Efficacy, immunogenicity, and safety of heterologous boosting with a novel chimera Chinese mRNA (RQ3013) SARS-CoV-2 vaccine: A randomized, double-blind, active-controlled trial

ABSTRACT

A randomized, double-blind, controlled phase 3 trial was conducted during a COVID-19 outbreak after the initial, stringent zero-Covid policy was relaxed in three provinces. Eligible adults aged ≥18 years who had received three doses of inactivated COVID-19 vaccines 6 months earlier were randomly assigned in a 1:1 ratio to receive either one intramuscular injection of RQ3013 or ZF2001 vaccine. The primary end point was protection against PCR-confirmed symptomatic SARS-CoV-2 infection with onset at least 7 days after the booster. A total of 3,167 and 3,169 eligible participants received one dose of RQ3013 or ZF2001 vaccine, respectively. COVID-19 illness was confirmed in 91 participants in the ZF2001 group (11.8 per 100 person years; 95% confidence interval [CI]: 9.6–14.6) and in 45 participants in the RQ3013 group (5.7 per 100 person-years; 95% CI: 4.3–7.7) during a 4-month follow-up, resulting in a relative efficacy of 51.7% (95% CI, 30.9–66.2%) (p < .001) in an intention-to-treat analysis. The RQ3013 vaccine was also found to be significantly more immunogenic against omicron BA.5 compared to the ZF2001 vaccine. Moderate, transient adverse reaction after vaccination occurred more frequently in the RQ3013 group than in the ZF2001 group. Serious adverse events (SAEs) were rare and occurred almost equally in two groups. All SAEs were not related to the vaccination. These findings suggest that a chimeric mRNA vaccine design involving multiple antigenic epitopes provides broader protection across subvariants and variants of SARS-CoV-2 than the subunit vaccine ZF2001.

Introduction

Although many countries discontinue COVID-19 specific reporting, the COVID-19 circulation never ceased, due to the high transmissibility of the virus and waning immunity in the population. SARS-CoV-2 evolves continuously, resulting in immune escape. Especially omicron variants have contributed to the continued transmission of SARS-CoV-2. Vaccination is the most efficient tool to prevent severe infection and thus there remains a continued need for novel and updated COVID-19 vaccines.

Since the beginning of the COVID-19 pandemic in December 2019, several vaccines were developed in China using various platforms including inactivated vaccines, sub-unit/protein vaccines, and vectored vaccines. The conditional licensed inactivated, subunit, and non-replicable adenovirus-vector vaccines, together with the Zero-Covid policy were implemented to prevent the transmission of COVID-19 in China.¹ Since December 7, 2022, the Omicron lineages BA.5 and BF.7 accounted for 67.0% and 29.5% of the SARS-CoV-2 infections in China, respectively,² with the increasing prevalence of less pathogenic COVID-19 strains China started to lift the draconian Zero-Covid restriction policies. During this transition period, a China-made mRNA chimera vaccine (RQ3013) developed by RNACure Inc. and Walvax Inc.³ was evaluated. We report here the safety, immunogenicity, and efficacy of a booster dose with the candidate COVID-19 mRNA vaccine RQ3013 in a double-blinded, randomized, controlled phase 3 trial.

Material and methods

Study vaccines

RQ3013 is a mRNA vaccine candidate with a chimeric design, which integrates modified epitope sequences of the spike S protein derived from SARS-CoV-2 South Africa strain⁴ and UK strain.⁵ The mRNA sequence was modified to reduce the innate immunity of mRNA molecules and enhance the translation efficiency of mRNA in vivo, ultimately improving the safety and efficacy of the vaccine candidate. The promising cross-neutralization potential of RQ3013 against various Omicron variants, including Omicron BA.5 was demonstrated in a preceding immunogenicity study.³ The RQ3013 vaccine was formulated in a 0.15 ml vial, containing 30 μg mRNA. The main excipients include cholesterol, DMG-PEG2000, DSPC, RL-151, trometamol, acetic acid, sodium acetate, and sucrose.³

The conditionally licensed protein subunit COVID-19 vaccine ZF2001 in China was employed as control. ZF2001 was developed using the tandem-repeat dimeric RBD of the SARS-CoV-2 spike protein (from the original Wuhan-Hu-1 strain) as the antigen.⁶ The antigen protein was produced in Chinese hamster ovary cells and aluminum hydroxide used as adjuvant. The protection was evaluated in a randomized, double-blind, placebo-controlled, phase 3 trial performed in 31 clinical centers across Uzbekistan, Indonesia, Pakistan, and Ecuador. A total of 28,873 adult participants (≥18 years of age) were randomly assigned in a 1:1 ratio to receive a total of three 25-μg doses (30 days apart) of ZF2001 or placebo. The efficacy against the occurrence of symptomatic COVID-19 was estimated to be 75.7% (95% confidence interval: 71.0%−79.8%) in a clinical trial conducted during the Delta variant dominant period in 2021.⁷

Participants and study design

This double-blind, active-controlled, randomized phase 3 clinical trial was conducted in Yunnan, Hunan, and Henan provinces, aimed to assess the efficacy, immunogenicity, and safety of heterologous boosting with one dose of RQ3013 in adults aged 18 years or older, who had received three doses of inactivated COVID-19 vaccines, produced by either Sinopharm⁸ or Sinovac⁹ six months earlier. Eligible participants were assigned randomly to receive either one dose of RQ3013 or one dose of ZF2001 in a ratio of 1:1 delivered in the deltoid muscle, stratified by age (18–59 years vs ≥60 years). The proportion of participants aged 60 years and older was set to be not less than 15% of the total trial population. To ensure that the characteristics of participants on age, residence, and participating in the immunogenicity study distributed evenly between the RQ3013 experimental group and the ZF2001 experimental group, a stratified block randomization was employed with stratification variable of age, study site, and immunogenicity subset (Supplementary Table S1). Unique random numbers were generated by computer with a block size of eight. A subset of 799 participants were selected randomly for an immunogenicity study. All eligible participants were followed actively by twice weekly SMS or telephone call from Day 0 throughout Day 90, and then once a week from Day 91 to Day 120, to collect information of suspected SARS-CoV-2 infections, adverse events (including SAEs, and AESIs), and pregnancy. All eligible participants were tested for anti-SARS-CoV-2 IgG antibodies on the day of vaccination, which served as one of the baseline characteristics of the trial participants. The protocol was approved by the ethics review committees of each study site, and written informed consent was obtained from each participant before enrollment. The study was registered with www.chictr.org.cn (ChiCTR2200067184) and carried out according to the Good Clinical Practice guidelines of the International Council Harmonization of Technical Requirements for Pharmaceuticals for Human Use.

Adults aged 18 years or older who were healthy or had stable chronic medical conditions in the previous 3 months, had been primed ≥6 months previously with three doses of SARS-CoV-2 inactivated vaccines, and had negative SARS-CoV-2 PCR test within the past 48 h were eligible for recruitment. The exclusion criteria included a confirmed COVID-19 infection previously; suspected of having SARS-CoV-2 infection within 2 months before enrollment; axillary temperature ≥37.3℃ on the day of vaccination; women who were pregnant or breastfeeding; a history of severe allergic reactions or allergic reactions to vaccines or drugs; received any vaccine within 28 days before enrollment; chronic diseases that affect the immune system, including using immunomodulatory drugs within 6 months before the enrollment.

Efficacy assessment

The primary endpoint was the protection against laboratory confirmed symptomatic SARS-CoV-2 infection (COVID-19) with onset at least 7 days post-vaccination. The secondary endpoint was laboratory confirmed severe, critical COVID-19, or death caused by SARS-CoV-2 with onset at least 14 days post-vaccination. Case definitions were established according to World Health Organization’s (WHO) guidelines.^10,11 Nasopharyngeal or oropharyngeal swabs were collected from each suspected case, and a SARS-CoV-2 RT – PCR test was carried out by the provincial Center for Disease Control, and Prevention (CDC). For those with confirmed SARS-CoV-2 infection, a weekly follow-up visit was performed to assess the severity until recovery. PCR-positive samples were sequenced at the Microbiology Laboratory of Hunan provincial CDC. A confirmed symptomatic SARS-CoV-2 infection was defined by a) a positive SARS-CoV-2 by PCR test, and (b) acute onset of one or more of the following signs or symptoms, lasting for 2 days or more (i.e., ≥48 h): fever, cough, shortness of breath or difficulty breathing, OR (c) acute onset of any one or more signs or symptoms of a respiratory tract infection: chills, malaise/fatigue, headache, muscle pain, dry/sore throat, nasal congestion/runny nose, anorexia/nausea/vomiting, diarrhea, new onset of abnormal smell/taste, and conjunctivitis; OR (d) clinical or radiographic evidence of pneumonia. Endpoint events were judged by an independent adjudication committee that was unaware of the vaccine assignment.

Safety assessment

The primary safety endpoints of this trial were the incidence rates of local or systemic adverse events/adverse reactions occurring within 30 min, 7 days, and 28 days after the receipt of RQ3013 or ZF2001. Study participants were observed on site for 30 min for immediate adverse events after vaccination. Reports of the local and systemic reactions were recorded daily on diary cards for 7 days. Other signs and symptoms occurring during a 28-day follow-up period were recorded as unsolicited adverse events. Serious adverse events (SAEs), adverse events of special interest (AESIs), and pregnancy-related events were recorded throughout the study period. Referring to the safety data of mRNA COVID-19 Vaccines produced by Moderna and BioNtech, AEFIs were preset as following: cerebral infarction, myocardial infarction, arrhythmia, sudden cardiac death, pericarditis, ischemic cardiomyopathy, arterial disease, acute liver/kidney function impairment, anemia, coagulation abnormalities, Guillain–Barré syndrome, tumor, and anosmia.

Immunogenicity assessment

The subset of participants for the immunogenicity study included 800 participants (~10% of entire sample size). Blood samples were obtained from each selected participant at baseline and on days 7, 14, 28, 90, and 180 after vaccination. The serum neutralizing antibody (nAbs) response against the Omicron BA.5 strain (GDCPP.2.00303) was quantified at the Guangzhou Institute of Respiration Health and Guangzhou Customs Technical Center. A microdose cytopathogenic effect assay was used for the measurement of nAbs.¹² A titer lower than the detection limit (1:8 dilution) was defined as 1:4. All serum samples were tested for anti-omicron specific IgG antibody using a commercial IgG ELISA kit (Vazyme, China).

Data management and statistical analysis

Assuming that the relative vaccine efficacy in comparison with the ZF2001 vaccine would be ≥60% with a superiority margin of 30%, a total of 154 cases of COVID-19 would be required to provide 90% power to reject the null hypothesis, with an overall one-sided error rate of 0.025. An interim analysis was planned at 70% of the target total number of COVID-19 cases, the O’Brien-Fleming consumption function was applied to control the type one error (estimated to be one-sided $\mathit{\alpha }$1 = 0.0074, $\mathit{\alpha }$2 = 0.0227), based on exact conditional test under Poisson assumption.¹³ Assuming the incidence of COVID-19 was 5,000 cases per 100,000 person-years, considering 30% drop-out rate, an estimated 3,150 participants in each study arm were required.

All data were double entered into custom-made data entry programs. The data management programs included range and consistency checks. An SAS program (SAS Institute Inc., Cary, NC, USA) was applied for statistical analysis, and a p-value <.05 (two-tailed) was considered statistically significant. A modified intention-to-treat dataset (mITT), and a per-protocol dataset (PPS) were used in the efficacy analysis. For the primary endpoint, mITT eligible participants met the following criteria: (a) they received one dose of the study vaccine (b) completed the follow-up visit one week after vaccination, and (c) were not attacked by SARS-CoV-2 virus (either symptomatic or asymptomatic infection) within 7 days after vaccination. The PPS included all study participants who did not experience any major protocol deviations.

A relative vaccine efficacy and confidence interval were calculated as 1 – adjusted relative risk (RQ3013 vs ZF2001) computed using a Poisson regression model adjusted for covariates.¹⁴ Cumulative incidence is presented using the Kaplan–Meier method. The safety analyses included all randomized participants who received a study agent. Those participants with administration errors were evaluated for safety as per the actual administrative status according to the ‘all participants as treated’ (ASaT) principle. Safety profiles were summarized according to terms in the Medical Dictionary for Regulatory Activities (Med-DRA), version 23.1. The analysis of immunogenicity was performed according to the ITT principle. The geometric mean titer (GMT) of neutralizing antibodies and their 95% confidence intervals were calculated. Antibody titers were logarithmically converted to allow assessment of GMTs.

Results

Summary of participants

Between December 28, 2022, and January 19, 2023, 7,475 participants were screened for eligibility. Of these, 3,167 and 3,169 eligible participants were assigned to the RQ3013 and ZF2001 groups, respectively. Before the vaccination, 6 participants withdrew their inform consent. Within 7 days after vaccination, 784 participants (426 vs 358 in ZF2001 and RQ3013 groups respectively) were reported baseline PCR positive after vaccination and thus were not followed up, due to time-consuming centralized testing of baseline PCR samples. In addition, 426 asymptomatic infections and 26 clinical infections occurred within 7 days of vaccination and were not followed-up. During a mean of 116.9 follow-up days (95% CI: 116.4 days−117.4 days), 46 major deviations from protocol were recorded, no participants were lost to follow-up. Eventually, 6,330, 5,094 and 5,044 participants were included in the safety, mITT and PP analyses, respectively (Figure 1). The baseline characteristics of the participants in RQ3013 and ZF2001 groups are shown in Table 1. The mean age of the participants was 48.0 years (range: 18 years to 88 years), with 16.5% of participants ≥60 years, 40.3% female, and 10.5% had chronic medical conditions.

Figure 1.

Assembly of participants.

Table 1.

Characteristics of participants at baseline according to a modified intention-to-treat (mITT) and a per protocol (PP) analysis.

Characteristics	mITT		PP
	ZF2001 (n = 2536)	RQ3013 (n = 2558)	ZF2001 (n = 2509)	RQ3013 (n = 2535)
Mean age (range) yr	46.4 (18, 84)	46.4 (18, 88)	46.3 (18, 84)	46.3 (18, 88)
Age group (n, %)
18–59 yr	2114 (83.4)	2139 (83.6)	2089 (83.3)	2119 (83.6)
≥60 yr	422 (16.6)	419 (16.4)	420 (16.7)	416 (16.4)
Gender (n, %)
male	1667 (61.9)	1707 (60.7)	1554 (61.9)	1566 (61.8)
female	955 (38.1)	969 (38.3)	955 (38.1)	969 (38.2)
Body-mass index (range) kg/m2	24.7 (13.5, 48.3)	24.7 (15.9, 45.9)	24.7 (13.5, 48.3)	24.7 (15.9, 45.9)
Body temperature(range) ℃	36.3 (35.4, 37.2)	36.3 (35.5, 37.2)	36.3 (35.4, 37.2)	36.3 (35.4, 37.2)
More than one comorbidity (n,%)	360 (14.2)	319 (12.5)	348 (13.9)	309 (12.2)
Province of volunteers allocated (n,%)
Yunnan	1054 (41.6)	1098 (42.9)	1036 (41.3)	1083 (42.7)
Hunan	577 (22.7)	577 (22.6)	570 (22.7)	571 (22.5)
Henan	905 (35.7)	883 (35.5)	903 (36.0)	881 (34.8)
Average follow-up days (range)	116.1 (9, 120)	107.7 (9, 120)	116.1 (9, 120)	117.7 (9, 120)
Previous COVID-19 vaccination history (n,%) 1st dose
Sinovac	1395 (55.1)	1400 (54.7)	1384 (55.2)	1393 (54.9)
Sinopharm	1122 (44.2)	1142 (44.6)	1107 (44.1)	1127 (44.5)
Other	19 (0.7)	16 (0.7)	18 (0.7)	15 (0.6)
2nd dose
Sinovac	1483 (58.5)	1486 (58.1)	1471 (58.6)	1473 (58.1)
Sinopharm	1038 (40.9)	1052 (41.1)	1024 (40.8)	1043 (41.1)
Other	15 (0.6)	20 (0.8)	14 (0.6)	19 (0.8)
3rd dose
Sinovac	1281 (50.5)	1281 (50.1)	1268 (50.5)	1270 (50.1)
Sinopharm	1226 (48.4)	1245 (48.6)	1213 (48.4)	1235 (48.7)
Other	29 (1.1)	32 (1.3)	28 (1.1)	30 (1.2)
Average days since the last vaccination (range)	361.7 (183.0, 522.0)	362.5 (191.0, 565.0)	361.5 (183.0, 457.0)	362.3 (191.0, 565.0)

Efficacy

Between December 28, 2022, and May 19, 2023, 546 symptomatic cases were confirmed as SARS-CoV-2 infections by RT-PCR assay. Of these, 136 cases and 80 cases presented 7 days and 14 days after administration, respectively. The relative efficacy was 51.7% (95% CI: 30.9%−66.2%) (p < .001) and 41.4% (95% CI: 7.9%−62.7%) (p = .02) for the primary and secondary endpoints, based on the mITT dataset (Figure 2a and Supplementary Figure S1a). In the per protocol analysis, the relative efficacy of RQ3013 was 52.8% (95% CI: 32.4%−67.1%) (p < .001) and 52.9% (95% CI: 32.5%−67.1%) (p < .001) for the primary and secondary endpoints, respectively (Figure 2b and Supplementary Figure S1b). A higher efficacy of RQ3013 (76.9%, 95% CI: 38.7%−92.3%) was observed in elderly, compared to the efficacy in adults aged 18 years to 59 years (44.1%, 95% CI: 17.6%−62.1%). The SARS-CoV-2 from nasopharyngeal or oropharyngeal swabs of the 136 COVID-19 cases presenting 7 days or more after vaccination were sequenced. Of these, 117 (86.1%) specimens were typeable. The most common subvariants detected was the DY strains (77 cases, 49 in ZF2001 vs 28 in RQ3013 groups), followed by BA.5 (22 cases, 14 in ZF2001 vs 8 in RQ3013 groups), and BF.7 (15 cases, 12 in ZF2001 vs 3 in RQ3013 groups). The DY-, BA.5- and BF.7-specific relative efficacies were 44.2% (95% CI: 11.2%−64.9%), 44.2% (95% CI: −33.1%−76.6%), and 75.6% (95% CI: 13.4%−93.1%) respectively. Omicron XBB was identified in three COVID-19 cases (2 in ZF2001 vs 1 in RQ3013 groups). All laboratory confirmed COVID-19 cases were uncomplicated, except for one severe case in the RQ3013 group. No SARS-CoV-2 associated death was recorded during the study period.

Figure 2a.

Efficacy of RQ3013 vs. COVID-19 in the mITT population.

Figure 2b.

Efficacy of RQ3013 vs. COVID-19 in the per-protocol population.

Immunogenicity analysis

Seven hundred and ninety-nine eligible participants were selected and assigned randomly to RQ3013 and ZF2001 groups. At the baseline, no significant differences in neutralizing antibodies positivity or antibodies titers were detected between the RQ3013 and ZF2001 groups. At baseline, the neutralizing antibody positive rate was 38.2% and 36.7% (p > .05), with a GMT of 14.5 (95% CI: 12.1–17.3) and 14.1 (95% CI: 11.8–16.9) in RQ3013 and ZF2001 groups, respectively (Figures 3a and 3b). Seven days after vaccination, a 14-fold increase (from 14.5 to 202.0) in neutralizing titers was measured in the RQ3013 group, compared to a 1.7-fold increase in the ZF2001 group, the neutralizing antibodies positivity increased correspondingly (93.0% in RQ3013 vs 60.8% in ZF2001 groups). At Day 14, the GMTs in both groups peaked (298.2 in RQ3013 vs 63.5 in ZF2001 groups), with a positivity of 98.3% and 84.9% in RQ3013 and ZF2001 groups, respectively. By day 28, a decline in antibodies titers (GMT: 165.7) was observed in the RQ3013 group and in the ZF2001 group (GMT: 59.8). No significant decline in antibody positivity (97.3% in RQ3013 vs 87.7% in ZF2001) was detected. Immunogenicity was further analyzed in omicron BA.5 susceptibles, and a similar trend was observed (Supplementary Table S2). The antibody positivity on Day 7, Day 14, and Day 28 was 91.7%, 99.1%, 97.3% in RQ3013 vaccinated participants and 37.1%, 76.7%, 81.5% in ZF2001 vaccinated participants, with a GMT of 142.6, 259.1 and 140.4 in the RQ3013 group, compared to a GMT of 8.4, 39.7, and 41.4 in the ZF2001 group. In the RQ3013 group, the peak antibody levels (day 14) were significantly higher in adults aged 18 years to 59 years (GMT: 308.4), compared to participants 60 years and older (GMT: 254.7) (p < .01).

Figure 3a.

Virus-neutralizing titers pre- and post-booster (all participants in immunogenicity subset).

Figure 3b.

Virus-neutralizing titers pre- and post-booster (susceptibles in immunogenicity subset).

Safety analysis

The overall incidence rate of any adverse events was 69.8% (95% CI: 68.2%−71.4%) and 44.9% (95% CI: 43.2%−46.7%) in the RQ3013 and ZF2001 groups, respectively. The occurrence rate of adverse events related to the investigational products (adverse reactions) was 58.6% (95% CI: 56.8%−60.3%) and 21.1% (95% CI: 19.6%−22.5%) in the RQ3013 and ZF2001 groups, respectively. Most of the AEs were defined as grade 1 and grade 2 (49.7% in RQ3013 group vs 34.4% in ZF2001 group). The rate of grade 3 adverse events was 7.8% and 2.2% in the RQ3013 and ZF2001 groups, respectively. Solicited local adverse reactions (35.2% vs 11.8%) and solicited systemic reactions (43.2% vs. 11.6%) were more frequent in the RQ3013 group compared to the ZF2001 group (Figure 4 and Supplementary Table S3). The most common solicited local reaction in both groups was pain at the injection site (33.7% vs. 10.4%), and the most common systemic adverse reactions were fever (34.9% vs. 6.2%), headache (12.6% vs. 4.6%), fatigue (10.8% vs. 2.8%), and muscle pain (10.1% vs. 3.8%) in the RQ3013 and ZF2001 groups, respectively (Figure 4).

Figure 4.

Solicited local and systemic adverse events.

Figure 4.

(Continued).

Unsolicited adverse reactions were observed in 4.4% of participants in the RQ3013 group compared to 1.7% the control ZF2001 group (Supplementary Table S3), all of which were grade 1 or grade 2. The incidence rates of serious adverse events (SAE) and adverse events of special interest (AESIs) were low and similar between the two groups. Thirty-six (1.1%) and 29 (0.9%) participants reported AESIs in the ZF2001 and the RQ3013 groups, respectively (p > .05). No cases of pericarditis, ischemic cardiomyopathy, Guillain–Barré syndrome, or anosmia were not reported. The most common AESIs reported in the RQ3013 group were abnormal liver function tests (0.25%), tumor (0.19%), and anemia (0.16%). No SAEs, or death related to the study products were reported (Supplementary Table S3).

Discussion

This was the first trial of a chimeric designed mRNA vaccine demonstrating the efficacy of a booster dose in a Chinese population. During the BA.5-dominant period, a 51.7% (95% CI: 30.9%−66.2%) reduction on symptomatic SARS-CoV-2 infection was observed in participants who were boosted with RQ3013 compared to participants who were boosted with ZF2001. The vaccine candidate was found to be significantly more immunogenic against omicron BA.5 compared to ZF2001, and both vaccines were more immunogenic against BA.5 in younger participants than in participants ≥60 years. Moreover, during the 4-month follow-up period, no waning of clinical protection was detected despite the observed decrease in neutralizing antibodies (Table 2). A similar trend has been reported previously in people who received a booster dose, either homologous or heterologous.^1,15–17

Table 2.

Efficacy in different time frame.

	ZF2001 (n = 2536)			RQ3013 (n = 2558)
Days after booster (day)	Cases	Person-yrs of exposure	Incidence Rate (95% CI) person-yr	Cases	Person-yrs of exposure	Incidence Rate (95% CI) person-yr	Vaccine Efficacy (95% CI) %	P value
8–21	56	97.3	57.6(44.3,74.8)	26	98.8	26.3(17.9,38.6)	54.3(27.3,71.3)	.0010
8–35	76	192.9	39.4(31.5,49.3)	36	196.7	18.3(13.2,25.4)	53.5(30.9,68.7)	.0002
8–49	82	288.1	28.5(22.9,35.3)	39	294.1	13.3(9.7,18.2)	53.4(31.8,68.2)	<.0001
8–63	87	382.9	22.7(18.4,28)	40	391.4	10.2(7.5,13.9)	55.0(34.6,69.1)	<.0001
8–77	89	477.6	18.6(15.1,22.9)	42	488.6	8.6(6.4,11.6)	53.9(33.4,68)	<.0001
8–91	89	572.3	15.6(12.6,19.1)	42	585.6	7.2(5.3,9.7)	53.9(33.4,68)	<.0001
8–105	89	666.7	13.3(10.8,16.4)	43	682.3	6.3(4.7,8.5)	52.8(32.1,67.2)	<.0001
8–120	91	767.9	11.8(9.6,14.6)	45	786.0	5.7(4.3,7.7)	51.7(30.9,66.2)	<.0001

There can be several interpretations for these findings. First, the control vaccine ZF2001 affords considerable protection against the BA.5 subvariant. Earlier studies found that a heterologous booster with ZF2001 in people primed with two doses of inactivated vaccine could significantly increase neutralization titers, evoke cross-antibody responses to different variants, and thus reduce the immune escape.^18–21 During the study period, the cumulative incidence rate was >50 cases per 100 person in the general population not involved in clinical trials,^22–25 while we observed an incidence of 11.9 cases per 100 person-year in ZF2001 group over the same period. During the first wave after zero-COVID policy adjustment, the effectiveness afforded by either a homologous or heterologous booster dose against SARS-CoV-2 omicron infection was estimated to be 49.0% within 3 months of booster vaccination.²² Second, a protection of BA.1-containing bivalent COVID-19 mRNA vaccines was reported to be 65% and that of BA.4/BA.5-containing bivalent mRNA vaccines 76% during the BA.5-dominant period in Japan.²⁶ The antigenic distance between alpha/beta variants and omicron BA.5 subvariant is far lower than that between subvariants in omicron variant.^21,27,28 Therefore, it stands to reason that the chimeric designed RQ3013 vaccine can provide broader protection across variants. Third, neutralizing antibody responses correlate with protection against COVID-19.^29,30 Compared to the immune response triggered by a booster with ZF2001, a booster with RQ3013 elicited a superior neutralizing antibody response against BA.5 subvariant as early as 7 days after the booster vaccination. Therefore, it is reasonable to assume that the chimeric vaccine design approach could elicit broader neutralization and confer better protection against omicron subvariants compared to the subunit vaccine. The exceptional efficacy of RQ3013 can be attributed to its multidimensional antigen design, which harmonizes precise structural engineering with an optimized immunological strategy to counter the challenges posed by emerging SARS‑CoV‑2 variants. Structurally, RQ3013 employs the spike (S) protein backbone from the Alpha variant (B.1.1.7) as a stable scaffold while incorporating critical mutations from the Beta variant (B.1.351) – notably K417N, E484K, and A701V – that have been linked to immune evasion. This chimeric approach preserves the native trimeric conformation of the S protein, as confirmed by cryo‑electron microscopy studies showing a stable prefusion state with an open receptor‑binding domain (RBD), potentially enhancing ACE2 binding.³¹ In addition, replacing the furin cleavage site with a GSAS motif prevents proteolytic processing, which further stabilizes the immunogen without compromising its immunogenicity. Immunologically, RQ3013 is designed to present a broad repertoire of epitopes that effectively prime the immune system. Preclinical evaluations in mice, hamsters, and nonhuman primates have demonstrated that RQ3013 elicits robust humoral responses with high titers of cross‑neutralizing antibodies against multiple SARS‑CoV‑2 variants – including wild‑type, Alpha, Beta, Delta, and various Omicron sublineages.^3,31 In summary, the integration of targeted mutations into the S protein, combined with modifications that ensure conformational stability and prevent premature cleavage, underpins the broad-spectrum efficacy of RQ3013.

The vaccine candidate RQ3013 was found to be safe. Few serious adverse events were reported in the RQ3013 and ZF2001 groups, and none of them were related to the vaccines. As expected, compared to booster with ZF2001, more solicited local and systemic adverse reactions were reported in the RQ3013 group. Reduced tolerability is a characteristic difference between mRNA- and protein-based COVID-19 vaccines. The reason behind this observation could the different immune mechanisms, as well as the presence of lipid nanoparticles (LNPs). It is worth noting that compared to internationally available mRNA COVID-19 vaccines, such as BNT162b2,³² mRNA-1273,³³ and bivalent mRNA-1273.214,³⁴ the signature fatigue and pain at injection site were less frequently incurred in recipients of RQ301. The most likely explanations for the improved tolerability could be, firstly, a different method was applied to modify mRNA encoding spike proteins. Pseudouridine was used with RQ3013, while N1-methylpseudourdine was used with BNT162b2 and mRNA-1273. Our study demonstrated that incorporation of N1-methylpseudouridine into mRNA resulted in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurred after vaccination.³⁵ Interestingly, Pseudouridine caused less frameshifting compared to N1-methylpseudourdine. Second, a lower dosage of RQ3013 mRNA was used compared to BNT162b2. However, fever (approximately 30%) was more common in recipients of RQ3013 than that observed in clinical trials of abovementioned three international available mRNA vaccines. This could be attributable to different definitions of fever. In China, a fever is defined as axillary temperature greater than or equal to 37.3°C,³⁶ while WHO’s definition is 38°C.³⁷ Nevertheless, fever needs to be monitored in RQ3013 recipients.

Findings derived from this study have several limitations. First, the end of “Zero-Covid” policy which featured as intensive RT-PCR screening started at the beginning of December 2022. The subsequent wave of COVID-19 peaked in China around December 20 and lasted until the end of January 2023.² Asymptomatic SARS-CoV-2 infections prior to enrollment could not be accounted for in this analysis, although the RT-PCR screening was performed during the enrollment. This subgroup who had experienced SARS-CoV-2 infection would decrease the risk of medically attended COVID-19 illness, and most likely led to an underestimate of rVE due to background immunity. Second, faced with the increase in COVID-19, most families reduced their contact with the outside world, and thereby reduced the risk of infection. Such a development may also result in an underestimate of the rVE. Third, longer follow-up of the protection against COVID-19 illness was not carried out. Hence, it was not possible to understand the longer immune persistence of RQ3013, as well as its ability conferring cross-protection against newer subvariants. During the first wave of nationwide sweeps after the ending of the zero-COVID policy, residents generally experienced natural infection (estimated infection rate ~85%, unpublished data) and longer immune persistence attributed to vaccination was difficult to estimate accurately, since the durability of immunity became much more complex, depending not only on how quickly immunity waned but also on how much the virus mutated. In addition, after 3 years of pandemic, residents became tired of the COVID-19 and COVID-19 vaccinations, which posed a challenge to compliance in long-term follow-up. In addition, protection against COVID-19 provided by booster doses fades faster when facing omicron subvariants compared to ancestral wild type.³⁸ It was reported that, after 20 weeks of a booster dose using Moderna and Pfizer – BioNTech bivalent vaccines, which contain spike messenger RNA from both the ancestral strain and omicron BA.4–BA.5 subvariants, the effectiveness against severe infection resulting in hospitalization or death, was 38.4% (95% CI, 13.4 to 56.1) during the period when omicron BQ.1 and XXB were predominant.³⁹ A study conducted in Israel recruited more than 10,000 health-care workers who had not previously been infected. All participants received either three or four doses of the Pfizer and/or BioNTech vaccines. Four months after boosting, the study found that four doses were not better than three doses at preventing infection.⁴⁰ Fourth, the study population may not adequately represent the elderly Chinese population, given that the life expectancy in China reached 77 years in 2020 but the mean age of participants ≥60 years was 66 years old.⁴¹ The rVE derived from this study should only be extrapolated to general elderly population with caution. Fifth, since only the neutralizing antibody against the dominant BA.5 subvariant during study period was quantified, protection against other variants need further study.

In summary, this study evaluated the performance of the novel mRNA SARS-CoV-2 vaccine RQ3013. Compared to the ZF2001 subunit vaccine, a booster with the mRNA vaccine RQ3013 provided significantly higher protection against BA.5 subvariant. The safety profile of the mRNA vaccine RQ3013 was found to be satisfactory. These findings suggest that a chimeric vaccine design involving multiple antigenic epitopes provides broader protection against subvariants and variants of SARS-CoV-2.

Biographies

Lin Yuan, General Manager of the Registration and Medical Center at Walvax Biotechnology Co., Ltd. Ms. Yuan has over 20 years of experience in vaccine process development, drug registration, and clinical research management. As the head of drug registration for the company, she has successfully led the registration of more than 20 vaccine products, with 9 vaccines obtaining production licenses and entering the market for sale. She has also participated as a core member in several major national projects or initiatives, including the “National High Technology Research and Development Program (863 Program)” guided project from the Ministry of Science and Technology, the “Major Science and Technology Special Project for Infectious Disease Prevention and Control” from the Ministry of Health, and the “2012 Protein-based Biopharmaceuticals and Vaccine Development Project” supported by the Ministry of Finance, the National Development and Reform Commission, the Ministry of Industry and Information Technology, and the Ministry of Health, among others.

Xuan-Yi Wang received medical training at Anhui Medical University, and a Ph.D. on epidemiology from the School of Public Health, followed by a postdoctoral training on immunology and molecular virology at Shanghai Medical College, Fudan University. Dr Wang is a research scientist in Fudan University, with an extensive experience in infectious disease research and vaccinology, combines academic knowledge of epidemiology with the crucial understanding of healthcare systems and infrastructure in remote areas of China. Currently, his team works on the R&D of rotavirus and norovirus vaccines, and is responsible for the clinical development of a therapeutic hepatitis B vaccine. He has involved in the clinical development of vaccines and immunization strategy against hepatitis A, B and E, rotavirus, EV71 in China. He serves as an external expert of the Center for Drug Evaluation, National Medical Products Administration, and a member of the National Immunization Advisory Committee in China.

Jin-Zhong Lin received B.S. degree from China Agricultural University, and Ph.D. degree from the Institute of Biophysics at Chinese Academy of Sciences, followed by a postdoctoral training at the Institute of Life Sciences in Beijing. Dr Lin is a research scientist in Fudan University. His main research interests include the molecular mechanisms of gene translation and regulation, the working and resistance mechanism of ribosome-targeted antibiotics.

Funding Statement

This work was supported by the Yunnan Provincial Science and Technology Department [grant no. 202002AA100007], and Shanghai Municipal Science and Technology Major Project [grant no. ZD2021CY001].

Disclosure statement

This trial was sponsored by Walvax Biotechnology. RQ3013 was co-developed by Walvax and RNACure. LY, JJC, HLF, XMY, SWT and WL are employees of Walvax. JL is employee of RNACure. HJY was supported by the National Natural Science Foundation of China, outside the submitted work. The other authors declare no competing interests.

Author contributions

XQL, TH, LLH, and MXZ served as the principal investigators for this clinical trial. XYW provided guidance on the study design and result analysis, as well as on the drafting and review of the manuscript. LY conceived and organized this clinical trial and participated in the interpretation of the results. YYQ was involved in the statistical analyses of this study and drafted and revised the manuscript. JJC contributed to the study design and analysis of the results. ZWJ offered expert advice on study design and data analysis. JZL, WL, JL organized the production of vaccines used in this trial. FS, QNZ, XLY, XZL, JBZ, ZHZ, GXC, HS, and W-MG organized the conduct and data collection of this trial. ZFW, JD, XYY and ZFZ organized the biological sample testing. YXW, JCH, YXZ, DFD, HLF, XMY, and SWT participated in quality control of this trial. LVS contributed to discussions and revised the manuscript. All authors critically revised, read and approved the final manuscript, and gave final approval of the version to be published.

Ethical approval statement

This trial protocol was reviewed and approved by the Ethics Committee of the First People’s Hospital of Anning City (No. IEC/AF/61/2-21-01.0).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21645515.2025.2502250