Enrolment and follow-up

Between 28 May 2021 and 2 July 2021, 132 individuals were screened (36 were enroled in each age cohort, with 12 per dose group in each age cohort) (Extended Data Figs. 1 and 2). All but one participant completed both vaccinations. The participant declining a second vaccination was from the adult 25 μg group and experienced moderate myalgia after the first vaccination, which resolved within 3 d and was accompanied by moderate creatinine phosphokinase elevation but normal cardiac enzymes, possibly related to strenuous physical labour. This participant was excluded from the per protocol immunogenicity analysis, as were two others: one participant from the adult 10 μg group who was diagnosed with asymptomatic COVID-19 on the sixth day after the second vaccination and one participant from the adult 50 μg group who had confirmed positive baseline anti-receptor binding domain (RBD) binding antibody test but was negative for SARS-CoV-2 molecular testing, indicating asymptomatic COVID-19 before enrolment. All participants contributed to the safety analysis.

Demographic and baseline clinical characteristics

Characteristics of the study participants are listed in Table 1. Mean ages(s.d.) in the adult and elderly cohorts were 35.8 (9.1) and 65.1 (5.4) yr, respectively; body mass indices were 23 (2.8) and 23.3 (3.3), and proportions of females were 67% and 53%, respectively.

Table 1 Demographic characteristics of the participants by dose age groupsSafety and tolerability of ChulaCov19

The most common local reaction was injection site pain. Its incidence was dose-dependent, more common in adults than the elderly, and after Dose 2. All local reactions were mild in the elderly; some moderate events occurred in adults. All participants recovered after 2.79 ± 1.7 and 1.91 ± 0.9 d on average in adults and the elderly after Dose 2, respectively. A single event of severe erythema in the adult 10 μg group after Dose 2 resolved within 6 d (Fig. 1).

Fig. 1: Local and systemic adverse events by dose group and age cohort.

a,b, Severity of solicited local and systemic reactions in the adult cohort (18–55 yr old) (a) and the elderly cohort (56–75 yr old) (b). Data on local and systemic reactions were collected using paper diaries for 7 d after each vaccination. Injection-site (local) reactions were: pain at injection site (mild: does not interfere with activity; moderate: interferes with activity; severe: prevents daily activity; and grade 4: emergency room visit or hospitalization), redness and swelling (mild: 2.5–5.0 cm in diameter; moderate: 5.1–10.0 cm in diameter; severe: >10.0 cm in diameter; and grade 4: necrosis or exfoliative dermatitis). Solicited systemic events were: fever (mild: 38.0–38.4 °C; moderate: 38.5–38.9 °C; severe: 39.0–40.0 °C; and grade 4: >40.0 °C), chills, headache, fatigue, myalgia and arthralgia (all were graded as mild and did not interfere with the daily activities of the participants), moderate (interferes with daily activities), severe (prevents daily activities) or grade 4 (led to an emergency department visit or hospitalization). The numbers above the bars show the overall percentage of the participants in each group who reported the specified systemic event.

Source data

The three most common systemic reactions were fever, headache and fatigue. Systemic reactions were more common at the 25 μg and 50 μg doses, after Dose 2, and in adults. Most reactions were mild to moderate and transient, with mean duration of 1.97 ± 1.2 d in adults and 1.39 ± 0.5 d in the elderly after Dose 2 (Fig. 1).

Immunogenicity of ChulaCov19SARS-CoV-2 RBD antibody responses

In the adult cohort 4 weeks after the second vaccination (day 50), the geometric mean titre (GMT) of anti-RBD antibody was significantly higher than for human convalescent sera 4 weeks after COVID-19 diagnosis (Fig. 2a), with geometric mean ratios (GMR) of 3.78 (95%CI 1.64–8.68), 3.23 (1.41–7.42) and 7.74 (3.37–17.77), respectively (P ≺ 0.01) (Supplementary Table 10). In the elderly cohort, anti-RBD GMTs were also dose-dependent but lower than observed in the adult cohort (Fig. 2a and Supplementary Table 6).

Fig. 2: Immunogenicity assay responses to ChulaCov.

a, The GMTs of anti-RBD antibody measured by ELISA. b, The GMTs of IgG antibody against S trimer protein of SARS-CoV-2 measured by ELISA. c, The GMTs of neutralizing antibody against wild-type SARS-CoV-2 (Micro-VNT50). *Fold-difference as compared to the adults’ GMT, **Comparison of GMTs of 50 μg dose in adults at day 29. d, The GMTs of pseudovirus neutralizing antibody against wild-type SARS-CoV-2 (PsVNT-50). The right side of a, c and d indicate two reference values from 27 Pfizer/BioNTech mRNA Malaysian vaccinees. The serum samples were collected on day 29 after the first dose. Thirty HCS were collected at 4 weeks after confirmed diagnosis. Error bars indicate 95% confidence intervals. The numbers above the bars show the GMT of each group.

Source data

In the adult cohort at day 50, ChulaCov19 at 50 μg elicited the highest anti-spike GMT at 24,493.9 binding antibody units ml−1 (P < 0.01). In the elderly cohort, the anti-spike GMT was lower than that of the adults at all three doses, while anti-spike GMT for the 50 μg dose was significantly higher than for the 10 μg dose (P < 0.001, Fig. 2b). GM of % RBD-ACE2 binding inhibition showed a dose-dependent pattern in both age cohorts at day 29 but not at day 50. All doses at day 50 in both age cohorts elicited GM > 90% inhibition. Inhibition (%) of the human convalescent serum (HCS) and Pfizer/BNT vaccinee’s (day 29) panels were 76% and 93%, respectively (Supplementary Fig. 1).

SARS-CoV-2-specific live-virus micro-neutralization tests (MicroVNT-50)

Seroconversion rate on MicroVNT-50 in all dose groups reached 100% by day 50. GMT of the responses against wild-type (WT) virus were both dose- and age-dependent, and markedly increased from day 29 to day 50 for the 10, 25 and 50 μg doses (Fig. 2c). In the adult cohort at day 50, ChulaCov19 at 10, 25 and 50 μg doses induced significantly higher MicroVNT-50 GMT than HCS at a ratio of 2.98 (95%CI 1.63–5.44), 2.59 (1.42–4.72) and 4.01 (2.2–7.31), respectively (P < 0.01), whereas in the elderly cohort the values were 0.48 (0.25–0.92), 1.26 (0.67–2.36) and 2.09 (1.12–3.91), respectively (Supplementary Table 7). ChulaCov19 at the 50 μg dose induced significantly higher MicroVNT-50 GMT against WT and 3 variants compared with the 10 μg dose in both age cohorts, while elicited MicroVNT-50 GMTs were lower for all doses in the elderly cohort compared with the adult cohort (Fig. 3a and Supplementary Table 8).

Fig. 3: SARS-CoV-2 neutralization responses against VOCs.

The serum samples were collected on day 29 after the first dose from participants in each cohort (adult n = 11 per dose, elderly n = 12 per dose). The numbers above the bars show the GMT in the group. Error bars indicate 95% confidence intervals. a, The GMTs of neutralizing antibody against the SARS-CoV-2 VOCs Alpha, Beta and Delta. b, The GMTs of pseudovirus neutralizing antibody against SARS-CoV-2 VOCs Alpha, Beta, Gamma and Delta. The right side of b indicates the reference values from 27 Pfizer/BioNTech mRNA Malaysian vaccinees. The serum samples were collected on day 29 after the first dose.

Source data

Pseudovirus neutralization tests (PsVNT-50)

In the adult cohort, PsVNT-50 GMTs (95% CI) of ChulaCov19 against WT at day 50 for 10, 25 and 50 μg and for the HCS panel were 885.3 (95%CI, 438.2–1,788.7), 902.9 (445.5–1,829.7), 1,273.1 (874.2–1,854) and 471.2 (290.5–764.5), respectively (Fig. 2d). The GMT ratio of 50 μg dose/HCS was 2.7 (P = 0.01). At day 29, ChulaCov19 at 50 μg dose induced significantly higher GMT than the Pfizer/BNT vaccinated panel against Alpha, Beta and Gamma variants at a ratio of 4.47, 11.94 and 4.23 (P = 0.01), respectively (Fig. 3b and Supplementary Table 9). PsVNT-50 test results for a new variant, Omicron, showed that two doses of ChulaCov19 at 50 mg elicited a substantial decline in GMT against the Omicron variant compared with WT (Supplementary Fig. 2).

SARS-Cov-2 spike-specific T-cell responses

At day 29, all participants in the adult and elderly cohorts at 10, 25 and 50 μg showed strong spike-specific T-cell responses measured by IFNγ-ELISpot tests. The responses were lower in the elderly at 10 and 25 μg than in adults (Fig. 4a and Supplementary Table 11). In adults, both spike-specific IFNγ+CD4+ and IL2+CD4+ T-cell percentages were notably higher in the 25 and 50 μg dose recipients compared with the 10 μg dose (Supplementary Table 12). ChulaCov19 elicited spike-specific Th1-dominated responses (Fig. 4b).

Fig. 4: Magnitude of ChulaCov-induced CD4+ and CD8+ T-cell responses.

PBMCs were obtained on day 29 after the first dose from participants in each cohort (adult n = 11 per dose, elderly n = 12 per dose). The numbers above the bars show the GMT in the group. Error bars indicate 95% confidence intervals. a, SARS-Cov-2 wild-type spike-specific IFN-γ ELISPOT T cells. *Comparison of the adults’ GMTs shown as fold-difference. b, SARS-CoV-2 spike-protein-specific CD4+ and CD8+ T-cell responses, and Th1/Th2 polarization responses quantified by intracellular cytokine staining.

Source data

Vaccine stability

We assessed ChulaCov19 appearance, pH, osmolality and lipid content under varying storage conditions. The key parameters related to vaccine stability are particle size, mRNA integrity and mRNA encapsulation. We detected no substantial change in these parameters at −75 °C for up to 9 months, at −20 °C for up to 6 months, or at 2–8 °C for up to 3 months. However, there was a small increase in particle size (72, 76 vs 78 nm, respectively; nevertheless, all remained within the acceptance criteria of 50–120 nm) when vaccine was stored at 2–8 °C at 6 months. Moreover, the mRNA integrity further decreased after 6 months at 2–8 °C, to 54% as compared to at −20 °C. Thus, all parameters remained in line with the vaccine specification at 6 months for all storage conditions and there were no substantial changes for any parameter tested at −75 °C and −20 °C. There were no remarkable changes in any parameter after 3 months storage at 2–8 °C and small changes after 6 months, but all the results remained within specification and where specifications are not set, the changes were minimal and would not be expected to have any impact on the functionality of the LNP (although note that this was not tested). These conclusions will be verified by future stability studies to be performed in future clinical development (Supplementary Table 15).