Introduction

Rabies, a fatal zoonotic disease caused by the rabies virus, is responsible for 59,000 of yearly deaths worldwide, according to the World Health Organization (WHO)1,2,3. The prophylactic administration of the rabies vaccine after exposure to the rabies virus is the only effective measure of controlling and preventing rabies. Since 2010, the WHO has recommended the administration of two intramuscular post-exposure prophylaxis (PEP) regimes: the Essen and Zagreb regimens4. One dose of the Essen regimen should be administered on days 0, 3, 7, 14, and 28, while two doses of the Zagreb regimen should be administered on day 0, followed by a single dose on days 7 and 214. The Zagreb regimen, which was developed in Zagreb, Croatia in 1984, has been used worldwide for over 20 years. Data from clinical studies and meta-analyses show that the Zagreb regimen is not inferior to the Essen regimen in terms of immunogenicity, and had a favorable safety profile5,6,7,8,9,10,11.

A vaccine booster is given to persons exposed to rabies who have completed the primary rabies vaccination course. The vaccine booster is usually administered at a lower dosage than the primary vaccine; its purpose is to rapidly raise the antibody titer and protect the exposed individual from the virus. However, the booster vaccination schedule varies among different countries. Most developed countries, such as the U.S., the U.K., and Canada, recommend giving two boosters intramuscularly on days 0 and 3 after exposure, regardless of the interval between exposure and primary vaccination12,13,14. Meanwhile, China only recommends giving two boosters intramuscularly if rabies exposure occurs at > 3 months after the primary vaccination15. Because of the cost-effectiveness of the intradermal injection, it is used by many developing countries, such as Thailand and the Philippines. Thailand recommends giving 0.1 mL of the vaccine intradermally on day 0 if the exposure occurs within 6 months after the primary vaccination, or on days 0 and 3 if the exposure occurs at > 6 months after the primary vaccination16. The Philippines recommends giving 0.1 mL of the vaccine on days 0 and 3 to people who are at a high risk of exposure to the rabies virus17. The variability in the booster vaccination schedules across countries may be associated with the limited persistence of the antibody response (‘immune persistence’) following primary vaccination. The antibody response elicited following rabies PEP is relatively short-lived18. Moreover, the antibody titer at the time of booster vaccination greatly affects the immunogenicity of the booster vaccination19,20,21,22. Thus, it is necessary to assess whether the booster vaccination can increase the immunogenicity of a vaccine, even if it is administered a long time after the primary vaccine. Vaccination with the Zagreb regimen involves administering a lower dosage of the vaccine over a shorter time period than vaccination with the Essen regimen; thus, the Zagreb regimen is associated with higher compliance rates than the Essen regimen and is more affordable23. Despite the widespread application of the Zagreb regimen, the immunogenicity and safety of the vaccine booster administered after vaccination with this regimen has not been thoroughly assessed. Thus, we conducted a randomized clinical trial to compare the immunogenicity and safety of the purified Vero cell rabies (Speeda) vaccine when used as a booster at 6 months after the primary vaccination with the Zagreb and Essen regimens.

Results

Participants

At day 0 (before the booster), a total of 767 participants who completed the primary vaccination in groups B, C, E, and F gave blood samples. During the booster vaccination phase, 12 participants dropped out of the trial (1, 4, 2, and 5 in groups B, C, E, and F, respectively), and 37 participants had severe protocol violations (9, 11, 8, and 9 in groups B, C, E, and F, respectively). Finally, 718 participants were included in the bPPS (187, 177, 181, and 173 in groups B, C, E, and F, respectively), and 760 participants were included in the bSS (196, 190, 190, and 184 in groups B, C, E, and F, respectively) (Fig. 1). At month 6, blood samples were collected from 100 participants in each group and included in the IPS. The demographic characteristics of the bPPS are shown in Table 1. All variables were well-balanced among the four groups (i.e., B, C, E, F; P > 0.05).

Fig. 1
figure 1

The booster vaccination study participant workflow. The booster safety set (bSS) included the participants receiving at least one booster. The booster per protocol set (bPPS) included the participants who completed the booster vaccination, provided blood samples before and at 14 days after receiving the booster, and had no severe protocol violations. The immune persistence set (IPS) included the participants who completed the booster vaccination and provided blood samples at 12 months after the primary vaccination. Blood samples were collected at 12 months after the primary vaccination from the first 100 participants in each group. 1st: first booster; 2nd: second booster.

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Table 1 Baseline patient characteristics in the bPPS.
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Immunogenicity

At day 0 (before the booster), the APR was 100% for each group, and the GMCs were 6.61, 6.74, 7.29, and 7.02 IU/mL for groups B, C, E, and F, respectively (Table 2). The APR (P = 1.0000) and GMC (P = 0.5124) were not significantly different among the four groups.

Table 2 Immunogenicity of the booster vaccination in the bPPS.
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At 14 days after the booster was administered, the antibody titer increased sharply (GMC = 48.80, 64.38, 34.25, and 42.89 IU/mL in groups B, C, E, and F, respectively, with corresponding GMFIR values of 70.05%, 74.01%, 56.35%, and 63.58%). We found that the GMCs of the two- booster regimen were significantly higher than those of the one-booster regimen (P = 0.0092 for group B vs. group C; P = 0.0004 for group E vs. group F) (Fig. 2). However, the GMFIR was similar between groups B and C (P = 0.4007), as well as between groups E and F (P = 0.1653) (Fig. 3). Moreover, the GMCs of the Zagreb regimen were significantly higher than those of the Essen regimen, regardless of the booster dose (P = 0.0004 for group B vs. group E; P < 0.0001 for group C vs. group F) (Fig. 2); this was also true for GMFIR (P = 0.0024 for group B vs. group E; P = 0.0352 for group C and group F) (Fig. 3).

Fig. 2
figure 2

The GMC of booster vaccination. Geometric mean concentration (GMC) was derived by transforming the antibody titer by log10. At day 0, the statistical comparison was between all four groups; at 14 days after booster and month 6, the statistical comparison was between the groups with the same primary vaccination regimen or the same booster dose. *P > 0.05, **P < 0.05, ***P < 0.001.

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Fig. 3
figure 3

The GMFIR at 14 days after booster. The geometric mean 4-fold increase rate (GMFIR) was defined as the proportion of participants who were antibody-positive before the booster and had a 4-fold antibody titer rise after the booster. The statistical comparison was between the groups with the same primary vaccination regimen or the same booster dose. *P > 0.05, **P < 0.05, ***P < 0.001.

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At month 6, the APR remained at 100% for each group, but the antibody titer decreased over time, as evidences by a GMCs of 20.51, 31.85, 17.00, and 22.32 IU/mL for groups B, C, E, and F, respectively. We found a significant difference in GMC between groups B and C (P = 0.0088) or between groups C and F (P = 0.0235); this difference was not significant between groups B and E (P = 0.2464) or between groups E and F (P = 0.0719).

Safety

The overall incidence of ARs occurring from the first booster dose to 30 days after final dose was 11.22%, 15.79%, 3.68%, and 10.87% for groups B, C, E, and F, respectively (Table 3). The incidence of ARs was significantly lower in group E than in the other three groups (Fig. 4).

Table 3 The frequency of adverse events in each group of the bSS.
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Fig. 4
figure 4

The incidence of AEs and ARs. The adverse reactions (ARs) were defined as the adverse events (AEs) associated with vaccination. The statistical comparison was between the groups with the same primary vaccination regimen or the same booster dose. *P > 0.05, **P < 0.05.

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In the present study, all solicited AEs were classes as ARs, based on the judgement of the study investigators. Most of these ARs occurred within 3 days after vaccination, and were mild and transient. The most common injection-site AE was pain, with incidences of 8.16%, 12.11%, 3.16%, and 1.63%, in groups B, C, E, and F, respectively. Meanwhile, the most common systemic AE was fever, with incidences of 2.55%, 3.16%, 1.05%, and 1.63% in groups B, C, E, and F, respectively.

Regardless of the booster dose, the incidences of injection-site AEs and systemic AEs were lower in the Essen groups than in the Zagreb groups in number. But the difference was only significant between group B and E (P = 0.0062) or group E and F (P = 0.0140) in the incidences of injection-site AEs (Table 3; Fig. 4). Additionally, the incidences of the other inject-site and systemic AE were not significantly different between groups B and C, or between groups E and F, with the exception of pain, which was significantly lower in group E (Fig. 4). No SAEs, which were either associated with vaccination or caused patients to exit the trial, were reported.

Discussion

The Zagreb regimen offers higher compliance rates and better cost-effectiveness than the Essen regimen. These features of the Zagreb regimen have popularized its adoption across the globe over the past 20 years; however, the regiment was not approved in China until 2010. The Speeda vaccine, which has been used for decades in China, is the only purified Vero cell rabies vaccine approved for use alongside the Zagreb regimen. Thus, it is important to assess the immunogenicity and safety of administering the booster after vaccination with the Zagreb regimen. In the present study, we showed that the booster vaccination after Zagreb regimen was both effective and safe. Specifically, we observed a significant rise in GMC values after the booster vaccination, and all ARs were mild and short-lived.

The anti-rabies-virus antibody titer of the groups vaccinated with the Zagreb regimen rose more prominently at 14 days after vaccination than that of the group vaccinated with the Essen regimen, irrespective of whether they received one or two boosters. Our findings align with those of other studies assessing the immunogenicity of booster vaccination after the Zagreb regimen22,24,25. However, few studies have compared the immunogenicity of booster vaccination after Zagreb regimen versus that after the Essen regimen. The immunogenicity of the booster vaccination relies on the antibody titer immediately before the booster is administered. When designing the study, we were concerned about whether the Zagreb regimen elicited a sufficiently long-lived antibody response to enable effective booster vaccination. The antibody titer prior to booster vaccination may also differ between various primary vaccination regimens. Previous studies have reported that the Speeda was comparable in terms of immune persistence between the Zagreb regimen and Essen regimen; in some cases, Zagreb regimen performed better than the Essen regimen26,27,28. In our study, the GMCs of the Zagreb groups before they received the booster were lower (albeit not significantly) than those of the Essen groups, but were higher than those of the Essen groups after they received the booster. This finding indicated that the immune persistence of antibodies elicited by the Zagreb regimen had little influence on the GMC of the booster vaccination, and that the immunogenicity of the booster vaccination after the Zagreb regimen was sufficiently high. However, several previous studies have reported GMC values < 0.5 IU/mL in male vaccinees, even after they had received the booster29,30,31. Perrine et al. found that male sex was independently associated with inadequately low antibody titers31, a finding echoed by others32,33,34,35. Thus, increasing the potency or the dosage of a rabies vaccine may be used to raise the GMC in vaccinees with low GMCs36; however, the optimal vaccine potency and dosage are still under debate.

In our study, administering one booster elicited a lower antibody titer than administering two boosters. However, even the one-booster regimen elicited significantly higher GMCs than 0.5 IU/mL, with a GMFIR that was not significantly different between groups (i.e., groups B vs. C or groups E vs. F). These findings indicate that one booster may be sufficient to protect the exposed individual from rabies. Given that rabies is an infectious disease associated with socioeconomic deprivation, the most cost-effective vaccination regimen is conducive to rabies control. Notably, delivering one booster instead of two, not only raised the GMC rapidly above 0.5 IU/mL, but is also the more affordable option for vaccinees.

The Speeda vaccine, which has been used for decades in China, has demonstrated a good safety profile in many patient populations (e.g., healthy subjects, young and old individuals, pregnant women)27,37,38. In our study, the incidence of ARs was higher in the Zagreb groups than in the Essen groups, irrespective of whether they received one or two boosters; however, all of these ARs were mild (most for grade 1) and short-lived. Previously reported SAEs associated with PVRV administration, such as acute disseminated encephalomyelitis39 and severe Henoch Schönlein purpura40, were not reported in our study.

One limitation of our study is that we only assessed the immunogenicity of the booster at 6 months after the administration of the Zagreb regimen. This interval between vaccination and boosting differs from the interval recommended by other vaccine manufacturers. For instance, boosting with the human diploid cell rabies vaccine (HDCV) 10 years after administering the Essen regimen elicited a high level of immunogenicity in another study19. In accordance, whether extending the interval between vaccination with the Zagreb regimen and boosting with the Speeda will increase immunogenicity awaits to be seen.

In conclusion, our study demonstrated that the immunogenicity of the booster was greater when administered 6 months after the Zagreb regimen than when given 6 months after the Essen regimen. Moreover, one booster may be sufficient to induce adequate antibody levels to protect the exposed individual from rabies.

Methods

Study design and participants

This study was a randomized, open-label, controlled, non-inferiority phase 3 clinical trial, conducted in Shangyu City and Shengzhou City (Zhejiang Province, China) between July 2021 and December 2022. All volunteers were screened before receiving the primary vaccination. Eligible participants were healthy volunteers aged 10–60 years, without a history of being bitten or scratched by a mammal capable of carrying rabies 6 months before vaccination, without a history of rabies vaccination or rabies immunoglobulin administration, and without a history of an acute allergic reaction. The details of inclusion and exclusion criteria for primary vaccination are provided in the Supplementary material (Supplementary page 1). Written informed consent was provided by the participants (if aged 18–60 years) or the participants (if aged 10–17 years) and their guardians prior to study screening. In addition, the participants who completed the primary vaccination and provided blood samples at 6 months after the primary vaccination in groups B, C, E, and F were eligible for the booster vaccination.

This phase 3 clinical trial included two phases: the primary vaccination phase and the booster vaccination phase. All eligible participants were randomly allocated (1:1:1:1:1:1) to six groups after being screened. The details of the randomization process are provided in the Supplementary material (Supplementary page 3). Participants in groups A, B, or C (the Zagreb group) received a primary vaccination with the Zagreb regimen, while participants in groups D, E, or F (the Essen group) received a primary vaccination with the Essen regimen. The booster vaccination was given 6 months after the primary vaccination. Participants in groups B and E received one dose of the booster on day 0, while those in groups C and F received two doses of the booster on days 0 and 3 (Fig. 5). Because of the different vaccination schedules, the investigators and participants were not blinded in this study.

Fig. 5
figure 5

The booster vaccination phase study design. Group B received the Zagreb regimen as the primary vaccination, followed by one dose of the booster vaccination. Group C received the Zagreb regimen as the primary vaccination, followed by two doses of the booster vaccination. Group E received the Essen regimen as the primary vaccination, followed by one dose of the booster vaccination. Group F received the Essen regimen as the primary vaccination, followed by two doses of the booster vaccination. The booster vaccination was conducted 6 months after the primary vaccination. The day on which blood samples were collected from individuals due to receive the first booster was defined as Day 0 in the booster vaccination phase. Blood samples were collected at 12 months after the primary vaccination, which was approximately 6 months after the booster was given; this timepoint was defined as Month 6 in the booster vaccination phase.

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This clinical trial was approved by Institutional Review Board (IRB) of Zhejiang Provincial Center for Disease Control and Prevention (approval number: 2021-001-01), and adhered to the Good Clinical Practice (GCP), Declaration of Helsinki, and Chinese regulatory requirements. The protocol was registered at www.chinadrugtrials.org.cn/index.html under the registration number CTR20210426 at 08/03/2021.

The vaccine

The study vaccine (Speeda) was a lyophilized, purified human rabies vaccine (PVRV), generated in Vero cell by Liaoning Cheng Da Co., Ltd (Shenyang, China). This vaccine was approved by the Chinese National Medical Products Administration (NMPA) in 2004. The immunogenic component of the Speeda vaccine is an inactivated rabies viral strain PV2061. The potency of the study vaccine is 5.4 IU/dose, which considerably exceeds the WHO requirement41. The vaccine was reconstituted in 0.5 mL of sterile water and then injected intramuscularly into the deltoid muscle of upper arm. During the primary vaccination phase, the participants were vaccinated according to their assigned group (i.e., the Zagreb regimen for groups A, B, and C; the Essen regimen for groups D, E, and F). During the booster vaccination phase (~ 6 months after the primary vaccination), groups B and E received one dose of the booster on day 0, while and groups C and F received two doses of the booster on days 0 and 3. Groups A and D did not receive the booster.

Immunogenicity assessment

In the booster vaccination phase, blood samples were collected at 6 months after the primary vaccination but before the booster (day 0), and at 14 days after the booster. In addition, 100 blood samples (the first half of each group’s total sample size) were collected at 12 months after the primary vaccination (month 6, 12 months after primary vaccination, was equivalent to approximately 6 months after the booster vaccination) to assess the persistence of the antibody response elicited by the booster vaccination (Fig. 5). The presence of a rabies neutralizing antibody response was determined using the rapid fluorescent focus inhibition test (RFFIT), performed by the National Institute for Food and Drug Control (NIFDC; Beijing, China); data were reported as geometric mean concentration (GMC) values. A GMC of ≥ 0.5 IU/mL was defined as a positive result in this study. Immunogenicity was assessed in terms of GMC values, the geometric mean 4-fold increase rate (GMFIR), and the APR at 14 days after booster. The immune persistence was assessed by the GMC and APR at 6 months after booster.

Safety monitoring

All participants were observed for 30 min after vaccination for any adverse events (AEs), and followed up for 7 days after vaccination to collect data on solicited AEs, including injection-site AEs (e.g., pain, induration, redness, swelling, itching, or a rash) and systemic AEs (e.g., fever, fatigue, headache, dizziness, vomiting, abdominal pain, diarrhea, myalgia, arthralgia, or an acute allergic reaction). The non-solicited AEs were collected from the first booster dose to 30 days after final dose. The SAEs were collected from the first booster dose to 6 months after the final dose. All AEs were evaluated by professional investigators, graded according to the requirements issued by the NMPA42, and recorded in the Medical Dictionary for Regulatory Activities (version 26.1). The safety was assessed by the incidence of AEs occurring from the first booster dose to 30 days after final dose and the SAEs occurring from the first booster dose to 6 months after final dose.

Sample size and statistical analysis

The sample size in the primary vaccination was 200 participants per group for groups A, B, C, D, E, and F; the same sample size was used in the booster vaccination phase for groups B, C, E, and F. Details of the sample size calculation are provided in the Supplementary material (Supplementary page 3). Additionally, the sample size for evaluating immune persistence in the booster vaccination groups at month 6 was 100 participants per group (half of the sample size of each group).

The safety analysis was performed on the booster safety set (bSS), which included participants who received at least one booster. The number and proportion of participants who had adverse reactions (ARs), defined as AEs associated with the vaccination (evaluated by professional investigators) after vaccination were reported. The immunogenicity analysis was performed on the booster per protocol set (bPPS), which included participants who completed the booster vaccination, gave blood samples before and 14 days after the booster, and had no severe protocol violations. The GMC was derived by transforming the antibody titer by log10. The APR was defined as the proportion of the participants who had an antibody titer ≥ 0.5 IU/mL after vaccination. The GMFIR was defined as the proportion of participants who were antibody-positive before the booster and had a 4-fold antibody titer rise after the booster. The immune persistence analysis was performed on the immune persistence set (IPS), which included participants who completed the booster vaccination, gave blood samples at month 6, and had available GMC and APR data. The Student’s t-distribution was used to calculate the 95% confidence interval (CI) from the GMC. The Clopper-Pearson method was used to calculate the 95% CI of the APR and GMFIR. The Student’s t-test and analysis of variance (ANOVA) were used to analyze continuous data, while the Pearson χ2 test and the Fisher’s exact test were used to analyze categorical data. The non-inferiority study design was only used in the primary vaccination phase. In the booster vaccination phase, the two-sided alpha value was set to 0.05 in all statistical comparisons. SAS software (version 9.4, Cary, USA) was used to perform all statistical analyses.