As shown in Fig. 1, 14,094 participants from Anfeng and Qindong were randomly assigned to either the HEV239 or placebo group, with each participant receiving at least one dose of the vaccine or placebo. Of these, 14,069 participants were tested for anti-HEV antibodies prior to the first administration, including 7037 in the HEV239 group and 7032 in the placebo group. The majority of participants were from Anfeng (81.21%, 11,426/14,069), while the remaining 2643 participants were from Qindong. Ultimately, 12,082 participants underwent anti-HEV antibody testing at month 7 and were included in the analysis set, comprising 6028 in the HEV239 group and 6054 in the placebo group.

14,094 participants from Anfeng and Qindong were randomly assigned to either the HEV239 or placebo group, each receiving at least one dose of the vaccine or placebo. In the HEV239 group, 11 missed anti-HEV result at 0 m and 1009 missed anti-HEV result at 7 m. In the placebo group, 14 missed anti-HEV result at 0 m and 978 missed anti-HEV result at 7 m. 12082 participants were included in the analysis set, with 6028 in the HEV239 group and 6054 in the placebo group.

In the HEV239 group of the analysis set, vaccine doses were distributed as follows: 88 participants received one dose, 373 participants received two doses (184 at months 0 and 1; 189 at months 0 and 6), and 5567 received three doses. Baseline characteristics such as age, township, and anti-HEV IgG seroprevalence were similar across these groups with different doses, but sex distribution differed between some groups (Table 1; Supplementary Table 1).

There was a distinct difference in the baseline seroprevalence of anti-HEV IgG between the two townships, with Qindong showing a higher baseline seroprevalence (59.64%) compared to Anfeng (44.11%) (p < 0.0001). Additionally, the unsynchronised sampling timepoints further precluded a combined analysis of immunogenicity persistence data from both townships. Therefore, we conducted separate analyses for each township.

As shown in Table 2, among baseline seronegative participants in Anfeng, 53.3% (8/15) of those who received a single dose remained seropositive at months 91. Meanwhile, seropositive rates were 61.2% (30/49) for two doses at months 0 and 1, 59.6% (34/57) for two doses at months 0 and 6, and 73.0% (1270/1740) for three doses, all significantly higher than the placebo group (p < 0.0001). Concerning baseline seropositive participants in Anfeng, all 14 individuals in the single-dose group maintained their serostatus at month 91 (Table 3). The seropositivity rate was maintained by 94.1% (32/34) at month 91 for two doses at months 0 and 1 and was 95.6% (43/45) for two doses at months 0 and 6. With the three-dose regimen, 97.9% (1409/1439) of recipients remained seropositive at month 91. In the placebo group with baseline seropositivity, 81.9% (1258/1536) remained seropositive at month 91, which was significantly lower than those observed in the two-dose group (the 0–6-month schedule) (p = 0.018) and the three-dose group (p < 0.0001).

In Qindong, due to the limited sample size, the seropositivity rates among baseline seronegative participants showed fluctuations across different dosage groups during the follow-up period, but the seropositivity rate in the three-dose group remained high (87.3%, 254/291) at month 103, which was significantly higher than that of the placebo group (p < 0.0001). Among participants seropositive for anti-HEV IgG at baseline, nearly all of them retained antibody positivity at month 103, regardless of the number of doses administered. In contrast, only 89.4% (470/526) of placebo recipients who were seropositive at baseline had detectable antibody at the 103-month mark.

The trends in anti-HEV IgG geometric mean concentration (GMC) dynamics exhibited similar patterns across the four vaccine dosage groups, showing a decline after reaching a peak at the month 7 and eventually stabilizing around the month 55 or 67. Although peak levels varied among the groups, the plateau antibody levels were relatively similar (Fig. 2).

Data are presented as GMC and 95% CI in the figure. Time points of the missing data were not listed in the figure (month 91 in Anfeng, and month 79 in Qindong). Comparisons of antibody dynamics between different dosage groups were conducted using the generalized estimating equation (GEE) model. # denotes no differences in GMC were observed between any of the dosage groups.

The comparative analyses of antibody dynamics across different dosage groups revealed noteworthy findings. Among the participants in Anfeng who were seronegative at baseline, an increase in the number of vaccine doses corresponded with a marked enhancement in antibody response, demonstrating a clear dose-response relationship. The GMC levels in the three-dose group were significantly higher than those observed in both two-dose groups (the 0–1- and 0–6-month schedules) and the single-dose group (all p-values < 0.0001). Similarly, the GMC levels in each two-dose group were significantly higher than those in the single-dose group, with p-values of 0.0237 and <0.0001 for the 0–1-month and 0–6-month schedules, respectively. Interestingly, between the two-dose regimens, the 0–6-month schedule induced significantly higher GMC levels than the 0–1-month schedule (p = 0.0086). A similar dose-response relationship was observed among baseline seronegative participants in Qingdong, but the sample sizes in the single-dose and two-dose groups limited the robustness of these observations (Supplementary Table 2).

Among participants who were seropositive for anti-HEV IgG at baseline, the GMC levels were comparable across different dosage groups, particularly after 43 months. Generally, for baseline seropositive participants, all dosages regimens induced similarly durable and high antibody responses, significantly exceeding their baseline levels. By month 67, the GMC levels of Anfeng participants with baseline seropositivity who received a single dose, two doses at months 0 and 1, two doses at months 0 and 6, and three doses were 1.17 WU/mL (95% CI 0.65–2.11), 1.60 WU/mL (95% CI 1.10–2.32), 1.72 WU/mL (95% CI 1.28–2.30) and 1.85 (95% CI 1.77, 1.94), respectively, indicating a 2.4- to 3.6-fold increase over baseline levels. Additionally, in Anfeng, the GMC in the three-dose group was significantly higher than in the one-dose group (p = 0.0446), with no statistical differences observed among the other groups. In contrast, no differences in GMC were observed between any of the dosage groups in Qindong (Supplementary Table 3).