Immunogenicity and safety of a new hexavalent rotavirus vaccine in Chinese infants: A randomized, double-blind, placebo-controlled phase 2 clinical trial

ABSTRACT

Rotavirus remains a major cause of diarrhea among 5-y-old children, and vaccination is currently the most effective and economical measure. We conducted a randomized, double-blind, placebo-controlled phase II clinical trial designed to determine the dosage, immunogenicity, and safety profile of a novel hexavalent rotavirus vaccine. In total, 480 eligible healthy infants, who were 6–12 weeks of age at the time of randomization were randomly allocated (1:1:1) to receive 10^5.5 focus-forming unit (FFU) or 10^6.5FFU of vaccine or placebo on a 0, 28 and 56-d schedule. Blood samples were collected 28 d after the third dose to assess rotavirus immunoglobulin A (IgA) antibody levels. Adverse events (AEs) up to 28 d after each dose and serious adverse events (SAEs) up to 6 months after the third dose were recorded as safety measurements. The anti-rotavirus IgA seroconversion rate of the vaccine groups reached more than 70.00%, ranging from 74.63% to 76.87%. The postdose 3 (PD3) geometric mean concentrations (GMCs) of anti-rotavirus IgA among vaccine recipients ranged from 76.97 U/ml to 84.46 U/ml. At least one solicited AE was recorded in 114 infants (71.25%) in the high-dose vaccine group, 106 infants (66.25%) in the low-dose vaccine group and 104 infants (65.00%) in the placebo group. The most frequently solicited AE was fever. The novel oral hexavalent rotavirus vaccine was safe and immunogenic in infants support the conclusion to advance the candidate vaccine for phase 3 efficacy trials.

KEYWORDS:

• Rotavirus
• vaccine
• safety
• immunogenicity

Introduction

Rotavirus infection is the leading cause of acute gastroenteritis (AGE) worldwide in children under 5 y of age and causes dehydration of varying degrees.^1,2 The WHO broadened its recommendation for rotavirus vaccination use in 2009 to cover all nations, with a focus on those with high rates of death from diarrhea.³ The introduction of oral rotavirus vaccines into national immunization programs has led to substantial reductions in rotavirus and all-cause acute gastroenteritis, hospital admissions and mortality among children younger than 5 y.⁴ In 2008, prior to the widespread implementation of rotavirus vaccines, rotavirus acute gastroenteritis (RAGE) led to estimated 453,000 deaths globally among children under 5 y of age.^5–7 Following the introduction of rotavirus vaccination, it is estimated that deaths due to RAGE dropped to 146,500 in 2015⁸ and 128,500 in 2016.⁹

As of November 2019, rotavirus vaccination was available or planned in 120 countries.¹⁰ Although rotavirus vaccines are commercially available, they are mostly unaffordable in developing countries. Children in low-income and middle-income countries are far more likely to be infected earlier in life than those in high-income countries. Because access to urgent care can be limited or unavailable in rural, impoverished settings, these children are more likely to develop severe disease and die.^11,12 However, in recent years, the predominant serotypes of epidemic rotavirus in China have been G1, G2, G3, G4, and G9.¹³ Based on the bovine rotavirus UK Compton strain, we developed a human-bovine reassortant hexavalent rotavirus vaccine (HRV) containing G1, G2, G3, G4, G8, and G9 serotypes, which was shown to be safe and highly immunogenic in animals and was well tolerated in a phase 1 clinical trial.¹⁴ The present study is a double-blinded, randomized controlled phase 2 clinical trial to assess the safety and immunogenicity of the hexavalent rotavirus vaccine.

Methods

Study design and participants

This phase 2 study was a single-center, randomized, double-blind, placebo-controlled clinical trial performed from June 2017, to July 2018 in Zhengding County, Hebei Province, China. This study was approved by the Institutional Review Board of the Hebei Center for Disease Control and Prevention. This study was conducted in accordance with good clinical practice, and the Declaration of Helsinki. Written informed consent was obtained from guardians before any study-related procedures. The clinical trial is registered at the National Medical Products Administration (NMPA) under number CRT20150878.

Healthy infants between 6 and 12 weeks of age were eligible for enrollment if they did not have a history of any confirmed or suspected immunosuppressive or immunodeficient condition, history of rotavirus gastroenteritis or intussusception (IS), or history of allergic disease. Vaccination was postponed in cases of acute febrile illness, vomiting, or diarrhea at the time of scheduled vaccination.

Eligible healthy infants aged 6–12 weeks were randomly assigned, in a 1:1:1 ratio, to receive three 2-ml oral doses of a high dose (l × 10^6.5 focus-forming unit (FFU)) of vaccine, low dose (l × 10^5.5 FFU) of vaccine or placebo on day 0, day 28, and day 56.

Randomization and blinding

Participants were randomly assigned (1:1:1) to receive a high-dose level, low-dose level of vaccine, or placebo only. The randomization codes were generated by the randomization statistician by means of block randomization using SAS software (version 9.2). A randomization code was assigned to each subject in sequence in the order of enrollment, and then the participants received the study vaccine labeled with the same code. Trial participants, study site staff, the data management team, and statisticians who performed the analysis remained blinded to the treatment assignment throughout this study.

Vaccines

The investigational vaccine (Wuhan Biological Products Research Institute Co. Ltd., Hubei, China) used in this study was a live attenuated, hexavalent rotavirus vaccine consisting of a mixture of bovine-human mono-reassortant viruses expressing the human VP7 genes encoding G1, G2, G3, G4, G8, and G9 on the genetic background of the bovine rotavirus UK Compton strain. A comprehensive description of the investigational products using indigenous manufacturing technology at the Wuhan National Biological Institute of China is described elsewhere.¹⁴ Two vaccine dose levels were assessed (low dose: 1 × 10^5.5 FFU, high dose: 1 × 10^6.5 FFU), according to the results of a phase 1 clinical trial. Each dose of the vaccine consisted of 2 ml. The placebo contained the same constituents as the vaccine but without the RVs.

Assessment of immunogenicity

The seroconversion rates and anti-rotavirus immunoglobulin A (IgA) antibody geometric mean concentrations (GMCs) at 28 d after receiving the full course of vaccination were the primary endpoints. To assess the serum immune response, approximately 2.5–3.0 ml of blood was collected before the first dose and 28 d after the third dose. Serum samples were isolated and stored at −20°C until evaluation. The rotavirus IgA antibody GMC was measured by the National Institute of Food and Drug Control (NIFDC) with an in-house-developed enzyme-linked immunosorbent assay kit; the method has been described elsewhere.¹⁴ The GMC was tested in the reference laboratory of the NIFDC, Beijing, China.

Assessment of safety

Participants were observed at the study site for at least 30 min after the administration of the vaccine to collect information on immediate adverse events. Thereafter, the subjects’ carers were given a thermometer and a diary card covering days 0–14 for safety follow-up. Parents or guardians of the participants were required to record any adverse events (e.g., fever, diarrhea, vomiting, anorexia, irritability/abnormal crying, drowsiness, and allergy) on the diary cards. On days 15 and 28, parents or guardians visited the study site for assessment by the study investigators who conducted face-to-face interviews to confirm subject safety. The incidence of solicited adverse events within 14 d, unsolicited adverse events within 28 d after each dose and serious adverse events (SAEs) reported during the 6 months after receiving the full course of vaccination were the secondary endpoints. The reported adverse events were graded according to the China National Medical Products Administration guidelines.¹⁵ The causal relationship between adverse events and vaccination was determined by the investigators.

Statistical analysis

All analyses were performed using SAS version 9.2. Hypothesis testing was two-sided and differences resulting in p values of .05 or less were considered statistically significant. We used the Pearson chi-square or Fisher’s exact test for the analysis of categorical outcomes. We calculated GMCs and corresponding 95% confidence intervals (CIs) on the basis of the standard normal distribution of the log-transformed antibody titer. We used ANOVA to compare the log-transformed antibody concentrations. Seroconversion rates were defined as anti-rotavirus IgA antibody concentrations ≥20 U/ml in initially seronegative infants, and a fourfold increase in the concentration from baseline to postdose 3 (PD3).¹⁴ An anti-rotavirus IgA antibody concentration <20 U/ml was calculated as 10 U/ml to be included in the GMC calculations.

Results

Demographic and other baseline characteristics

In total, 593 volunteers participated in interviews and physical examinations for this trial. Of these volunteers, 113 were excluded due to failure to sign the informed consent form, meeting the exclusion criteria or other reasons, and 480 subjects were enrolled in the trial (Figure 1). Overall, 480 subjects (100.0%) received at least one dose of vaccine or placebo; 449 (93.54%) received three doses and were followed up for safety for 30 d after the third dose; and 395 (82.29%) were included in the per-protocol population for immunogenic evaluation on day 28 after the third dose. The demographic characteristics of subjects were similar across treatment groups in terms of sex, mean age, length, and height (Table 1). The mean age of the subjects at the time of entry was 8.66 ± 1.89 weeks.

Figure 1. Trial profile.

Table 1. Summary of demographic characteristics.

	High-dose vaccine group(N = 160)	Low-dose vaccine group（N = 160）	Placebo group（N = 160）	P
Sex, n (%)				.28
Men	79 (49.38)	74 (46.25)	88 (55.00)
Women	81(50.64)	86 (53.75)	72 (45.00)
Age, weeks				.74
Mean (SD)	8.57 (1.72)	8.69(1.95)	8.73 (2.00)
Race, n (%)				*1.00
Han	160 (100.00)	160 (100.00)	159 (99.38)
Other	0 (0.00)	0 (0.00)	1 (0.63)
Length, cm				.98
Mean (SD)	58.69 (2.531)	58.64 (2.38)	58.69 (2.44)
Weight, kg				.23
Mean (SD)	5.76 (0.79)	5.62 (0.75)	5.74 (0.84)

N: total number of vaccinations; Data are mean (SD) or n (%); *Fisher’s exact test.

Immunogenicity

The anti-rotavirus IgA seroconversion rate of the vaccine groups reached more than 70.00%. The seroconversion rates ranged from 74.63% to 76.87% in the two vaccine groups. The seroconversion rates ranged from 74. 63% (G2) to 76.87% (G4 and G9) in the high-dose vaccine group and from 74.63% (G3) to 76.87% (G4 and G8) in the low-dose vaccine group. A significant difference was not observed between the low-dose and high-dose groups (Figure 2).

Figure 2. Seroconversion rates of serum 28d after last vaccination in vaccine and placebo groups.

The baseline GMCs of anti-rotavirus IgA were comparable between the groups. The GMC ranged from 79.25 U/ml (G4) to 84.46 U/ml (G1) in the high-dose vaccine group, and from 76.97 U/ml (G3) to 81.88 U/ml (G9) in the low-dose vaccine group, whereas it was approximately 10.0 U/ml in the placebo group. A significant difference was not observed between the low-dose and the high-dose vaccine group (Table 2).

Table 2. Summary of IgA response to serotypes G1, G2, G3, G4, G8, and G9 in the per-protocol immunogenicity population.

Serotype	Parameter	High-dose vaccine group (n = 134)		Low-dose vaccine group(n = 134)		Placebo group (n = 127)		P
		GMC	95%CI	GMC	95%CI	GMC	95%CI
G1	Baseline	11.64	10.65–12.73	10.98	10.27–11.74	12.44	11.11–13.94	.16
	PD3	84.46	65.89–108.26	78.16	60.85–100.41	12.35	10.91–13.97	< .0001
G2	Baseline	11.45	10.56–12.40	11.02	10.28–11.82	11.61	10.54–12.78	.66
	PD3	81.47	63.19–105.04	79.81	62.10–102.55	12.28	10.84–13.91	< .0001
G3	Baseline	11.48	10.55–12.49	11.12	10.36–11.94	12.66	11.28–14.20	.12
	PD3	79.47	61.98–101.88	76.97	60.10–98.56	12.33	10.88–13.99	< .0001
G4	Baseline	11.74	10.67–12.92	11.52	10.63–12.47	13.12	11.51–14.96	.17
	PD3	79.25	61.97–101.35	78.40	61.31–100.25	12.71	11.17–14.46	< .0001
G8	Baseline	11.22	10.44–12.06	10.74	10.13–11.39	11.96	10.79–13.26	.17
	PD3	80.76	62.95–103.59	79.26	61.83–101.60	12.32	10.90–13.94	< .0001
G9	Baseline	11.6	10.57–12.73	11.27	10.45–12.17	13.07	11.54–14.80	.09
	PD3	84.17	65.83–107.60	81.88	63.70–105.24	12.53	11.05–14.22	< .0001

GMC: geometric mean concentrations; PD3: post-dose 3; n: in the per-protocol population for immunogenic evaluation on day 28 after the third dose; P: ANOVA.

Vaccine safety

During the 28-d postvaccination follow-up period, AEs (solicited and unsolicited) occurred at a similar rate among both vaccine recipients (84.75% in the high-dose group; 85.63% in the low-dose group) and placebo (80.00%) recipients. The incidences of vaccine-related adverse events in the three groups were 71.88%, 66.25%, and 65.63%, respectively. All adverse reactions were mild or moderate in severity, and most resolved shortly after their onset. At least one solicited AE was recorded in 114 infants (71.25%) of the high dose of vaccine group, 106 infants (66.25%) of the low dose of vaccine group and 104 infants (65.00%) of the placebo group. At least one unsolicited AE was recorded in 86 infants (53.75%) of the high dose of vaccine group, 91 infants (56.88%) of the low dose of vaccine group and 92 infants (57.50%) of the placebo group. Upper respiratory tract infection (28.75% in the high-dose group, 35.00% in the low-dose group, and 32.50% in the placebo group) was the most common unsolicited AE reported in the groups (Table 3).

Table 3. Summary of adverse events (AEs).

	High-dose vaccine group (N = 160)		Low-dose vaccine group（N = 160）		Placebo group （N = 160）		P
	n	%	n	%	n	%
All AE	134	83.75	137	85.63	128	80.00	.41
With vaccination-related AE	115	71.88	106	66.25	105	65.63	.43
Without vaccination-related AE	85	53.13	91	56.88	92	57.50	.72
Discontinued due to an AE	10	6.25	4	2.50	6	3.75
With serious AE	2	1.25	5	3.13	2	1.25	.52
Solicited AEs	114	71.25	106	66.25	104	65.00	.45
Unsolicited Aes	86	53.75	91	56.88	92	57.50	.78

N: total number of vaccinations; n: the total number of participants who had adverse reactions; %: n/N; P: Fisher’s exact test.

During the 6-month postvaccination follow-up period, serious adverse events (SAEs) were reported in 2 of the 160 high-dose vaccine recipients (1.25%), 5 of 160 low doses of vaccine recipients (3.13%) and 2 of 160 placebo recipients (1.25%). During the entire study period, no fatal events were reported. None of the SAEs recorded were causally related to vaccination. No intussusception was reported (Table 3).

AEs of special interest are shown in Table 4. Fever (58.13% in the high-dose group, 51.88% in low-dose group, and 52.50% in placebo group) and diarrhea (23.13% in high-dose group, 25.63% in low-dose group, and 25.00% in placebo group) were the most common solicited AEs reported in the groups. There was no statistically significant difference among the three groups with respect to the incidence of fever, diarrhea, vomiting, anorexia, irritability/abnormal crying, or drowsiness. However, a higher frequency of allergy reported by participants in the low-dose group was observed than among participants in the high-dose group and in the placebo group (p = .04). All four allergic reactions occurred in the low-dose group.

Table 4. Summary of solicited adverse events (AEs).

	High-dose vaccine group			Low-dose vaccine group			Placebo group			P
	N	n	%	N	n	%	N	n	%
Fever
Post1 Dose	160	69	43.13	160	58	36.25	160	54	33.75	.20
Post2 Dose	147	46	31.29	155	44	28.39	150	37	24.67	.44
Post3 Dose	145	25	17.24	155	31	20.00	149	33	22.15	.58
Post any Dose	160	93	58.13	160	83	51.88	160	84	52.50	.47
Diarrhoea
Post1 Dose	160	21	13.13	160	22	13.75	160	19	11.88	.91
Post2 Dose	147	10	6.80	155	12	7.74	150	19	12.67	.19
Post3 Dose	145	12	8.28	155	15	9.68	149	10	6.71	.65
Post any Dose	160	37	23.13	160	41	25.63	160	40	25.00	.89
Vomiting
Post1 Dose	160	8	5.00	160	6	3.75	160	6	3.75	.88
Post2 Dose	147	2	1.36	155	3	1.94	150	2	1.33	1.00
Post3 Dose	145	1	0.69	155	2	1.29	149	3	2.01	.70
Post any Dose	160	9	5.63	160	9	5.63	160	9	5.63	1.00
Anorexia
Post1 Dose	160	7	4.38	160	2	1.25	160	3	1.88	.26
Post2 Dose	147	2	1.36	155	0	0.00	150	0	0.00	.11
Post3 Dose	145	2	1.38	155	1	0.65	149	5	3.36	.21
Post any Dose	160	9	5.63	160	3	1.88	160	7	4.38	.20
Irritability/abnormal crying
Post1 Dose	160	18	11.25	160	8	5.00	160	10	6.25	.10
Post2 Dose	147	2	1.36	155	2	1.29	150	5	3.33	.48
Post3 Dose	145	4	2.76	155	3	1.94	149	3	2.01	.85
Post any Dose	160	20	12.50	160	12	7.50	160	15	9.38	.35
Drowsiness
Post1 Dose	160	5	3.13	160	5	3.13	160	3	1.88	.83
Post2 Dose	147	0	0.00	155	3	1.94	150	1	0.67	.33
Post3 Dose	145	1	0.69	155	0	0.00	149	3	2.01	.17
Post any Dose	160	6	3.75	160	8	5.00	160	5	3.13	.77
Allergy
Post1 Dose	160	0	0.00	160	3	1.88	160	0	0.00	.11
Post2 Dose	147	0	0.00	155	1	0.65	150	0	0.00	1.00
Post any Dose	160	0	0.00	160	4	2.50	160	0	0.00	.04

N: total number of vaccinations; n: the total number of participants who had adverse reactions; %: n/N; P: Fisher’s exact test.

Discussion

Rotavirus vaccines have been shown to be safe and effective in preventing deaths from rotavirus gastroenteritis infection.¹² In low-income and middle-income countries, including China, issues such as the cost, purchase, and supply of vaccine stocks have always been points of concern.¹⁶ Therefore, cost-effective and safe rotavirus vaccines for the countries that need them the most remain an overwhelming need.¹⁴ Recently, a novel hexavalent rotavirus vaccine (serotypes G1, G2, G3, G4, G8, and G9) candidate was developed by the Wuhan Institute of Biological Products Co., Ltd. (WIBP) in China and was evaluated for safety and preliminary immunogenicity in phase I trials.¹⁴ We therefore aimed to further evaluate the immunogenicity and safety of the vaccine and to determine the dose for a planned phase 3 efficacy trial.

In this study, our findings showed that the hexavalent rotavirus vaccine was well tolerated at both high and low vaccine doses, without identifiable safety concerns. During the 28-d postvaccination follow-up period, AEs (solicited and unsolicited) occurred at a similar rate among both vaccine recipients (84.75% in high-dose group; 85.63% in low-dose group) and placebo (80.00%) recipients. The incidence of adverse reactions in different dose groups was similar, indicating that there were no dose-related aggravation concerns regarding safety. Moreover, most adverse reactions were mild and transient, and most resolved shortly after their onset. The results were similar to those of our study on a phase 1 clinical trial¹⁴ as well as those of other studies on rotavirus vaccines.^17–25 Fever was the most frequently reported symptom. Nine serious adverse events were reported during the trial, and none of the SAEs recorded were causally related to vaccination. Correspondingly, no intussusception or death was reported. There were four cases of allergy in the low-dose group (one subject had systemic red miliary papules; one subject had cheek eczema; and one subject had systemic rash, which appeared 3 d after receipt of the vaccine, mostly on the chest, back, and elbows, and the rest appeared scattered and untreated, lasting for 7 d; and one subject had systemic water blisters), but the same allergy was not observed in the groups and did not increase with the time since inoculation. Nevertheless, we will still focus on this adverse event in further large-scale population research.

The results of our study suggest that the hexavalent rotavirus vaccine is strongly immunogenic with a positive cumulative effect, with evidence that it might provide protection against rotavirus in infants. The IgA antibody responses observed in the infants who had received three vaccine doses of 1 × 10^5.5 FFU or 1 × 10^6.5 FFU were similar. Although it was not possible to establish a direct correlation with protection^26–31 because serological correlates of protection were lacking, this vaccine can be expected to be effective on the basis of the excellent vaccine response found in the present study. The immunogenicity of rotavirus vaccines has been evaluated in numerous phase I, II, and III studies.^32–36 Rong-Cheng Li et al. showed that the anti-rotavirus IgA seroconversion rate was 74.7%,³⁴ whereas the study by Zhaojun Mo et al. showed that the anti-rotavirus IgA seroconversion rate was 89.4%. In our study, the anti-rotavirus IgA seroconversion rate of the vaccine groups (high-dose group and low-dose group) reached more than 70.00%, and the seroconversion rates ranged from 74.63% to 76.87% in the two vaccine groups. The PD3 GMCs of anti-rotavirus IgA among vaccine recipients were higher than those among placebo recipients; additionally, a significant difference was not observed between the low-dose group and the high-dose group. The 10^5.5 FFU dosage formulation was selected for further investigation of efficacy.

There are some limitations to this study. First, the coadministration of the study vaccine with other vaccines could have affected the assessment of the safety and immunogenicity of the study vaccine. Although the subjects were required to be immunized with other vaccines 2 weeks after oral administration of the study vaccine, overlap might still have occurred, so further study on simultaneous administration must be conducted. In addition, the relatively small number of subjects in this study could have resulted in some bias or uncertainty, and since the sample size was small, it was not possible to identify potentially increased risks of some rare but severe adverse reactions. However, further studies, including an efficacy trial of the vaccine currently underway in China, will assist in addressing these important questions.

In conclusion, the novel oral hexavalent rotavirus vaccine was safe and immunogenic in infants aged 6–12 weeks, supporting the advancement of the vaccine candidate to phase 3 efficacy trials.

Acknowledgments

We greatly appreciate all the researchers who were involved in this clinical trial and the participants and their parents or legal guardians who participated in this study.

Disclosure statement

Qing-Liang Li, Kai Duan, Wei Chen, Ge-Lin Xu, Biao Yang, Ben Dong, and Jiu-Wei Zhang are currently employees of the Wuhan Institute of Biological Products Co., Ltd. Xiao-Ming Yang is currently an employee of China National Biotec Group Co., Ltd., Beijing, China. The other authors declare that they have no conflicts of interest.

Additional information

Funding

This study was sponsored and funded by the Wuhan Institute of Biological Products Co., Ltd., Hubei, China. The funders had no role in data collection, analysis, or interpretation.