clinical laboratory findings (hematology, coagulation panel, biochemistry, inflammatory panel, and urine analysis), vital signs (respiratory rate, heart rate, axillary temperature, blood pressure, and peripheral oxygen saturation), and physical examinations. Participants with a history of being infected with the COVID-19 (confirmed with realtime polymerase chain reaction assay or serological assay or clinical symptoms) were excluded at the beginning of the study. COVID-19 compatible clinical symptoms were defined as fever (axillary temperature more than 37°C), cough, myalgia, rhinorrhea, sore throat, headache, diarrhea, chest pain, dyspnea, anosmia, and ageusia.
Participants with a history of close contact with COVID-19 confirmed cases during the past 2 weeks were also excluded. The pregnancy status was checked with the assay of blood β-HCG level for women participants in the screening phase and during the study. Other exclusion criteria included a history of hypersensitivity reactions to any ingredient in the vaccine, history of seizures or psychiatric illness, congenital malformations, congenital or acquired immune diseases, growth disorders, malnourishment, kidney impairment, liver impairment, serious chronic disease, uncontrolled hypertension, heart failure, asthma, diabetes, morbid obesity, splenectomy, history of malignancy, coagulation impairment, tuberculosis, acute or chronic viral diseases, history of receiving any blood products or immunoglobulins in the previous 3 months, history of receiving any vaccines in the previous or future 3 months, health care providers, and a failure to comply with the study schedule. Participants with a history of the below conditions were also excluded before the injection of the second or third dose of vaccine: positive pregnancy test, high-grade fever (axillary temperature more than 39°C for more than 3 days), receiving steroids or immunoglobulins, and the development of

| Randomization and blinding
As a safety measure, 14 volunteers were initially randomly allocated in a 3:3:1 ratio to the 80 μg, 120 μg, and placebo groups, respectively.
They were observed for reactogenicity for 12 h in the trial center.
After the approval of the data monitoring and safety board (the DSMB, consists of members from the Iranian food and drug organization), the remaining participants were randomly allocated (3:3:1) into three groups to receive three doses of the vaccine (80 μg or 120 μg doses) or placebo on Day 0, 21, and 35. Vaccine regimens and trial timeline has been illustrated in Figure 1.
A random sequence of length 70 (the size of the sample size) was generated by the online system (SealedEnvelope.com). For this purpose, 10 random blocks with size seven were produced. Three cases with low doses (80 μg), three cases with high doses (120 μg), and one case with placebo. The generated codes were labeled on the vaccine vials before the start of the study and were assigned to the candidates during the study by the study software. The appearance, color, and viscosity were indistinguishable across all vials. Each vial was labeled with a unique code. Investigators and statisticians were

| Safety assessments (primary outcome)
Volunteers were observed for 60 min after each injection in the observing room of the clinical trial center for any probable reactogenicity and immediate adverse events. After each vaccination, any adverse events were recorded through daily telenursing visits. The nurses responsible, called the participants daily for up to 7 days and any local (pain, redness, and induration) and systemic (fatigue, myalgia, headache, flushing, drowsiness, sore throat, aphthous, and chest discomfort) adverse events were recorded as solicited adverse events.
Laboratory safety tests including hematology, coagulation panel, biochemistry, inflammatory panel, and urine analysis were obtained to evaluate any toxicity after each vaccination. Unsolicited adverse events were also documented during the study. Serious adverse events and those events which needed medical interventions were also recorded. The safety results were graded according to the latest Food and Drug Administration (FDA) toxicity grading scale. 19 Any serious adverse events which may occur after a year of the first dose injection, and also follow-up are continuing.

| Immunogenicity assessments (secondary outcome)
Blood samples were obtained to assess the humoral and cellular immunity. Anti-RBD IgG antibody and neutralizing antibody were measured by ELISA kits (PishtazTeb Co.) on Days 0, 21, 28, 35, 42, and 49. In addition, VNT was performed on Day 49. Briefly, to analyze the VNT, we treated Vero cells with serial dilutions of both the virus and pseudo-virus, and the IC50 dilution was determined.
The convalescent human sera as well as vaccinated and placebo groups were incubated with 100 TCID50 and exposed to Vero cell culture. In addition, the neutralizing antibody was measured by Abnova ELISA kit (no. KA6111; Abnova Corporation) according to the supplier's protocol.
The seroconversion was also defined as the rise of RBD IgG and neutralizing antibodies based on the ELISA kit cutoff (>5 μg/ml and >2.5 μg/ml, respectively). Cellular immunity was measured by measuring the interleukin (IL) 4, IL-10, IL-12, and interferongamma (IFN-γ) cytokines released from peripheral blood monocyte on Day 49.
ELISA was performed to assess the IL-4, IL-10, IL-12, and IFN-γ according to the manufacturer's instructions (R&D System). In brief, on Day 49, peripheral blood mononuclear cells from vaccinated and placebo volunteers were isolated and exposed to 20 μg/ml Noora vaccine in the cell culture. After 48 h, the supernatant culture was collected and used for cytokines assay.

| Flow cytometry analysis
On day 49, peripheral blood mononuclear cells from vaccinated and placebo volunteers were isolated and exposed to 20 μg/ml Noora vaccine in the cell culture. According to the same channel of CFSE and IFN-γ-FITC antibody in the Flow cytometry test, the PBMC samples were cultured in two parts. In one part the cell was labeled with CFSE for proliferation detection and in another part did not use CFSE and these wells were used for CD4 + , CD8 + , and IFN-γ finding in the Flow cytometry test. The cells were cultured completely in the same condition, media volume, cell number, mitogen, and vaccine concentrations were similar together in both parts. After 48 h, the CD3 + , CD4 + , CD8 + , and IFN-γ T cells (BD company, 337184 and 346048) were counted using the fluorescence-activated cell sorting (FACS) calibur flow cytometer (Becton Dickinson). Flowjo7 software was used to analyze the results.

| Statistical analysis
The safety results were analyzed with descriptive statistics and comparison was done using the χ 2 test and the Fisher's exact probability method. The immunogenicity outcomes were reported with seroconversion rates and Geometric Mean Titers (GMTs).
Cellular immunogenicity was also calculated with GMTs. The SPSS software version 24 (IBM Corporation) and Prism software version 9.2.0 (GraphPad) were used for statistical analysis.

| RESULTS
In this Phase 1 trial, a total of 70 cases were included and finally, 60 cases completed the study. During the study, 10 cases (one positive pregnancy test, three symptomatic cases, and seven positive RT-PCT cases) were excluded. The CONSORT diagram of the trial is shown in Figure 2.
The age of the participants was 31.78 ± 7.35 years old indicating no statistically significant differences across the three groups (p = 0.60) of which 39 (65%) cases were male. The gender of participants was also not statistically different across the three groups (p = 0.97). Table 1 shows the demographic data of participants.
In this trial, all cases in three groups experienced at least one adverse event through 1 month after injection, and no significant difference was seen across all three groups.  Surprisingly, the raising of neutralizing antibody titer was seen in all groups with no statistical differences (p = 0.29). Figure 5 has illustrated the results of immunogenicity.
The results of flow cytometry analysis are also illustrated in

F I G U R E 3
The frequency of immediate adverse events across the three groups during the study (after each injection). Local pain at the injection site was the only reported adverse effect. No significant difference was seen across groups. After first dose, 62.5% of the placebo group, 85.19% of the 80 μg group, and 76% of the 120 μg group reported local pain at the injection site (p = 0.366). After second dose, local pain at the injection site was reported in 62.5% of the placebo group, 81.48% of the 80 μg group, and 68% of the 120 μg group (p = 0.414). After 3rd dose, local pain at the injection site was reported in 62.5% of the placebo group, 81.48% of the 80 μg group, and 88% of the 120 μg group (p = 0.640). The bar charts represented the percentage of cases that developed adverse events.
group (p = 0.48) (Figure 8). The results of VNT showed that among the 28 volunteer sera, 23 sera (78%) of vaccinated cases in both groups (80 μg and 120 μg doses) were able to reutilize the wild and pseudo-type virus. No significant differences were observed between the positive sera (p = 0.62) (Figure 9). The immunogenicity results also showed both 80 μg and 120 μg elicited the humoral and cellular immunities. Hence, there are no differences between these two doses.

| DISCUSSION
In this study, we evaluated the safety and immunogenicity of a Severe local pain was also reported with only one case that needed acetaminophen administration. Notably, the pain resolved after medical intervention and did not appear in the next doses. These adverse events are expected for protein subunit vaccines because they use Alum adjuvant in their formulation. 25 It is expected that these adverse events resolve after a few days of vaccination. These F I G U R E 4 The solicited adverse events during the study. Pain at the injection site was the most frequent local adverse event (74.1% of the 80 μg group and 80% of the 120 μg group). Redness and induration of the injection site also occurred in less than 10% of participants. Myalgia, fatigue, headache, flushing, drowsiness, sore throat, aphthous, and chest discompose were among the reported systemic adverse events. The frequency of these adverse events was not different across the studied groups and they occurred in less than 10% of participants (p = 0.72). The result represented the percentage of cases that developed adverse events. The bar charts represented the percentage of cases that developed adverse events. vaccine receipts also experienced headache (68%) and fever (71%). 30 In comparison with NVX-CoV2373, another protein subunit vaccine, the incidence of fatigue, myalgia, and headache were also lower in the Noora vaccine. 22 However, safety results in the clinical trials may be exaggerated through individual factors, such as the personal feelings of volunteers. Hence, the severity of pain, headache, or other subjective variables may differ among participants.
Humoral and cellular immune responses against RBD protein showed significant increases in immune responses after vaccination.
Up to now, different vaccine candidates have been developed.
Among them, RBD base platforms have shown notable F I G U R E 5 The immunogenicity results of the studied groups and seroconversion status. Anti-RBD IgG increased significantly in both 80 μg and 120 μg groups in comparison with the placebo group (p < 0.001 and p = 0.002, respectively). No significant difference between the 80 μg and 120 μg groups (p = 0.74). 88% and 84% of cases in the 80 μg and 120 μg group showed seroconversion for anti-RBD IgG (p < 0.001). Neutralizing antibody titer also increased but no significant difference was seen across the studied groups (p = 0.29). The seroconversion was also defined as the rise of RBD IgG and neutralizing antibodies based on the ELISA kit cutoff (>5 μg/ml and >2.5 μg/ml, respectively). ELISA, enzyme-linked immunosorbent assay; RBD, receptor-binding domain The flow cytometry analysis for gating IFN-γ-produceing CD4 + and CD8 + T cells. On Day 49, peripheral blood mononuclear cells from vaccinated and placebo volunteers were isolated and exposed to 20 μg/ml Noora vaccine in the cell culture. After 48h, the cells were stained for IFN-γ producing CD4 + and CD8 + T cells. On gated CD4 + and CD8 + T cells, IFN-γ-producing T cells were identified based on the presence or absence of IFN-γ. Numbers within the plots indicate the relative percentage of the IFN-γ producing T cells population in the control samples. IFN-γ, interferon gamma F I G U R E 7 The analysis of IFN-γ producing CD4 + and CD8 + T cells populations in the vaccinated and placebo groups. On Day 49, in the vaccinated group, RBD-specific CD4 + and CD8 + T cell was proliferated and increased significantly in comparison to control which were not exposed to RBD antigen (CTR) (80 μg group; CD4 + : p = 0.03 and CD8 + : p = 0.04, 120 μg group; CD4 + : p = 0.04 and CD8 + : p = 0.04). The placebo group did not show a significant increase. Also, the RBD-specific CD4 + T cells and RBD-specific CD8 + T cells significantly produced IFN-γ in comparison to the control which was not exposed to RBD antigen (CTR The analysis of the TH1/TH2 cytokines panel in the vaccinated and placebo group. After RBD antigen exposure, the supernatants of cultured PMBC were evaluated by assessing the IL4, IL-10, IL-12, and IFN-γ at day 49. IL-4 cytokine was not changed after RBD exposure in the vaccinated and placebo group (p = 0.12 for 80 μg dose group, p = 0.83 for 120 μg dose group, and p = 0.58 for placebo). The IL-10 cytokine tend to increase in the vaccinated group and had a significant change in 80 μg dose and 120 μg doses (p = 0.02 for 80 μg dose group, p = 0.007 for 120 μg dose group, and p = 0.48 for placebo). The IL-12 cytokine was increased in the 80 μg group and 120 μg group (p = 0.04 for 80 μg dose group, p = 0.01 for 120 μg dose group, and p = 0.62 for placebo). The IFN-γ cytokine also showed an increase in vaccinated participants and no significant changes were detected in the placebo (p = 0.04 for 80 μg dose group, p = 0.001 for 120 μg dose group, and p = 0.42 for placebo). CTR, Control; IFN-γ, interferon gamma; PHA, phytohemagglutinin; RBD, receptor-binding domain seroprotected. After exposure of PBMC of the vaccinated group with RBD protein, Both RBD-specific CD4 + and CD8 + T cells with their intracellular IFN-γ increased. Also, IFN-γ and IL-12 cytokines were released after RBD exposure. These results showed that cellmediated immunity (Th1) response was inducted in response to RBD vaccination. However, IL-4 did not change after vaccination.
BNT162b1 vaccine, induced functional CD4 + and CD8 + responses in all vaccinated cases, mainly Th1 helper responses. 35 The mRNA1273 vaccines also induced TH1 helper response with minimal Th2 response. 36 The rAd26-S and rAd5-S vaccine also increased the 100% formation of CD4 + and CD8 + cells and increased the level of IFN-γ. 27 NVX-CoV2373 induced Th1 phenotype response with the increased levels of IFN-γ, IL-2, and TNF-α with slight induction in Th2 responses which was measured by IL-5 and IL-13 cytokines. 37,38 Other vaccines also did not assess cellular immunogenicity and it is not feasible to compare their results with the current study.
BIV1-CovIran, an inactivated whole virus particle vaccine, showed acceptable safety results, and only mild to moderate transient adverse effects were reported. Similar to the current study, the seroconversion for neutralizing and anti-RBD antibodies was 82.8%. 39 The results of SpikoGen, a subunit COVID-19 vaccine, showed no serious adverse events. They reported that the seroconversion rate against S1 was 63.55%. 40 It seems in response to inflammatory cytokines, IL-10 as an anti-inflammatory cytokine was increased to regulate the cytokine network. Additionally, the 120 μg dose showed no better immu-

AUTHOR CONTRIBUTIONS
All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Jafar Salimian, Ali Ahmadi, Jafar Amania, Gholamreza Oladd, Gholamhossein Alishiri, and Hassan Abolghasemi. The first draft of the manuscript was written by Ali Saffaei and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data set is available on request. F I G U R E 9 The results of the virus neutralizing test (VNT). It was performed on Day 49. Briefly, Vero cells were treated with the serial dilutions of both the virus and pseudo-virus, and the IC50 dilution was determined. The convalescent human sera as well as vaccinated and placebo groups were incubated with 100 TCID50 and exposed to Vero cell culture. About 78% of the vaccinated cases in both vaccinated groups were able to neutralize the wild and pseudo-type virus. No significant difference was seen between the two vaccinated groups (p = 0.62). Despite a bite increase in anti-RBD Ig in the placebo group, the sera were not able to neutralize the virus. Each bar indicates each participant's serum. ND, not determined