Zhaotong, which is the city where the serum samples were collected, is located at the intersection of Yunnan, Guizhou and Sichuan (Fig. 1). The hypsography is southern high and northern low-lying with an average altitude of 1, 685 meters, and the area is located between north 26°–28° latitude and east 102°–105° longitude. The areas have a typical continental monsoon climate that is characterized by wet and rainy summers, which is conductive to viral spread and transmission by vectors. According to data from the Yunnan Institute of Parasitic Diseases, JE cases were first reported in 1955 in Zhaotong. The annual incidence was 1.32/100, 000 from 1955 to 2010, which was two times the province average and was listed in the province-wide top three locations. JE has been sporadically spreading in this area and has a high mortality rate. Cases in children less than 14 years of age account for more than 87% of the total, which is far above the world or national average (70%–75%) (Campbell et al. 2011). August is the peak month for the disease in Zhaotong.
The seropositive rates to JEV detected by ELISA are shown in Fig. 2A and Supplementary Table S1. The variation trend in IgG was assessed by year post-booster vaccination (by 1-year classes) in this study. Generally, the overall seroprevalence of IgG to JEV was 46% (138/300) in samples from 300 vaccinated children. No significant differences were observed in overall seropositivity between males and females (χ2 = 0.006, P = 1.000, Supplementary Table S1), indicating that gender was not a crucial factor for the IgG seropositive prevalence to JEV.
Figure 2. Seropositive rates of anti-JEV antibodies detected by A ELISA and B a PRNT50 ≥ 1:10 in different years post-booster dose (by 1-year classes). Asterisks indicate significant differences among the overall total subjects, ** P < 0.01.
A linear association was found between time elapsed after the booster vaccination and waning of the seropositive rates of JEV-specific IgG (Fig. 2A, Supplementary Table S1). Waning of the seroprevalence of anti-JEV IgG was found with the elapse of time after the booster (χ2 for linear trend = 7.447, P = 0.006). As shown in Fig. 2A, the peak anti-JEV IgG seroprevalence was observed in the first 0.5–1.5 years (up to 82%), markedly decreased at approximately 2.5 years and then presented a plateau-like trend to 7.5–8.5 years with the lowest seroprevalence of 30% after booster vaccination (Fig. 2A). Remarkably, the anti-JEV IgG seroprevalence displayed linear trends when the waning trend was analysed for male or female vaccinated children alone, indicating that gender was not a crucial factor for the IgG seropositive prevalence to JEV (Fig. 2A).
In this study, the PRNT was performed to evaluate the seroprevalence of anti-JEV nAbs. As shown in Fig. 2B and Supplementary Table S2, the overall nAb seropositive rate was 36% (107/300), and no obvious differences were observed for nAb seroprevalence by gender (χ2 = 0.158, P = 0.718, Supplementary Table S2). The seroprevalence of anti-JEV nAbs was 82% at 0.5–1.5 years and then decreased gradually over time to 25% at 7.5–8.5 years after booster vaccination, suggesting an overall linear trend between the waning and the elapsed time after booster vaccination (χ2 = 10.103, P = 0.001, Fig. 2B, Supplementary Table S2). Likewise, the anti-JEV nAb seroprevalence in male or female vaccinated children displayed significant linear associations with the year post-booster (Fig. 2B).
In this study, we also analysed changes in the 50% plaque reduction (PRNT50) titres against JEV among 300 serum samples grouped by year post-booster (Fig. 3, Supplementary Table S3). High (> 1:160) anti-JEV nAb levels were not found in any of the time period groups. Medium (1:40–160) levels were detected in 23% of the serum samples collected in the first 0.5–1.5 years after the booster dose.Then, the JEV-specific nAb seroconversion rate and the nAb titres decreased with the time elapsed post-booster vaccination (Fig. 3, Supplementary Table S3). Similarly, the GMT of the anti-JEV nAbs fell gradually from 1:28.9 at 0.5–1.5 years to 1:7.3 at 7.5–8.5 years.
Factors associated with the seroprevalence to anti-JEV antibodies were analysed based on the ELISA or PRNT50 criteria. As shown in Table 1, the anti-JEV antibody seropositivity detected by ELISA was higher than that defined by a PRNT50 ≥ 1:10. Although a gap existed between positive seroconversion determined by the two methods (Fig. 2, Supplementary Table S1, S2), generally the changed trend observed by ELISA was consistent with that of the PRNT50. When the year post-booster was used as an index measurement, their respective highest (0.5–1.5-year group) and lowest (7.5–8.5-year group) values had good coincidence (Fig. 2). In addition, the anti-JEV IgG seropositivity rate (46%) detected by ELISA was higher than the overall anti-JEV nAb seropositive rate (36%). In other words, none of anti-JEV nAb seropositive subjects determined by PRNT50 were anti-JEV IgG seronegative by ELISA. This finding indicated that the seroprevalence of anti-JEV nAbs had higher reliability in evaluating the risk for JEV infection than anti-JEV IgG.
Table 1. Sensitivity differences between ELISA and PRNT50 for the determination of seropositivity against Japanese encephalitis virus.
The vaccinated sera were pooled into three groups according to the nAb level (negative, low and medium) during different periods based on the year post-booster (by 2-year classes, Table 2). To more precisely elucidate the association between the nAb titre and substantial protective efficacy, pooled nAb titres were evaluated and compared to the calculated input nAb titre before passive transfer into mice. As shown in Table 2, the pooled nAb titres are basically consistent with the input nAb values (Table 2). The medium pooled nAb titres in sera from the 0.5–2.5, 2.5–4.5, and 4.5–6.5-year periods were 1:105.6, 1:91.9, and 1:80.0, respectively (Table 2). Mice that received those sera showed no significant body weight changes and 100% of them survived when challenged with 50 LD50 of JEV, indicating effective protection (Fig. 4A, 4B). Surprisingly, sera collected from the 0.5–2.5-year period, which had a low nAb titre of 1:34.8, also showed 100% protection, indicating that other factors were involved in protection during this period. The low pooled nAb titres in the sera from 2.5–4.5 and 4.5–6.5 years were 1:30.3 and 1:26.4, respectively (Table 2); mice that received those sera had slight body weight losses (Fig. 4A) and 60% and 80% of them survived, respectively (Fig. 4A, 4B). A low nAb titre of 1:23.0 was detected in sera collected from the 6.5–8.5-year period, and no protection was observed when mice were passively immunized with the sera, similar to the protection level provided by the nAb seronegative sera. The median survival for the group with the low titre (6.5–8.5) was 10 days, which was slightly longer than that of all seronegative groups (7–8 days, Table 2). These results suggest that sera with medium nAb titres in all year periods and with low nAb titres in the early year periods could provide full protection from JEV challenge in mice. Conversely, long-term sera with low nAb titres or seronegativity exhibited limited or no protection. Additionally, a positive correlation was found between the pooled nAb titre (lg) and the final survival rate at day 21 (coefficient of determination 0.8122, Fig. 4C). Furthermore, the EPT50 value, which is the nAb titre in silico required to effectively protect half of the mice (50% survival rate) after JEV challenge during the observation period, was calculated from the slopes of the curves using regression analysis. As shown in Fig. 4C, an EPT50 of ~1:21.3 for nAbs was necessary in our study, which was higher than the recommended value in the WHO position paper (1:10).
Figure 4. Protective efficacy of human vaccinated sera based on passive transfer in mice. Groups of BALB/c mice (n = 10) were treated with human sera from vaccinated children and challenged with JEV (Beijing-1 strain) 12 h later. The mice were observed daily for 21 days. A Body weight changes and B survival curves. C Correlation of pooled nAb titres and the probability of survival against JEV. The pooled titre was lg transformed and plotted against final survival at day 21. R2 represents the coefficient of determination. The blue dotted line shows that the calculated EPT50 for nAbs of 1:21.3. Asterisks indicate significant differences among the overall total subjects.
Table 2. Experimental groups and survival data from mice passively inoculated with the sera/JEV mixture.
Spatial and Epidemiological Features of the Study Site
Waning Trend of Seropositive Rates to JEV after the Booster Dose
Waning Trend of Anti-JEV nAbs after Booster Vaccination
Reduction of Anti-JEV nAb Titres after Booster Vaccination
Seroprevalence of Anti-JEV nAbs Had Higher Reliability in Evaluating the Risk for JEV Infection
Vaccinated Sera with Different nAb Titres Showed Varying in Vivo Protective Efficacies in Mice
Table S1. Seroprevalence of antibodies against Japanese encephalitis virus detected by ELISA.
Table S2. Seroprevalence of antibodies against Japanese encephalitis virus detected by PRNT50.
Table S3. Categories of anti-JEV nAb titres against Japanese encephalitis virus.