Safety of Cov2-Bio COVID-19 Vaccine in Healthy Populations Aged 18 years and Above in Indonesia

doi:10.21203/rs.3.rs-2579054/v1

Background

According to WHO Target Product Profile for COVID-19 Vaccines, vaccine development should be indicated for active immunization in all populations, including the elderly, in conjunction with other control measures to curtail or end an outbreak. In this study, we evaluated the safety of SARS-CoV-2 protein subunit recombinant vaccine (CoV2-Bio) in healthy population aged 18 years and above in Indonesia.

Methods

This study is an observer-blinded, randomized, and controlled prospective phase 1 intervention study about the safety of CoV2-Bio COVID-19 vaccine. We evaluated the safety in two age groups: adult (18–59 years old) and elderly (above 60 years old). There were 54 healthy participants in total, of which 30 were adults and 24 were elderly. Each group was then randomized to receive either three doses of CoV2-Bio COVID-19 or two doses of Sinovac and one placebo. The vaccines in both groups were administered approximately 28 days apart. Subjects were followed up to approximately 3 months. We analyzed the data using IBM SPSS 25.

Results

A total of 54 healthy participants divided into adult (n = 30) and elderly (n = 24) groups were enrolled. There was no significant difference of any local or systemic adverse event in terms of incidence rate or intensity in control group and vaccine group. No serious adverse events were deemed related to vaccine administration.

Conclusion

CoV2-Bio COVID-19 vaccine is safe in healthy populations aged 18 years old and above.

Trial registration:

This study has been registered in ClinicalTrials.gov ID: NCT05067894 on October 5, 2021.

Safety

SARS-CoV-2 protein subunit recombinant vaccine

CoV2-Bio COVID-19 vaccine

CoronaVac

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently the most consequential global health problem. The investment in SARS-CoV-2 vaccine development contributes to the supply of safe, affordable COVID-19 vaccines for the global market, especially for low- and middle-income countries. In Indonesia, the target population for vaccination is still limited due to the less availability of the vaccine for mass campaigns.^1–3

The current vaccines under emergency use authorization are not sufficient yet to accomplish the vaccination target for all populations. The supply of COVID-19 vaccines in Indonesia mostly relies on imports from overseas with the consequent high cost. Producing domestic vaccine and implementing a safe and effective administration would be the propitious ways to increase vaccine coverage and optimize COVID-19 mitigation cost.^1–3

Several receptor-binding domain (RBD-based) COVID-19 vaccines have been evaluated in clinical trials and showed advantages,^4–6 including temperature stability.^7–10 However, the most pertinent point is its potential to protect against new variants of SARS-CoV-2. In addition, RBD is considered a key biomarker, which is a dominant target in the elicitation of neutralizing antibodies (NAbs) following natural infection or vaccination processes.

In terms of mass production, the use of P. pastoris, a low-cost expression platform, results in high-yield expressed antigens with high purity and well-defined structure, thus easing the transfer from pilot to industrial-scale manufacturing. The antigen is created under cGMP and can be put into vials to produce 20,000 to 200,000 doses of vaccines. This is supported by the availability of expertise in fermentation technology using P. pastoris in developing countries’ vaccine manufacturers, including in Indonesia. Besides, the production of RBD-based vaccines requires low cost. Therefore, to help overcome the issue, Bio Farma Pharmaceutical Company develops a vaccine in the subunit protein recombinant platform.^{1, 8–10}

The subunit protein vaccine that we currently manufacture, CoV2-Bio COVID-19, does not require genetic material, making it non-infectious or non-viable. We believe in the safety of this vaccine and its favorability in mass production.⁷ Hence, the present study aimed to evaluate the safety of the CoV2-Bio COVID-19 vaccine in healthy populations aged 18 years and above in Indonesia.

Study design

Generally, phase I clinical trial, including a vaccine trial, is a small-scale study with the primary focus to identify the clinical tolerance and safety of the intervention. The present study is an observer-blinded, comparative and randomized phase I clinical trial.

Sample size

The final sample was a total of 54 healthy participants divided into two age groups: 18 to 59 years old (adult) and 60 years old and above (elderly). After the first vaccine was administered to the adults, we followed them up for seven days. Then, we recruited the elderly group. The treatment in each intervention and control group of adults and elderly was presented in Table 1.

Table 1

Overview of treatment arms
Age	Group	No. of subject	No. of dose
Adults (18–59 years)	I	15	3 doses of vaccine, dose 50 µg
Adults (18–59 years)	II	15	3 doses of control (2 doses of active control and 1 dose of placebo)
Elderly (60 years and above)	II	12	3 doses of vaccine, dose 50 µg
Elderly (60 years and above)	III	12	3 doses of control (2 doses of active control and 1 dose of placebo)

Participants enrolment

Before subject enrollment, we performed an initial screening for all candidates to determine whether they were eligible to participate in the study. All candidates underwent the following safety examinations: routine biochemical and hematological tests, urine test, chest X-ray, ECG, and SARS-CoV-2 antigen detection. Then, we analyzed the results based on the exclusion criteria.

The exclusion criteria of this study were having a history of vaccination with any investigational product against COVID-19 during or 6 months prior to enrollment; past COVID-19 in the last 3 months; positive result for COVID-19 rapid antigen test; history of immunodeficiency or uncontrolled chronic disease; women who were lactating, pregnant, or planning a pregnancy during the study period; having abnormal hematology and biochemical test results; history of asthma; and those with a history of allergy to vaccines or vaccine ingredients.

For each included subject, numbers from 001 to 054 and randomization codes (A/B) were allocated by the unblinded team. The team randomized and vaccinated the participants with the doses shown in Table 1. The randomization list was made using automatically generated randomization provided by the website www.sealedenvelope.com.

The recruitment process was conducted using the age-escalating method, starting with the adult group (n = 30). We assessed and reviewed the vaccine safety during the first seven days after the first vaccination, which turned out to show good results. The study then continued with the recruitment of the elderly group (n = 24).

The vaccine group received the candidate vaccine currently developed, CoV2-Bio COVID-19, and the control group received the CoronaVac vaccine. Three doses of either vaccine were administered on the first visit or V1 (day 0), the second visit or V2 [(V1 + 28 days (-4/+7 days) ], and the third visit or V3 [(V2 + 28 days (-4/+7 days)].

Safety evaluation

All subjects were observed for 30 minutes following vaccine administration. They were also advised to record daily any adverse events, such as local or systemic reactions, in the diary card for 28 days after the last dose, with special attention within the first three days. The adverse events that should be recorded were listed in the diary card (solicited adverse events), although they could also note the medical reactions not listed (unsolicited adverse events).

Safety data of each visit from the diary card were evaluated at the next visits (V2, V3, and V4). The local reaction was examined on the vaccination site. The systemic event assessment involved daily axillary temperature measurement using a thermometer given to the patients along with instruction on how to use it. The presence of weakness should also be recorded as a systemic event. Any medical office visit, emergency room visit, or hospitalization for any reason was recorded throughout the trial period. In case of serious adverse events, participants must report immediately and it would be documented in the case report form (CRF). Then, all the data were recorded in the electronic case report form (eCRF) and given to the ethics committee. The serious adverse event was reviewed by the Data Safety Monitoring Board (DSMB). Finally, we analysed the data using SPSS software.

Study intervention

Candidate vaccine

The dose of CoV2-Bio COVID-19 for all participants was 0.5 ml injected three times with an interval of 28 days. Every 0.5 ml of vaccine contained 50 µg of SARS-CoV-2 RBD subunit recombinant protein, 200 µg of aluminum as an adjuvant, 2.742 mg of normal saline, and 1.137 mg of tris(hydroxymethyl)aminomethane with excipients Alhydrogel® adjuvant, sodium chloride, and tris(hydroxymethyl)aminomethane. The products were packaged in single-dose prefilled syringes with a minimum recoverable volume of 0.5 mL (0.5 ml/dose).

SARS-CoV-2 subunit recombinant protein vaccine had SARS-CoV-2 RBD as an antigen. The clone of RBD protein was generated by Texas Children’s Hospital Center for Vaccine Development at Baylor College of Medicine (BCM), USA. The protein was developed based on the sequence of the wild-type SARS-CoV-2 RBD amino acid, representing residues 331–549 of the spike (S) protein (GenBank: QHD43416.1) of the Wuhan-Hu-1 isolate (GenBank: MN908947.3).

SARS-CoV-2 subunit recombinant protein vaccine was a non-infectious recombinant RBD protein of SARS-CoV-2. The recombinant proteins were produced through fermentation in recombinant Pichia pastoris. The fermentation process involved the growth of P. pastoris on chemically defined fermentation media which contained glycerol, vitamins, and mineral salts. The recombinant proteins were collected from the supernatant of centrifuged culture and purified by a series of chemical and physical methods, e.g. chromatography and diafiltration. The purified RBD recombinant proteins were aseptically formulated with buffer, and aluminum hydroxide adjuvant was subsequently added into single-dose prefilled syringes to finalize the product, which was a sterile white liquid suspension.

Comparator vaccine

The control product was a combination of the COVID-19 Vaccine Bio Farma (CoronaVac) and placebo. CoronaVac was administered at dose 1 and dose 2, followed by placebo at dose 3. The product was manufactured through inoculation of Novel Coronavirus (CZ02 Strain) into African Green Monkey Kidney Cell (Vero Cell). Then, the viruses were successfully incubated, harvested, inactivated, concentrated, purified, and adsorbed by aluminum hydroxide. The result was a milky white suspension, which was stratified by precipitation and easy to shake. The products were packaged in multidose vials (0.5 mL/dose).

Each dose of CoronaVac in the control product contained 600 SU/0.5 mL (3 ug/0.5 mL) of SARS-CoV-2 antigen with excipients aluminum hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, etc.¹¹ Meanwhile, the placebo, also produced by Bio Farma, was a BCG vaccine solvent consisting of normal saline injection. The product was colorless, odorless, and tasteless liquid packaged in multidose vials (0.5 mL/dose).

Demographics of the study participants

A total of 54 healthy participants were divided into two age groups: adult group aged 18 to 59 years (n=30) and elderly group aged >60 years (n=24). All of them consented to participate in this vaccine evaluation study. Fifty of 54 participants (92.60%) were monitored successfully up to day 28 after the third dose. There were four dropouts, one of which was from the vaccine group and three from the control group. The reason for the participants’ discontinuation was described in Table 2.

Table 2. Reasons for participants with early discontinuation

No	Inclusion no.	Age group	Intervention group	Reason
1	030	Adult	Vaccine group	Change of domicile
2	022	Adult	Control group	Lost-to-follow-up
3	025	Adult	Control group	Discontinuation by investigator due to an adverse event, which was COVID-19
4	042	Elderly	Control group	Discontinuation by investigator due to serious adverse event

Of the 54 participants, 29 were female (53.7%). The rest of the demographic characteristics of participants were shown in Table 3.

Table 3. Demographic characteristics of participants

Parameter	Vaccine group *(n=27)**	Control group (n=27)	Total (n=54)
Mean age [years] (SD)^T	47.33 (± 18.381)	51.89 (± 18.408)	49.61 (± 18.365)
Adult group	33.20 (± 11.761)	38.33 (± 12.799)	35.77 (± 12.356)
Elderly group	65.00 (± 3.104)	68.83 (± 5.357)	66.92 (± 4.708)
Sex n(%)
Male Adult group Elderly group	12 (44.44) 10 (37.04) 2 (7.41)	13 (48.14) 7 (25.92) 6 (22.22)	25 (46.29) 17 (31.48) 8 (14.81)
Female Adult group Elderly group History of COVID-19 vaccination prior to study >6 months Yes Adult group Elderly group No Adult group Elderly group History of COVID-19 prior to study >3 months Yes Adult group Elderly group No Adult group Elderly group History of controlled comorbidity Hypertension Adult group Elderly group Diabetes mellitus Adult group Elderly group Stroke Adult group Elderly group	15 (51.70) 5 (18.52) 10 (37.04) 18 (66.67) 8 (29.63) 10 (37.04) 9 (33.33) 7 (25.92) 2 (7.41) 5 (18.52) 3 (11.11) 2 (7.41) 22 (81.48) 12 (44.44) 10 (37.04) 5 (18.52) 0 (0.00) 5 (18.52) 1 (3.70) 0 (0.00) 1 (3.70) 0 (0.00) 0 (0.00) 0 (0.00)	14 (48.30) 8 (29.62) 6 (22.22) 16 (59.26) 7 (25.92) 9 (33.33) 11 40.74) 8 (29.63) 3 (11.11) 1 (3.70) 1 (3.70) 0 (0.00) 26 (96.30) 14 (51.85) 12 (44.44) 9 (16.67) 1 (3.70) 8 (29.63) 1 (3.70) 0 (0.00) 1 (3.70) 1 (3.70) 0 (0.00) 1 (3.70)	29 (53.70) 13 (24.07) 16 (29.62) 34 (62.96) 15 (27.78) 19 (79.17) 20 (37.04) 15 (27.78) 5 (9.26) 6 (11.11) 4 (7.41) 2 (3.70) 48 (88.89) 26 (48.15) 22 (40.74) 14 (25.92) 1 (3.70) 13 (24.07) 2 (3.70) 0 (0.00) 2 (3.70) 1 (3.70) 0 (0.00) 1 (3.70)

*n= number of participants; ^TSD = standard deviation

The safety of CoV2-Bio COVID-19 vaccine

The overall incidence of adverse events was 62.97% in the period from the first dose to 28 days after the last dose administration. The incidence rate of adverse events in the vaccine group and control group were 66.67% and 59.26%, respectively. There was no significant difference in incidence rates between groups (P=0.573). Most of the adverse events were reported within seven days after the injection (P=0.573).

Based on age group, the overall incidence of adverse events in the adult group and the elderly group were 80.00% and 41.67%, respectively. There was no remarkable difference in incidence rates between vaccine and control group in the adult group (P=0.651) and the elderly group (P>0.999).

The most frequent solicited adverse events were local pain and myalgia, while influenza was reported the most as the unsolicited adverse event. The intensity of most adverse events was mild. The local reactions, swelling and induration, were categorized as severe intensity based on plastic bangle measurements. However, these local reactions were transient and self-limiting. There was one moderate unsolicited adverse event recorded from the control group which was a subcutaneous hematoma, although it was considered unrelated to the vaccination. A participant in the control group noted a serious adverse event deemed unlinked to the vaccination based on the examination and expertise of a specialized doctor and DSMB. The proportion of adverse events after each vaccination dose in the vaccine and control groups was presented in Table 4 through Table 18.

Table 4. Incidence of adverse events after the first injection

Vaccine group (n=27)			Control group (n=27)		Total (n=54)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse Events	27	16 (59.25)	30	14 (51.85)	57	30 (55.56)	0.584
Local	7	6 (22.22)	8	6 (22.22)	15	12 (22.22)	>0.999
Systemic	20	13 (48.14)	22	12 (44.44)	44	25 (46.30)	0.785
Solicited	18	12 (44.44)	23	13 (48.14)	41	25 (46.30)	0.785
Local***	7	6 (22.22)	7	6 (22.22)	14	12 (22.22)	>0.999
Systemic****	11	9 (33.33)	16	11 (40.74)	27	20 (37.03)	0.573
Unsolicited	9	8 (29.62)	7	4 (14.81)	16	12 (22.22)	0.191
Local	0	0 (0.00)	1	1 (3.70)	1	1 (1.85)	>0.999**
Systemic	9	8 (29.62)	6	3 (11.11)	15	11 (20.38)	0.09
Within 30 mins	8	7 (25.92)	6	6 (22.22)	14	13 (24.07)	0.750
0-7 days	16	9 (33.33)	21	11 (40.74)	37	20 (37.03)	0.573
>7 days	3	3 (11.11)	3	2 (7.40)	6	6 (11.11)	>0.999**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 5. Incidence of adverse events after the first injection in the adult group

Vaccine group (n=15)			Control group (n=15)		Total (n=30)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse Events	17	12 (80.00)	19	9 (60.00)	36	21 (70.00)	0.427
Local	3	3 (20.00)	4	4 (26.67)	7	7 (23.33)	>0.999**
Systemic	14	10 (66.67)	15	8 (53.33)	29	18 (60.00)	0.456
Solicited	10	9 (60.00)	16	9 (60.00)	26	18 (60.00)	>0.999
Local***	3	3 (20.00)	4	4 (26.67)	7	7 (23.33)	>0.999**
Systemic****	7	7 (46.67)	12	8 (53.33)	19	15 (50.00)	0.715
Unsolicited	7	6 (40.00)	3	1 (6.67)	10	7 (23.33)	0.08
Local	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	>0.999
Systemic	7	6 (40.00)	3	1 (6.67)	10	7 (23.33)	0.08
Within 30 mins	7	6 (40.00)	5	5 (33.33)	12	11 (36.67)	0.705
0-7 days	8	6 (40.00)	14	7 (46.67)	22	13 (43.44)	0.713
>7 days	2	2 (13.33)	0	0 (0.00)	2	2 (6.67)	0.483

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 6. Incidence of adverse events after the first injection in the elderly group

Vaccine group (n=12)			Control group (n=12)		Total (n=24)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse events	10	4 (33.33)	11	5 (41.67)	21	9 (37.50)	>0.999**
Local	4	3 (25.00)	4	2 (16.67)	8	5 (20.83)	>0.999**
Systemic	6	3 (25.00)	7	4 (33.33)	13	7 (29.17)	>0.999**
Solicited	8	3 (25.00)	7	4 (33.33)	15	7 (29.17)	>0.999**
Local***	4	3 (25.00)	3	2 (16.67)	7	5 (20.83)	>0.999**
Systemic****	4	2 (16.67)	4	3 (25.00)	8	5 (20.83)	>0.999**
Unsolicited	2	2 (16.67)	4	3 (25.00)	6	5 (20.83)	>0.999**
Local	0	0 (0.00)	1	1 (8.33)	1	1 (4.17)	>0.999**
Systemic	2	2 (16.67)	3	2 (16.67)	5	4 (16.67)	>0.999
Within 30 mins	1	1 (8.33)	1	1 (8.33)	2	2 (8.33)	>0.999
0-7 days	8	3 (25.00)	7	4 (33.33)	15	7 (29.17)	>0.999**
>7 days	1	1 (8.33)	3	2 (16.67)	4	3 (12.50)	>0.999**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 7. Incidence of adverse events after the second injection

Vaccine group (n=26)			Control group (n=25)		Total (n=51)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse events	7	5 (19.23)	12	6 (24.00)	19	11 (21.57)	0.679
Local	2	2 (7.70)	7	4 (16.00)	9	6 (11.77)	0.630**
Systemic	5	5 (19.23)	5	4 (16.00)	10	9 (17.64)	>0.999**
Solicited	6	4 (15.39)	10	5 (20.00)	16	9 (17.64)	0.947**
Local***	2	2 (7.70)	7	4 (16.00)	9	6 (11.77)	0.630
Systemic****	4	4 (15.39)	3	3 (12.00)	7	7 (13.72)	>0.999**
Unsolicited	1	1 (3.84)	2	2 (8.00)	3	3 (5.89)	0.970
Local	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0
Systemic	1	1 (3.84)	2	2 (8.00)	3	3 (5.89)	0.970
Within 30 mins	1	1 (3.84)	4	3 (12.00)	5	4 (7.84)	0.580
0-7 days	5	3 (11.53)	7	4 (16.00)	12	7 (13.72)	0.954
>7 days	1	1 (3.84)	1	1 (4.00)	2	2 (3.92)	>0.999**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 8. Incidence of adverse events after the second injection in the adult group

Vaccine group (n=14)			Control group (n=14)		Total (n=28)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	p-value^*
Overall adverse events	8	4 (28.58)	5	4 (28.58)	13	8 (28.58)	>0.999**
Local	3	2 (14.29)	1	1 (7.14)	4	3 (10.71)	>0.999**
Systemic	5	4 (28.58)	4	4 (28.58)	9	8 (28.58)	>0.999**
Solicited	6	3 (21.42)	4	3 (21.42)	10	6 (21.42)	>0.999**
Local***	3	2 (14.29)	1	1 (7.14)	4	3 (10.71)	>0.999**
Systemic****	3	3 (21.42)	3	3 (28.58)	6	6 (21.42)	>0.999**
Unsolicited	2	2 (14.29)	1	1 (7.14)	3	3 (10.71)	>0.999**
Local	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0
Systemic	2	2 (14.29)	1	1 (7.14)	3	3 (10.71)	>0.999**
Within 30 mins	4	3 (21.42)	1	1 (7.14)	5	4 (14.29)	0.596
0-7 days	3	2 (14.29)	3	2 (14.29)	6	4 (14.29)	>0.999**
>7 days	1	1 (7.14)	1	1 (7.14)	2	2 (7.14)	>0.999**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 9. Incidence of adverse events after the second injection in the elderly group

Vaccine group (n=12)			Control group (n=12)		Total (n=23)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse events	2	1 (8.33)	4	2 (9.10)	6	3 (13.04)	0.932
Local	1	1 (8.33)	4	2 (9.10)	5	3 (13.04)	0.932
Systemic	1	1 (8.33)	0	0 (0.00)	1	1 (4.35)	>0.999**
Solicited	2	1 (8.33)	4	2 (9.10)	6	3 (13.04)	0.932
Local***	1	1 (8.33)	4	2 (9.10)	5	3 (13.04)	0.932
Systemic****	1	1 (8.33)	0	0 (0.00)	1	1 (4.35)	>0.999**
Unsolicited	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0
Local	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0
Systemic	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0
Within 30 mins	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0
0-7 days	2	1 (8.33)	4	2 (9.10)	6	3 (13.04)	0.932
>7 days	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 10. Incidence of adverse events after the third injection

Vaccine group (n=26)			Control group (n=24)		Total (n=50)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse events	10	7 (26.92)	7	6 (25.00)	17	13 (26.00)	0.877
Local	6	4 (15.39)	3	3 (12.50)	9	7 (14.00)	>0.999**
Systemic	4	4 (15.39)	4	4 (16.67)	8	8 (16.00)	>0.999
Solicited	8	5 (19.32)	3	3 (12.50)	11	8 (16.00)	0.797
Local***	6	4 (15.39)	2	2 (8.33)	8	6 (12.00)	0.747
Systemic****	2	2 (7.70)	1	1 (4.17)	3	3 (6.00)	>0.999**
Unsolicited	2	2 (7.70)	4	3 (12.50)	6	5 (10.00)	0.922
Local	0	0 (0.00)	1	1 (4.17)	1	1 (2.00)	0.960**
Systemic	2	2 (7.70)	3	3 (12.50)	5	5 (10.00)	0.922
Within 30 mins	1	1 (3.84)	2	2 (8.33)	3	3 (6.00)	>0.999**
0-7 days	8	6 (23.08)	4	3 (12.50)	12	9 (18.00)	0.549
>7 days	1	1 (3.84)	1	1 (4.17)	2	2 (4.00)	>0.999**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 11. Incidence of adverse events after the third injection in the adult group

Vaccine group (n=14)			Control group (n=13)		Total (n=27)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse events	8	5 (35.71)	7	6 (46.15)	15	11 (40.74)	0.581
Local	6	4 (28.58)	3	3 (23.08)	9	7 (25.92)	>0.999**
Systemic	2	2 (14.29)	4	4 (30.76)	6	8 (22.22)	0.573
Solicited	8	5 (35.71)	3	3 (23.08)	11	8 (29.62)	0.770
Local***	6	4 (28.58)	2	2 (15.39)	8	6 (22.22)	0.724
Systemic****	2	2 (14.29)	1	1 (7.70)	3	3 (11.11)	>0.999**
Unsolicited	0	0 (0.00)	4	3 (23.08)	4	3 (11.11)	0.196
Local	0	0 (0.00)	1	1 (7.70)	1	1 (3.70)	0.963**
Systemic	0	0 (0.00)	3	3 (23.08)	3	3 (11.11)	0.196
Within 30 mins	1	1 (7.14)	2	2 (15.39)	3	3 (11.11)	>0.999**
0-7 days	7	5 (35.71)	4	3 (23.08)	11	8 (29.62)	0.770
>7 days	0	0 (0.00)	1	1 (7.70)	2	1 (3.70)	0.963**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 12. Incidence of adverse events after the third injection in the elderly group

Vaccine group (n=12)			Control group (n=11)		Total (n=23)
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	P-value^*
Overall adverse events	2	2 (16.67)	0	0 (0.00)	2	2 (8.70)	0.522**
Local	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	-
Systemic	2	2 (16.67)	0	0 (0.00)	2	2 (8.70)	0.522**
Solicited	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	-
Local***	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	-
Systemic****	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	-
Unsolicited	2	2 (16.67)	0	0 (0.00)	2	2 (8.70)	0.522**
Local	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	-
Systemic	2	2 (16.67)	0	0 (0.00)	2	2 (8.70)	0.522**
Within 30 mins	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	-
0-7 days	1	1 (8.33)	0	0 (0.00)	1	1 (4.34)	>0.999**
>7 days	1	1 (8.33)	0	0 (0.00)	1	1 (4.34)	>0.999**

*P-value was calculated using Chi-square test **P-value was calculated using Fisher Exact test

***Solicited local adverse events were pain, redness, induration, and swelling; ****Solicited Systemic adverse events were fever, fatigue, and myalgia.

Table 13. Intensity of adverse events in the vaccine group after the whole dose (n=27)

Mild			Moderate		Severe		Total
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)
Total Adverse Events	42	18 (66.67)	0	0 (0.00)	2	1 (3.70)	44	18 (66.67)
Total Solicited Adverse Events	30	13 (48.15)	0	0 (0.00)	2	1 (3.70)	32	14 (51.85)
Local reactions	13	8 (29.63)	0	0 (0.00)	2	1 (3.70)	15	9 (33.33)
Local pain	10	8 (29.63)	0	0 (0.00)	0	0 (0.00)	10	8 (29.63)
Redness	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Induration	2	2 (7.40)	0	0 (0.00)	1	1 (3.70)	3	3 (11.11)
Swelling	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)	1	1 (3.70)
Systemic events	17	10 (37.03)	0	0 (0.00)	0	0 (0.00)	17	10 (37.03)
Fever	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Fatigue	2	2 (7.40)	0	0 (0.00)	0	0 (0.00)	2	2 (7.40)
Myalgia	15	9 (33.33)	0	0 (0.00)	0	0 (0.00)	15	9 (33.33)
Total Unsolicited Adverse Events	12	11 (40.74)	0	0 (0.00)	0	0 (0.00)	12	11 (40.74)
Respiratory, thoracic and mediastinal disorders	7	6 (22.22)	0	0 (0.00)	0	0 (0.00)	7	6 (22.22)
Cough	2	2 (7.40)	0	0 (0.00)	0	0 (0.00)	2	2 (7.40)
Influenza	4	4 (14.81)	0	0 (0.00)	0	0 (0.00)	4	4 (14.81)
Oropharyngeal pain	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Gastrointestinal disorders	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Diarrhea	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Eye disorders	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Eye pain	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Skin and subcutaneous tissue disorders	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Pruritus	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Nervous system diseases	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Dizziness	2	2 (7.40)	0	0 (0.00)	0	0 (0.00)	2	2 (7.40)

The solicited local adverse events were pain, redness, induration, and swelling. Pain was graded into mild (pain at injection site when touched), moderate (pain with movements), and severe (significant pain at rest). Redness, induration, and swelling intensity were measured using a plastic bangle and categorized into mild (< 5 cm), moderate (5 – 10 cm), and severe (> 10 cm). Other local events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

The solicited systemic adverse events were fever, fatigue, and myalgia. Fever was graded into mild (38.0–38.4° C), moderate (38.5–38.9° C), and severe (≥ 39.0° C). Fatigue, myalgia, and other systemic events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

Table 14. Intensity of adverse events in the vaccine group of adults after the whole dose (n=15)

Mild			Moderate		Severe		Total
Adverse Events	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)	No. of events	No. of subjects (%)
Total Adverse Events	28	13 (86.67)	0	0 (0.00)	2	1 (6.67)	30	13 (86.67)
Total Solicited Adverse Events	20	10 (66.67)	0	0 (0.00)	2	1 (6.67)	22	11 (46.67)
Local reactions	8	5 (33.33)	0	0 (0.00)	2	1 (6.67)	10	6 (40.00)
Local pain	6	5 (33.33)	0	0 (0.00)	0	0 (0.00)	6	5 (33.33)
Redness	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Induration	1	1 (6.67)	0	0 (0.00)	1	1 (6.67)	2	2 (13.33)
Swelling	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)	1	1 (6.67)
Systemic events	12	8 (53.33)	0	0 (0.00)	0	0 (0.00)	12	8 (53.55)
Fever	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Fatigue	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Myalgia	11	7 (46.67)	0	0 (0.00)	0	0 (0.00)	11	7 (46.67)
Total Unsolicited Adverse Events	8	7 (46.67)	0	0 (0.00)	0	0 (0.00)	8	7 (46.67)
Respiratory, thoracic and mediastinal disorders	6	5 (33.33)	0	0 (0.00)	0	0 (0.00)	6	5 (33.33)
Cough	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Influenza	4	4 (26.67)	0	0 (0.00)	0	0 (0.00)	4	4 (26.67)
Oropharyngeal pain	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Eye disorders	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Eye pain	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Nervous system diseases	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Dizziness	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)

The solicited local adverse events were pain, redness, induration, and swelling. Pain was graded into mild (pain at injection site when touched), moderate (pain with movements), and severe (significant pain at rest). Redness, induration, and swelling intensity were measured using a plastic bangle and categorized into mild (< 5 cm), moderate (5 – 10 cm), and severe (> 10 cm). Other local events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

The solicited systemic adverse events were fever, fatigue, and myalgia. Fever was graded into mild (38.0–38.4° C), moderate (38.5–38.9° C), and severe (≥ 39.0° C). Fatigue, myalgia, and other systemic events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

Table 15. Intensity of adverse events in the vaccine group of elderly after the whole dose (n=12)

Mild			Moderate		Severe		Total
Adverse Events	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)
Total Adverse Events	14	5 (41.67)	0	0 (0.00)	0	0 (0.00)	14	5 (41.67)
Total Solicited Adverse Events	10	3 (25.00)	0	0 (0.00)	0	0 (0.00)	10	3 (25.00)
Local reactions	5	3 (25.00)	0	0 (0.00)	0	0 (0.00)	5	3 (25.00)
Local pain	4	3 (25.00)	0	0 (0.00)	0	0 (0.00)	4	3 (25.00)
Redness	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Induration	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Swelling	0	0 (0.00	0	0 (0.00)	0	0 (0.00)	0	0 (0.00
Systemic events	5	2 (16.67)	0	0 (0.00)	0	0 (0.00)	5	2 (16.67)
Fever	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Fatigue	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Myalgia	4	2 (16.67)	0	0 (0.00)	0	0 (0.00)	4	2 (16.67)
Total Unsolicited Adverse Events	4	4 (33.33)	0	0 (0.00)	0	0 (0.00)	4	4 (33.33)
Respiratory, thoracic, and mediastinal disorders	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Cough	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Gastrointestinal disorders	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Diarrhea	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Skin and subcutaneous tissue disorders	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Pruritus	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Nervous system diseases	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Dizziness	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)

The solicited local adverse events were pain, redness, induration, and swelling. Pain was graded into mild (pain at injection site when touched), moderate (pain with movements), and severe (significant pain at rest). Redness, induration, and swelling intensity were measured using a plastic bangle and categorized into mild (< 5 cm), moderate (5 – 10 cm), and severe (> 10 cm). Other local events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

The solicited systemic adverse events were fever, fatigue, and myalgia. Fever was graded into mild (38.0–38.4° C), moderate (38.5–38.9° C), and severe (≥ 39.0° C). Fatigue, myalgia, and other systemic events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

Table 16. Intensity of adverse events in the control group after the whole dose (n=27)

Mild			Moderate		Severe		Total
Adverse Events	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)
Total Adverse Events	48	16 (59.26)	1	1 (3.70)	0	0 (0.00)	49	16 (59.26)
Total Solicited Adverse Events	36	15 (55.55)	0	0 (0.00)	0	0 (0.00)	36	15 (55.55)
Local reactions	16	10 (37.03)	0	0 (0.00)	0	0 (0.00)	16	10 (37.03)
Local pain	13	10 (37.03)	0	0 (0.00)	0	0 (0.00)	13	10 (37.03)
Redness	2	2 (7.40)	0	0 (0.00)	0	0 (0.00)	2	2 (7.40)
Induration	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Swelling	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Systemic events	20	12 (44.44)	0	0 (0.00)	0	0 (0.00)	20	12 (44.44)
Fever	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Fatigue	7	6 (22.22)	0	0 (0.00)	0	0 (0.00)	7	6 (22.22)
Myalgia	13	10 (37.03)	0	0 (0.00)	0	0 (0.00)	13	10 (37.03)
Total Unsolicited Adverse Events	12	8 (29.62)	1	1 (3.70)	0	0 (0.00)	13	9 (33.33)
Respiratory, thoracic and mediastinal disorder	5	5 (18.51)	0	0 (0.00)	0	0 (0.00)	5	5 (18.51)
Influenza	3	3 (11.11)	0	0 (0.00)	0	0 (0.00)	3	3 (11.11)
Oropharyngeal pain	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Dyspnea	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Infections and infestations	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
COVID-19	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Gastrointestinal disorders	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Diarrhea	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Musculoskeletal and connective tissue diseases	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Backpain	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Skin and subcutaneous tissue disorders	1	1 (3.70)	1	1 (3.70)	0	0 (0.00)	1	1 (3.70)
Pruritus	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Subcutaneous hematoma	0	0 (0.00)	1	1 (3.70)	0	0 (0.00)	1	1 (3.70)
Nervous system diseases	2	2 (7.40)	0	0 (0.00)	0	0 (0.00)	2	2 (7.40)
Headache	2	2 (7.40)	0	0 (0.00)	0	0 (0.00)	2	2 (7.40)
Other general disorders	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)
Chills	1	1 (3.70)	0	0 (0.00)	0	0 (0.00)	1	1 (3.70)

The solicited local adverse events were pain, redness, induration, and swelling. Pain was graded into mild (pain at injection site when touched), moderate (pain with movements), and severe (significant pain at rest). Redness, induration, and swelling intensity were measured using a plastic bangle and categorized into mild (< 5 cm), moderate (5 – 10 cm), and severe (> 10 cm). Other local events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

The solicited systemic adverse events were fever, fatigue, and myalgia. Fever was graded into mild (38.0–38.4° C), moderate (38.5–38.9° C), and severe (≥ 39.0° C). Fatigue, myalgia, and other systemic events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

Table 17. Intensity of adverse events in the control group of adults after the whole dose (n=15)

Mild			Moderate		Severe		Total
Adverse Events	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)

Total Adverse Events	34	11 (73.33)	0	0 (0.00)	0	0 (0.00)	34	11 (73.33)
Total Solicited Adverse Events	25	11 (73.33)	0	0 (0.00)	0	0 (0.00)	25	11 (73.33)
Local reactions	9	6 (40.00)	0	0 (0.00)	0	0 (0.00)	9	6 (40.00)
Local pain	9	6 (40.00)	0	0 (0.00)	0	0 (0.00)	9	6 (40.00)
Redness	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Induration	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Swelling	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Systemic events	16	9 (60.00)	0	0 (0.00)	0	0 (0.00)	16	9 (60.00)
Fever	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Fatigue	6	5 (33.33)	0	0 (0.00)	0	0 (0.00)	6	5 (33.33)
Myalgia	10	7 (46.67)	0	0 (0.00)	0	0 (0.00)	10	7 (46.67)
Total Unsolicited Adverse Events	9	6 (40.00)	0	0 (0.00)	0	0 (0.00)	9	6 (40.00)
Respiratory, thoracic and mediastinal disorders	4	4 (26.67)	0	0 (0.00)	0	0 (0.00)	4	4 (26.67)
Influenza	2	2 (13.33)	0	0 (0.00)	0	0 (0.00)	2	2 (13.33)
Oropharyngeal pain	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Dyspnea	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Infections and infestations	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
COVID-19	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Gastrointestinal disorders	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Diarrhea	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Skin and subcutaneous tissue disorders	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Pruritus	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Nervous system diseases	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Headache	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Other general disorders	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)
Chills	1	1 (6.67)	0	0 (0.00)	0	0 (0.00)	1	1 (6.67)

The solicited local adverse events were pain, redness, induration, and swelling. Pain was graded into mild (pain at injection site when touched), moderate (pain with movements), and severe (significant pain at rest). Redness, induration, and swelling intensity were measured using a plastic bangle and categorized into mild (< 5 cm), moderate (5 – 10 cm), and severe (> 10 cm). Other local events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

The solicited systemic adverse events were fever, fatigue, and myalgia. Fever was graded into mild (38.0–38.4° C), moderate (38.5–38.9° C), and severe (≥ 39.0° C). Fatigue, myalgia, and other systemic events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

Table 18. Intensity of adverse events in the control group of elderly after the whole dose (n=12)

Mild			Moderate		Severe		Total
Adverse Events	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)	No. of events	No. of Subjects (%)
Total Adverse Events	14	5 (41.67)	1	1 (8.33)	0	0 (0.00)	15	5 (41.67)
Total Solicited Adverse Events	11	4 (33.33)	0	0 (0.00)	0	0 (0.0)	11	4 (33.33)
Local reactions	7	4 (33.33)	0	0 (0.00)	0	0 (0.00)	7	4 (33.33)
Local pain	4	4 (33.33)	0	0 (0.00)	0	0 (0.00)	4	4 (33.33)
Redness	2	2 (16.67)	0	0 (0.00)	0	0 (0.00)	2	2 (16.67)
Induration	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Swelling	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Systemic events	4	3 (25)	0	0 (0.00)	0	0 (0.00)	4	3 (25)
Fever	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)	0	0 (0.00)
Fatigue	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Myalgia	3	3 (25)	0	0 (0.00)	0	0 (0.00)	3	3 (25)
Total Unsolicited Adverse Events	3	2 (16.67)	1	1 (8.33)	0	0 (0.00)	4	3 (25)
Respiratory, thoracic and mediastinal disorders	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Influenza	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Musculoskeletal and connective tissue diseases	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Backpain	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Skin and subcutaneous tissue disorders	0	0 (0.00)	1	1 (8.33)	0	0 (0.00)	1	1 (8.33)
Subcutaneous hematoma	0	0 (0.00)	1	1 (8.33)	0	0 (0.00)	1	1 (8.33)
Nervous system diseases	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)
Headache	1	1 (8.33)	0	0 (0.00)	0	0 (0.00)	1	1 (8.33)

The solicited local adverse events were pain, redness, induration, and swelling. Pain was graded into mild (pain at injection site when touched), moderate (pain with movements), and severe (significant pain at rest). Redness, induration, and swelling intensity were measured using a plastic bangle and categorized into mild (< 5 cm), moderate (5 – 10 cm), and severe (> 10 cm). Other local events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

The solicited systemic adverse events were fever, fatigue, and myalgia. Fever was graded into mild (38.0–38.4° C), moderate (38.5–38.9° C), and severe (≥ 39.0° C). Fatigue, myalgia, and other systemic events were graded into mild (no interference with activity), moderate (some interference with activity not requiring medical intervention), and severe (limited daily activity, requiring medical intervention).

This observer-blinded, comparative, and randomized phase I clinical trial was conducted in 54 healthy participants of Indonesian population, who were divided into adult (n = 30) and elderly (n = 24) groups.

In the present study, we found that a three-dose regimen of the candidate vaccine, CoV2-Bio COVID-19, was well tolerated in healthy adults and elderly. The incidence rate of adverse events in both age groups was not significantly different (P = 0.573).

The most frequent local reaction was local pain (33.33%), whereas myalgia was the most common systemic event (35.18%). This result was in accordance with the previous findings about other COVID-19 vaccines.^5–7 Additionally, we noticed two severe local reactions in the vaccine group: swelling and induration. Although, those events were transient and self-limiting. There was one moderate unsolicited adverse event in the control group, which was a subcutaneous hematoma. However, it was considered not related to the vaccine according to the examination. On the other hand, one participant in the control group had a serious adverse event, but based on the examination and expertise of a specialized doctor and DSMB, we decided that it was not associated with the vaccination.

In terms of severity, the majority of adverse events were mild and resolved spontaneously within the first 24–48 hours after onset.

Based on the safety described in this study, it can be concluded that the three doses of CoV2-Bio COVID-19 vaccine were safe in healthy adults and elderly.

We concluded that the CoV2-Bio COVID-19 vaccine was well tolerated and safe in Indonesian populations, including healthy adults and elderly.

RBD = Receptor binding domain
CoV2-Bio COVID-19 = SARS-CoV-2 protein subunit recombinant vaccine developed by BioFarma Pharmaceutical Company
CRF = Case report form
DSMB = Data Safety Monitoring Board

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The study protocol and all amendments were reviewed and approved by Health Research Ethics Committee of Faculty of Medicine, Universitas Indonesia – Cipto Mangunkusumo Hospital (KET-845/UN2.F1/ETIK/2021) in compliance with local law (No. 845/UN2.F1/ETIK/PPM.00.02/2021). This study has also been registered in ClinicalTrials.gov ID: NCT05067894 on October 5, 2021.

The investigator or delegate obtained the written informed consent from all subjects prior to any study-related procedures were performed and after the subjects had been informed of the nature of the trials, the potential risks, and subjects’ obligations.

This trial was conducted in accordance with the latest Edinburg, Scotland revision of the Declaration of Helsinki, ICH Good Clinical Practice guidelines, and local regulatory requirements.

CONSENT FOR PUBLICATION

Not applicable

AVAILABILITY OF DATA AND MATERIALS

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

COMPETING INTERESTS

This study was funded by PT Bio Farma, Indonesia. Data analysis and interpretation were solely done by investigators and the funding body was not involved in this process. The authors declared no competing interests.

FUNDING

All steps of this study, starting from study planning, recruitment, study continuity, to the salaries of investigators and research team were funded by PT Bio Farma, Indonesia.

AUTHORS’ CONTRIBUTIONS

SK designed and planned the study, composed the initial manuscript draft, reviewed, and edited the final manuscript. SM, RS, SS, BEM, IY, WI, AW, WR, and CO were involved in the data collection and subjects recruitment, and critically reviewed the manuscript. RMS designed the study, provided the vaccines, analyzed the blood samples, and critically reviewed the manuscript. NPA reviewed the design of the study and critically reviewed the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGEMENTS

We acknowledge the following colleagues for their contributions to this study: dr. Otty Mitha Sevianti, SpA.; dr. Meita Dwi Utami, SpA.; dr. Agnes Tri Harjaningrum, MsC, SpA.; dr. Andini Striratnaputri, SpA.; Meidyna Dwi Puspa, SKM.; Yuni Yudha Aprilia, Amd. Keb.; Raden Roro Maulidya Arifianti Ningtyas, SKM.; Ardhya Ridha Pranada Siagian Putri, SKM.; and Kevin Sebastian Santoso, SKM. We would also like to thank the staffs of Pancoran Mas primary health center: dr. Sih Mahayanti; dr. Chandra Suryani; Eta Rahmawati; Widia Hastuti; and Zulfa Majidah. For the kind support for the trial, we would like to thank dr. Rini Mulia Sari and the staffs of Bio Farma; and Ade Risman and Prodia Laboratory’s staffs. Lastly, we would like to thank the National Institute of Health Research & Development (NIHRD)/Litbangkes for the helpful support.

AUTHORS INFORMATION

SK and SM are affiliated with Department of Internal Medicine, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo General National Hospital, Jakarta, Indonesia
RS, SS, BEM, IY, WI, AW, WR, and CO are affiliated with Department of Child Health, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mengunkusumo General National Hospital, Jakarta, Indonesia
RMS is affiliated with PT. Bio Farma, Bandung, Indonesia 4 National Institute of Health Research and Development, Ministry of Health Republic of Indonesia
NPA is affiliated with Department of Community Medicine, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo General National Hospital, Jakarta, Indonesia

Hotez PJ, Bottazzi ME. Developing a low-cost and accessible COVID-19 vaccine for global health.PLoS Negl Trop Dis. 2020 Jul29;14(7):e0008548.
Dong Y, Dai T, Wei Y, Zhang L, Zheng M, Zhou F. A systematic review of SARS-CoV-2 vaccine candidates. Signal Transduct Target Ther. 2020 Oct;13:5:237.
To KKW, Tsang OTY, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020 May;20(5):565–74.
Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.Science. 2020 Mar27;367(6485):1444–8.
Cai Y, Zhang J, Xiao T, Peng H, Sterling SM, Walsh RM et al. Distinct conformational states of SARS-CoV-2 spike protein. Science. 2020 Jul 21;eabd4251.
Dai L, Gao GF. Viral targets for vaccines against COVID-19. Nat Rev Immunol. 2021;21(2):73–82.
Pormohammad A, Zarei M, Ghorbani S, Mohammadi M, Razizadeh MH, Turner DL et al. Efficacy and safety of COVID-19 vaccines: a systematic review and meta-analysis of randomized clinical trials. Vaccines (Basel). 2021 May;9(5):467.
Karbalaei M, Rezaee SA, Farsiani H. Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol. 2020 Sep;235(9):5867–81.
Kumar R, Kumar P. Yeast-based vaccines: new perspective in vaccine development and application. FEMS Yeast Res. 2019 Mar 1;19(2):foz007.
Kleanthous H, Silverman JM, Makar KW, Yoon IK, Jackson N, Vaughn DW. Scientific rationale for developing potent RBD-based vaccines targeting COVID-19. NPJ Vaccines. 2021 Oct;28(1):1–10.
Wu Z, Hu Y, Xu M, Chen Z, Yang W, Jiang Z et al. Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine (CoronaVac) in healthy adults aged 60 years and older: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial. The Lancet Infectious Diseases. 2021 Jun 1;21(6):803–12.

No competing interests reported.

Safety of Cov2-Bio COVID-19 Vaccine in Healthy Populations Aged 18 years and Above in Indonesia

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Trial registration:

Background

Methods

Study design

Sample size

Participants enrolment

Safety evaluation

Study intervention

Candidate vaccine

Comparator vaccine

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1