Trial Population

In both studies, the demographic and baseline clinical characteristics of the subjects were comparable across vaccination groups (Table 1). In the phase I study, the mean age was 31.4 years and 61.3 years among younger and older adults, with a comparable gender distribution in both age groups. Most subjects were White (55.0%) and Asian (35.0%). All 100 subjects except for one in the older group received at least one study vaccination and 98 subjects completed the full vaccination. In the phase II study, 347 subjects were randomized: 66.5% were male, all were Asians of Philippine descent, and the mean age was 31.8 years. About half (51.4%) of the enrolled subjects were SARS-CoV-2 IgM or IgG positive at baseline due to the COVID-19 surge during the enrollment period. All subjects except one received at least one study vaccination and 338 subjects completed the full vaccination. Subjects were followed up regularly throughout the study period, and any individual feeling unwell and/or suspected of being infected with the SARS-CoV-2 virus (presenting with fever, cough, and other respiratory symptoms) was tested for SARS-CoV-2 nucleic acid to confirm infection. No COVID-19-positive individuals were identified. Figure 1 presents flow diagrams for phase I and phase II studies.

Table 1 Demographic characteristics of subjects in phase I and phase II studiesSafety Outcomes

In both studies, no vaccination-related SAE, AESI, AE leading to early discontinuation, or clinically significant abnormalities in vital signs or laboratory results related to study vaccination were reported.

In the phase I study, the incidences of AEs were generally higher in ReCOV groups than those in the placebo group (normal saline). In the pooled ReCOV group and placebo group, the incidences of solicited local AEs were 65.0% and 10.0% among younger adults and 69.2% and 10.0% among older adults, respectively; the incidences of solicited systemic AEs were 60.0% and 30.0% among younger adults and 51.3% and 50.0% among older adults, respectively (Fig. 2A, B); the incidences of unsolicited AEs were 65.0% and 20.0% among younger adults and 59.0% and 70.0% in older adults (Table S1 in the supplementary material). Unexpectedly, the incidences of AEs in the 40 μg ReCOV group were remarkably lower in the phase II study than in the phase I study, and appeared to be similar to placebo group (all compositions in ReCOV vaccine except for the antigen): the incidences of solicited local AEs, solicited systemic AEs, and unsolicited AEs were 13.4%, 19.0%, and 7.8% in the ReCOV group and 16.5%, 13.9%, and 8.6% in the placebo group, respectively (Fig. 2C, Table S1).

Fig. 2

Frequency of solicited local and systemic AEs after the first or second dosing with two dose levels of ReCOV among younger adults (A) and older adults (B) in the phase I study and with 40 μg ReCOV among adults in the phase II study (C)

In the phase I study, although the incidences of solicited local AEs tended to be higher in the 40 μg ReCOV group (younger adults 70.0%, older adults 84.2%) than those in the 20 μg ReCOV group (younger adults 60.0%, older adults 55.0%), the incidences of solicited systemic AEs appeared unrelated to the dose level (Fig. 2), and unsolicited AEs were similar between the two dose level groups (Table S1).

The incidences of solicited AEs after the first and second dosing were compared. In the phase I study, the incidences of solicited AEs after the second dosing tended to be higher than those post the first dosing. In the pooled ReCOV group, the incidences of solicited local and systemic AEs were respectively 48.1% and 21.5% after the first dosing and 58.2% and 49.4% after the second dosing (Fig. 2A, B). However, different data trends in the ReCOV group were observed in the phase II study: the incidences of solicited local and systemic AEs were respectively 10.7% and 13.8% after the first dosing and 5.4% and 8.6% after the second dosing (Fig. 2C).

Consistent common AEs in ReCOV groups were observed in the phase I and phase II studies. In the phase I study, the common (≥ 10%) solicited local AEs were injection site reactions (first dose 48.1%, second dose 58.2%); the common (≥ 10%) solicited systemic AEs were fatigue (first dose 13.9%, second dose 34.2%), myalgia (first dose 6.3%, second dose 30.4%), headache (first dose 8.9%, second dose 27.8%), and pyrexia (first dose 0, second dose 12.7%) (Fig. 2A, B); all solicited AEs in ReCOV groups were transient (median duration 4 days, pooled ReCOV group) and mild in severity, except for one young adult in the 20 µg ReCOV group who developed moderate pyrexia (Tables S2 and S3 in the supplementary material). In the phase II study, the common (≥ 5%) solicited AEs in the ReCOV group were injection site pain (11.6%), headache (8.9%), and pyrexia (8.0%), as shown in Fig. 2C; all solicited AEs were transient (median duration 1.0–4.0 days) and mild or moderate in severity, except for one subject in the ReCOV group who experienced one grade 3 injection site rash and three subjects in the ReCOV group who experienced three grade 3 events of pyrexia (Table S4 in the supplementary material). Similarly, almost all unsolicited AEs were mild to moderate in severity in both studies (Table S1). In the phase I study, the common (≥ 10%) unsolicited AEs in ReCOV groups were upper respiratory tract infection. In the phase II study, no common unsolicited AE was detected in the ReCOV group.

Immunogenicity Outcomes

Robust neutralization against SARS-CoV-2 prototype was consistently shown in ReCOV groups for both phase I and phase II, starting from 21 days post the first dosing, peaked at 14 days post the second dosing, and persisted till the end of the studies, i.e., 6 months post the second dosing. In addition, strong cross-neutralization against the prototype, Omicron BA.2, and BA.4/5 were observed in the phase II study. The phase I study also proved that ReCOV elicited persistent Th1 biased cellular response.

Neutralizing Antibody Response Against SARS-CoV-2 Prototype

In the phase I study, live-virus NAb against the prototype was elicited in most subjects at 21 days after the first dosing of both 20 μg and 40 μg ReCOV, with an SCR of 95.0% and 100% in younger adults and 70.0% and 94.4% in older adults, respectively. After the second ReCOV dosing, the SCR reached 100% at 14 days and remained at this level till the end of the study, i.e., at least 6 months after the dosing, irrespective of dose level and age group. No subject in pooled placebo groups was seropositive at all timepoints tested (Table S5 in the supplementary material). The high seroconversion elicited by 40 μg ReCOV was confirmed in the phase II study. Among subjects seronegative for SARS-CoV-2 IgG/IgM at baseline, a similar population to that in the phase I study, the SCR was significantly higher in the ReCOV group (99.1%) than that in the placebo group (5.8%) at 14 days post the second dosing, and remained at 88.8% in the ReCOV group at the end of the study (Table S6 in the supplementary material).

Both phase I and phase II studies showed that the GMT of live-virus NAb against the prototype reached peak level at 14 days post the second dosing, then decreased over time while remaining at a high level till the end of the studies (Figs. 3 and 4). In the phase I study, high levels of GMTs were demonstrated in both younger and older adults, although the GMTs tended to be lower in older groups as expected. Both 20 μg and 40 μg ReCOV induced high levels of NAb throughout the study period, 40 μg ReCOV tended to elicit higher levels post the first dosing, while 20 μg ReCOV led a stronger response post the second dosing. At 21 days post the first dosing, the GMTs in 20 μg and 40 μg ReCOV groups were 118.0 IU/mL (95% CI 70.5, 197.3) and 135.5 IU/mL (95% CI 88.4, 207.7) among younger adults and 59.0 IU/mL (95% CI 28.5, 122.2) and 78.7 IU/mL (95% CI 47.0, 131.8) among older adults, respectively. The peak GMTs, at 14 days post the second dosing in 20 μg and 40 μg ReCOV groups, were 1643.2 IU/mL (95% CI 1188.5, 2271.9) and 1289.2 IU/mL (95% CI 868.3, 1914.1) among younger adults, with corresponding GMIs of 265.0 and 208.0, while among older adults, the peak GMTs were 1122.3 IU/mL (95% CI 722.6, 1743.1) and 680.3 IU/mL (95% CI 440.2, 1051.4), with corresponding GMIs of 174.9 and 109.7, respectively. At end of the study, 6 months post the second dosing, the GMTs in the two ReCOV groups were still above the level post the first dosing, i.e., 357.6 IU/mL (95% CI 251.1, 509.2) and 293.0 IU/mL (95% CI 168.1, 510.7) among younger adults and 178.8 IU/mL (95% CI 125.6, 254.6) and 268.7 IU/mL (95% CI 131.7, 548.3) among older adults, respectively (Fig. 3).

Fig. 3

GMTs of neutralizing antibody against SARS-CoV-2 prototype in the phase I study. The GMTs of neutralizing antibody at baseline, before the second vaccination (21p1), 14 days (14p2), 30 days (30p2), 3 months (3mp2), and 6 months (6mp2) post the second vaccination, respectively, are illustrated for younger adults (A) and older adults (B)

Fig. 4

GMTs of neutralizing antibody against SARS-CoV-2 prototype in the phase II study. The GMTs of neutralizing antibody at baseline, 14 days (14p2) and 6 months (6mp2) post the second vaccination are illustrated for the overall study population (Overall), subjects with SARS-CoV-2 seronegative status at baseline (Seronegative), and subjects with SARS-CoV-2 seropositive status at baseline (Seropositive)

The strong and persistent live-virus NAb elicited by ReCOV was further demonstrated in the phase II study (Fig. 4). At baseline, the GMTs were comparable between vaccination groups; however, they were at least 20 times higher in subjects with SARS-CoV-2 seropositive status at baseline (936.0 IU/mL, 95% CI 704.8, 1242.9) than those in subjects with seronegative status at baseline (44.4 IU/mL, 95% CI 34.7, 56.8). At 14 days post the second dosing, the GMTs in ReCOV groups were significantly higher than those in placebo groups, irrespective of serostatus at baseline: in seronegative subjects, the GMTs were 3741.0 IU/mL (95% CI 3113.4, 4495.0) and 41.7 IU/mL (95% CI 30.4, 57.1), with GMIs of 84.3 and 1.1, respectively, while in seropositive subjects, the GMTs were 6138.3 IU/mL (95% CI 5255.1, 7169.9) and 540.3 IU/mL (95% CI 391.0, 746.8), with GMIs of 6.6 and 0.8, respectively (all p values < 0.0001). In the ReCOV group, the NAbs remained at high level at the end of the study, with GMTs of 1094.9 IU/mL (95% CI 894.6, 1340.2) and 1569.8 IU/mL (95% CI 1315.4, 1873.5) in seronegative and seropositive subjects, respectively.

In addition, pseudovirus NAbs against SARS-CoV-2 prototype was also evaluated in the phase II study (Fig. 5), with results consistent with those of the live-virus neutralization. A significant correlation in titers of NAbs measured by live-virus and pseudovirus-based tests was established (Fig. 6), with an overall Pearson’s correlation coefficient of 0.95 (p < 0.0001).

Fig. 5

GMTs of pseudovirus neutralizing antibody against SARS-CoV-2 prototype and Omicron variants in the phase II study. The GMTs of pseudovirus NAb against SARS-CoV-2 prototype, Omicron BA.2, and BA.4/5 at baseline, 14 days (14p2) and 6 months (6mp2) post the second vaccination respectively are illustrated for the overall study population (Overall), subjects with SARS-CoV-2 seronegative status at baseline (Seronegative), and subjects with SARS-CoV-2 seropositive status at baseline (Seropositive)

Fig. 6

Correlation in titers of neutralizing antibody measured by live-virus and pseudovirus neutralizing antibody tests in the phase II study. The overall Pearson’s correlation coefficient was 0.95 (p < 0.0001), indicating a significant correlation in titers of NAb measured by live-virus and pseudovirus-based methodologies among all tested timepoints, i.e., at baseline, 14 days and 6 months post the second vaccination

Neutralizing Antibody Response Against Omicron Variants

Pseudovirus NAbs against the prototype, Omicron BA.2, and BA.4/5 were evaluated in the phase II study. Among seronegative subjects at baseline, the SCRs for Omicron BA.2 and BA.4/5 were 91.7% and 91.7% with GMIs of 56.3 and 59.9, respectively, in the ReCOV group, while in the placebo group, the SCRs were 11.5% and 11.5% with GMIs of 1.3 and 1.3, respectively. Among seropositive subjects at baseline, the SCRs for Omicron BA.2 and BA.4/5 were 50.5% and 58.6% with GMIs of 6.3 and 6.1 in the ReCOV group and 11.7% and 10.0% with GMIs of 1.0 and 0.8 in the placebo group, respectively (Table S6).

At baseline, the GMTs for both Omicron strains were similar between vaccination groups, but were around 15–20 times higher in seropositive subjects than those in seronegative subjects, as shown in Fig. 5. At 14 days post the second dosing, the levels of NAb against both Omicron strains in ReCOV groups were significantly higher than those in placebo groups, irrespective of serostatus at baseline. Among seronegative subjects, the GMTs against the prototype, BA.2, and BA.4/5 were 8856.6 (95% CI 7136.7, 10,991.1), 4441.0 (95% CI 3348.8, 5889.4), and 2644.3 (95% CI 1990.4, 3513.0) in the ReCOV group and 88.6 (95% CI 57.1, 137.2), 76.8 (95% CI 43.7, 134.8), and 43.0 (95% CI 25.6, 72.2) in the placebo group, respectively; among seropositive subjects, the GMTs were 15,667.3 (95% CI 13,511.5, 18,167.0), 7334.3 (95% CI 6160.1, 8732.4), and 4478.8 (95% CI 3829.8, 5237.9) in the ReCOV group and 1548.0 (95% CI 1067.0, 2245.9), 968.4 (95% CI 653.2, 1435.7), and 526.0 (95% CI 357.7, 773.5) in the placebo group, respectively (all p values < 0.0001) (Table S6). Among subjects receiving ReCOV, the GMTs were only around 2.1-fold lower for BA.2 and around 3.5-fold lower for BA.4/5, comparing to the levels for the prototype, irrespective of serostatus at baseline (Fig. 5). Consistent with the results of the prototype, at 6 months post the second dosing, the GMTs against Omicron BA.2 and BA.4/5 remained at high levels in the ReCOV group, i.e., 1447.2 (95% CI 1120.98, 1868.39) and 1052.6 (95% CI 828.6, 1337.3), respectively, among seronegative subjects and 1474.9 (95% CI 1227.1, 1772.7) and 985.9 (95% CI 829.2, 1172.3), respectively, among seropositive subjects, only 2.5-fold to 5.2-fold lower compared to the corresponding peak levels at 14 days post the second dosing (Fig. 5, Table S6).

Spike RBD- and NTD-Specific IgGs

In the phase I study, consistently low levels of RBD- and NTD-specific IgGs were observed in all study groups at baseline. Comparable peak GMTs of RBD- and NTD-specific IgGs were shown in 20 μg and 40 μg ReCOV groups, irrespective of age groups. Among older adults, the peak GMTs tended to be 1.3–1.7 times lower compared to that among younger adults; however, the antibody levels were still high and persisted throughout all testing timepoints tested. In 20 μg and 40 μg ReCOV groups, the peak GMTs of RBD- and NTD-specific IgGs were 278,596.1 AU/mL (95% CI 206,532.0, 375,805.1) and 11,232.4 AU/mL (95% CI 8273.6, 15,249.4) in the 20 μg ReCOV group and 271,838.2 AU/mL (95% CI 216471.5, 341,366.0) and 11,266.5 AU/mL (95% CI 8420.5, 15,074.4) in the 40 μg ReCOV group among younger adults, while they were 185,337.6 AU/mL (95% CI 136,635.2, 251,399.5) and 9422.8 AU/mL (95% CI 6846.1, 12,969.3) in the 20 μg ReCOV group and 203,829.7 AU/mL (95% CI 145,482.5, 285,577.6) and 6705.9 AU/mL (95% CI 4657.3, 9655.6) in the 40 μg ReCOV group among older groups, respectively. At 6 months post the second dosing, the GMTs of RBD- and NTD-specific IgGs were still above the level of post the first dosing, irrespective of dose levels and age groups (Fig. 7).

Fig. 7

GMTs of SARS-CoV-2 Spike RBD- and NTD-specific IgG in the phase I study. The GMTs of SARS-CoV-2 Spike RBD- and NTD-specific IgG at baseline, before the second vaccination (21p1), 14 days (14p2), 30 days (30p2), 3 months (3mp2), and 6 months (6mp2) post the second vaccination are illustrated for the pooled placebo group, 20 µg ReCOV group, and 40 µg ReCOV group

Cellular Immune Responses

The cellular immune response was evaluated in the phase I study. At baseline, very low levels (≤ 0.06%) in average percentage of CD4+ T cells secreting antigen-specific IL-2 or IFNγ were observed at baseline among all study groups. In younger subjects, the average percentage of CD4+ T cells with IL-2 or IFNγ secretion increased to 0.3% (20 μg ReCOV) and 0.2% (40 μg ReCOV) at 14 days post second dosing and remained at 0.2% (20 μg ReCOV) and 0.1% (40 μg ReCOV) at 6 months post the second dosing, while in older subjects, the level increased to 0.2% (20 μg ReCOV) and 0.2% (40 μg ReCOV) at 14 days post second dosing and remained at 0.2% (20 μg ReCOV) and 0.16% (40 μg ReCOV) at 3 months post the second dosing. In placebo groups, the average percentage of CD4+ T cells secreting antigen-specific IL-2 or IFNγ remained at low levels at all timepoints tested. The peak cellular responses among older adults tended to be lower than that among younger adults.

In contrast to the Th1 biased cellular immune responses, no obvious increase in Th2 cytokine (IL-4 or IL-5) secretions was observed in both age groups receiving 20 μg and 40 μg ReCOV (Fig. 8). In addition, no obvious CD8+ T cell responses were observed across age groups and dose levels (data not shown).

Fig. 8

Th1 and Th2 cytokine responses in the phase I study. The observation was considered as an outlier if the value was less than 1.5 × interquartile range (IQR) below Q1 or greater than 1.5 × IQR above Q3. Data show the percentage of CD4+ T cells for Th1 (IL-2 or IFNγ) and Th2 (IL-4 or IL-5) cell responses at baseline, 14 days (14p2), 30 days (30p2), 3 months (3mp2), and 6 months (6mp2) post the second vaccination of pooled placebo group, 20 µg ReCOV group, and 40 µg ReCOV group