Summary
Background
In India, two inactivated Japanese encephalitis (JE) vaccines, JENVAC and JEEV, are used in childhood immunization program as a two-dose schedule. Vaccine supply interruptions and population mobility can result in children receiving different products for their first and second doses. We assessed immunogenicity and safety of mixed-product vaccine schedules.
Methods
In this multicentric, open-label, phase 4, non-inferiority randomized trial conducted during November 11, 2023–September 2, 2025, infants aged 9–12 months were assigned (1:1:1:1) to JENVAC–JEEV, JENVAC–JENVAC, JEEV–JENVAC, or JEEV–JEEV. Second dose was given at the age of 16–24 months. The primary outcome was seroconversion assessed by 50% plaque reduction neutralization test titer against wild-type JE virus 28 days after dose-2. The secondary outcomes included frequency of solicited and unsolicited adverse events, and serious adverse events following JE vaccine schedules. Non-inferiority was concluded if the lower bound of the 95% CI for difference in seroconversion proportion between the mixed and same-product vaccine schedule exceeded −10% and was less than 0%. Registered with Clinical Trials Registry—India (CTRI/2023/07/055155).
Findings
Between November 11, 2023, and June 8, 2024, 911 infants were randomly assigned (1:1:1:1) to JENVAC–JEEV (n = 228), JENVAC–JENVAC (n = 228), JEEV–JENVAC (n = 227), and JEEV–JEEV (n = 228). In modified intention-to-treat analysis, seroconversion after second dose was 89.8% (194/216), 90.2% (184/204), 90.9% (189/208), and 89.2% (190/213) respectively. The difference in seroconversion between mixed-product and same-product vaccine schedules was −0.38% (95% CI −6.1 to 5.4) among JENVAC-primed and 1.66% (−4.1 to 7.4) among JEEV-primed infants. The corresponding estimates in per-protocol analysis were −1.11% (95% CI −6.7 to 4.5) and 0.89% (−4.9 to 6.6) respectively. Solicited adverse events occurred in 35.5–37.9% infants across the study groups. None of the serious adverse events was vaccine related.
Interpretation
Mixed-product JENVAC and JEEV schedules were non-inferior to same-product schedules for short-term neutralizing antibody seroconversion and showed no safety signals. These findings support programme flexibility when children cannot complete JE vaccination with the same-product.
Funding
The trial was funded by Indian Council of Medical Research, New Delhi, India.
Keywords: Japanese encephalitis, Inactivated vaccines, Immunogenicity, Vaccine interchangeability, India
Research in context.
Evidence before this study
In India, two inactivated Vero-cell derived Japanese encephalitis (JE) vaccines, JENVAC and JEEV are used in JE endemic districts as a two-dose schedule administered at 9–12 months and 16–24 months of age. Given inter-state migration and procurement delays, it is possible that children who received the first dose of a particular JE vaccine type might receive the second dose with another type of JE vaccine. Evidence on safety and immunogenicity of such mixed-product schedules is therefore needed. We searched PubMed on 01/05/2026 by using the combination of keywords such as “Japanese encephalitis”, “JE Vaccines”, “Interchangeability”, “immunogenicity” and “safety”. We identified 11 studies evaluating JE vaccine interchangeability or heterologous schedules. Most studies assessed combinations involving live-attenuated, chimeric, inactivated mouse-brain-derived, or IXIARO/IC51 vaccines, often in booster contexts or with shorter intervals between doses. We found no trial evaluating mixed-product schedules of JENVAC and JEEV under India's routine two-dose immunization schedule.
Added value of this study
We did a multicenter, phase 4, non-inferiority, randomized controlled trial to generate evidence on immunogenicity and safety of interchanging JEEV and JENVAC as a part of a two-dose schedule. Mixed-product schedules (JENVAC–JEEV and JEEV–JENVAC) were non-inferior to the corresponding same-product schedules for seroconversion based on 50% plaque reduction neutralization test (PRNT50) titers against JE virus 28 days after the second dose, with about 90% seroconversion across all groups. No safety signals were identified, and no serious adverse events were assessed as related to vaccination. Our findings indicated that JENVAC and JEEV vaccines can be interchanged within the two dose schedule without compromising short-term immunogenicity or safety.
Implications of all the available evidence
In large-scale vaccination settings, children may not always receive the same vaccine for both doses due to migration, vaccine shortages, changes in procurement, or supply issues. Our findings provide evidence that JENVAC and JEEV can be used interchangeably for the two dose JE vaccination schedule without affecting safety or immune responses. This flexibility could prevent missed vaccine doses, especially in JE-endemic areas, where high two-dose vaccination coverage is needed to prevent the disease.
Introduction
Japanese encephalitis (JE) is a mosquito-borne viral encephalitis caused by Japanese encephalitis virus (JEV), a flavivirus transmitted predominantly by Culex mosquitoes.1 It remains the leading vaccine-preventable cause of viral encephalitis in the Asia–Pacific region. An estimated 3 billion people residing in 24 countries are at risk of JEV infection.2,3 In 2019, an estimated 57,000 JE cases (95% CI: 18,003–184,525) and 20,642 deaths (95% CI: 2252–77,204) occurred globally, with India contributing the largest share (39.1%) of burden, followed by Bangladesh (18%) and China (14%).4 Although most infections are asymptomatic, about 1 in 250 infected people develop severe neuroinvasive disease. Among those who develop encephalitis, case fatality rates can range between 20–30% and 30–50% of the survivors have long-term neurological, cognitive, or psychiatric sequelae.5, 6, 7 Childhood vaccination remains the cornerstone of JE prevention and control in endemic countries.
Several JE vaccines based on live attenuated, inactivated mouse brain–derived, and inactivated Vero cell–derived platforms are currently used across Asia, with substantial variation in immunization schedules between countries. Inactivated JE vaccines are used in many national immunization programmes.8 In India, JE is endemic in 357 districts across 24 states and Union Territories with more than 10,000 laboratory-confirmed JE cases and 984 deaths reported during 2018–2025.9 Under India's Universal Immunization Programme (UIP), two inactivated Vero cell-derived JE vaccines are currently used: JENVAC (Bharat Biotech International Ltd., Hyderabad, India), developed from an Indian JE virus strain (Kolar-821564XY), and JEEV (Biological E. Ltd, Hyderabad, India), derived from the SA 14-14-2 strain that forms the basis of several internationally used JE vaccines.10,11 JEEV received regulatory approval for use in India in 2011, and JENVAC received its first marketing authorization in 2016.12,13
In UIP, both vaccines are administered as a two-dose intramuscular schedule, with the primary dose given at 9–12 months of age and a second dose at 16–24 months age.14 Although individual states generally receive a single type of JE vaccine for routine childhood immunization programmes, programmatic realities, such as vaccine supply interruptions, procurement changes and population mobility, can result in some children receiving the second dose of another type of JE vaccine. Evidence on the immunogenicity and safety of interchangeable use of different inactivated JE vaccines is limited,15 particularly for two-dose schedule with a longer interval between doses. Such evidence could help sustain vaccination coverage during supply disruptions in endemic settings.
We therefore conducted a multicentre, randomized, non-inferiority trial to compare the immunogenicity and safety of mixed-product JE vaccination schedules (JENVAC–JEEV or JEEV–JENVAC) with same-product schedules (JENVAC–JENVAC or JEEV–JEEV) among Indian children vaccinated according to the national immunization schedule.
Methods
Study design and participants
This phase-4, open-label, randomized, non-inferiority trial was conducted during November 11, 2023–September 2, 2025 at five medical college hospitals located in five different regions of India (Kolkata, West Bengal; Wardha, Maharashtra; Bhubaneswar, Odisha; Bibinagar, Telangana and Patna, Bihar).
Healthy infants aged 9–12 months attending immunization clinics of the participating sites were eligible for enrollment. Eligibility was assessed by a study pediatrician on the basis of medical history and clinical examination. Infants with severe malnutrition, defined as weight-for-age z-score < −3 SD of the median according to WHO Child Growth standards, a known systemic disorder, history of epilepsy or other diagnosed neurological disorder, or prior receipt of a JE vaccine were excluded from the study. Infants with a history of febrile illness 72 h before or at enrollment were temporarily excluded and reassessed after 7 days for eligibility.
Randomization and allocation concealment
All eligible infants were randomly assigned in a 1:1:1:1 ratio to receive one of the four vaccination schedules: JENVAC–JEEV, JENVAC–JENVAC, JEEV–JENVAC, or JEEV–JEEV. Randomization was performed centrally, using a computer-generated permuted block sequence with variable block sizes of 4, 8, and 16, generated by an independent statistician using the ralloc package in STATA SE version 17.0. The randomization list was uploaded into a secure, web-based application. Study staff, who had no access to the allocation sequence, enrolled participants and assigned interventions using this system after obtaining written informed consent. As this was an open-label study, caregivers and site staff were aware of the vaccine product administered. However, laboratory staff performing immunogenicity assays were masked to treatment allocation. Data were collected and managed using Redcap version 14.0.
Procedures
JENVAC and JEEV vaccines were supplied to the study sites as multidose vials and stored at 2–8 °C until administration. Each 0.5 mL dose of JENVAC contained 5 μg of purified inactivated JE virus protein from JEV Kolar strain 821564-XY, 0.25 mg of aluminum as aluminum hydroxide gel, 0.025 mg of thiomersal, and phosphate-buffered saline. Each 0.5 mL dose of JEEV contained 3 μg of purified inactivated Japanese encephalitis virus derived from SA-14-14-2 strain, 0.1% w/v aluminum as aluminum hydroxide, and phosphate-buffered saline.12,16
On the day of randomization, participants received 0.5 mL of the assigned vaccine intramuscularly in the anterolateral aspect of the left mid-thigh. The second dose of the assigned JE vaccine was administered at 16–24 months of age in the anterolateral aspect of the right mid-thigh according to the national immunization schedule. Concomitant routine vaccines administered along with the first dose of JE vaccine included Measles Rubella containing vaccine (MRCV) administered subcutaneously in right upper arm, the third dose of fractional inactivated polio vaccine (fIPV) administered intradermally in left upper arm and a booster dose of pneumococcal conjugate vaccine (PCV) administered in the anterolateral aspect of right mid-thigh. The second dose of JE vaccine was administered along with second dose of MRCV and DPT booster given in the anterolateral aspect of the left mid-thigh.14
Participants were observed for at least 30 min after each vaccination for immediate adverse events. Parents or caregivers were given diary cards to record any adverse event and information about the adverse event was also collected during the follow up visits. They were contacted by telephone twice during the first week after each vaccine dose (day 2 + 2 and day 6 ± 2), weekly for the subsequent three weeks, and monthly thereafter for safety assessment, with follow-up continuing until 12 weeks after the second dose. Parents or caregivers were advised to contact the study team or bring the child to the study site for any illness or adverse event. Children with adverse events were monitored until resolution of event.
Solicited local and systemic adverse events were predefined and recorded for 7 days after each vaccine dose. Unsolicited adverse events were all other untoward medical events reported after vaccination and were collected throughout follow-up. A serious adverse event (SAE) was defined as any untoward medical occurrence, resulting in death or required inpatient hospitalization or prolonged an existing hospitalization or resulted in persistent or significant disability/incapacity. SAEs were collected from enrollment until the final study visit. Adverse events (AEs) were graded for severity according to the Division of AIDS (DAIDS), National Institutes of Health toxicity grading scale based on clinical intensity, functional limitation, and need for medical intervention.17 Causality assessment of SAEs was performed according to the World Health Organization Adverse Events Following Immunization (AEFI) causality assessment algorithm and classified as consistent with immunization, indeterminate, inconsistent with immunization, or unclassifiable.18
3 mL venous blood sample was collected from each participant before vaccination, 28 (−2 to +7) days after dose-1, immediately before dose-2, and 28 (−2 to +7) days after dose-2. Sera were tested for 50% plaque reduction neutralization test antibody titers (PRNT50) against a wild-type JEV strain (JE 057434) at the ICMR–National Institute of Virology, Pune, according to a standardized assay protocol (Appendix p 3). This strain, which belongs to genotype III, was isolated during a large outbreak in 2005 in Uttar Pradesh, India.19,20 In a subset of randomly selected participants, PRNT50 assays were also performed against the Kolar-821564XY (homologous to JENVAC) and SA-14-14-2 (homologous to JEEV) strains at the same four time points.
Outcomes
The primary outcome was the proportion of participants with seroconversion 28 days after completion of the two-dose vaccination schedule. Seroconversion was defined as a PRNT50 antibody titer against JEV of <1:10 at baseline and ≥1:10 after vaccination, or a ≥4-fold rise in PRNT50 antibody titer among participants with a baseline antibody titer ≥1:10. A PRNT50 antibody titer against JEV of ≥1:10 was considered indicative of seroprotection.21 The secondary outcomes included solicited and unsolicited adverse events, and SAEs following mixed-product and same-product JE vaccine schedules.
Statistical analysis
The trial was designed to test whether mixed-product JE vaccine schedules were non-inferior to the same-product schedules for seroconversion after completion of the two-dose schedule. The two prespecified primary comparisons were JENVAC–JEEV versus JENVAC–JENVAC among JENVAC-primed participants, and JEEV–JENVAC versus JEEV–JEEV among JEEV-primed participants.
Seroconversion of up to 90% after two doses of either JENVAC or JEEV have been reported in earlier trials.22, 23, 24 Assuming a seroconversion of 90% with two-dose JE vaccination, non-inferiority margin of 10%, one-sided alpha of 0.025, 90% power, and 15% loss to follow-up, a sample size of 225 participants per group was required. This resulted in a total sample size of 900, enabling two independent comparisons: JENVAC–JEEV versus JENVAC–JENVAC, and JEEV–JEEV versus JEEV–JENVAC.
Immunogenicity analyses were done in both the modified intention-to-treat (mITT) and per-protocol (PP) populations. The mITT population included all participants who received the first assigned vaccine dose and had evaluable blood samples at baseline and 28 days after the second dose, irrespective of whether the post-dose-2 sample was collected within the prespecified visit window. The PP population included participants who received both doses according to random allocation, adhered to protocol specified visit windows, and had evaluable blood samples collected at baseline and 28 days after the second dose. The safety analysis set comprised all participants who received at least one dose of the assigned JE vaccine. For the primary and other outcomes, missing data were not imputed. The analyses were conducted using an available-case approach, the denominator for each analysis was based on the number of participants with available data for the respective outcomes.
We calculated the differences in the seroconversion proportion in the mixed-product vaccine schedule and the seroconversion proportion in same-product vaccine schedule within the corresponding priming stratum. Non-inferiority was concluded if the lower bound of the two-sided 95% CI for the difference was greater than −10% and was less than 0%. Seroconversion, geometric mean titers (GMT), and adverse events were summarized with corresponding 95% CIs. Geometric mean titers were calculated after log transformation and presented with 95% CIs after back-transformation. Statistical analyses were done using Stata SE version 17.0.
Ethics
The trial was approved by the Institutional Ethics Committees of ICMR National Institute of Epidemiology, Chennai (Ethics reference number: NIE/IHEC/202201-03) and all participating sites. Written informed consent was obtained from the parent or legal guardian before enrollment. The trial was registered with the Clinical Trials Registry–India (Registration number: CTRI/2023/07/055155). A Data and Safety Monitoring Board (DSMB) was constituted to oversee the safety of participants and the integrity of trial data. The study protocol and analysis plan are provided in a separate supplementary file.
Role of the funding source
The trial was funded by Indian Council of Medical Research, New Delhi, India. The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.
Results
Between November 11, 2023, and June 8, 2024, a total of 937 infants were screened, of whom 26 were found ineligible. The remaining 911 eligible infants were randomly assigned to JENVAC–JEEV (n = 228), JENVAC–JENVAC (n = 228), JEEV–JENVAC (n = 227), and JEEV–JEEV (n = 228) (Fig. 1).
Fig. 1.
Study flow diagram.
At enrollment, the mean (SD) age of participants across the four groups was 9.5 (0.77), 9.5 (0.73), 9.4 (0.69), and 9.5 (0.77) months, respectively. The corresponding mean (SD) weight was 7.9 (1.1), 7.9 (1.0), 7.9 (0.9), and 8.0 (0.9) kg, and the mean (SD) length was 70.9 (3.2), 70.7 (2.8), 70.7 (3.1), and 71.2 (3.0) cm respectively (Table 1).
Table 1.
Baseline characteristics of study participants.
| Characteristics | Vaccine schedules |
|||
|---|---|---|---|---|
| JENVAC–JEEV (n = 228) no. (%) | JENVAC–JENVAC (n = 228) no. (%) | JEEV–JENVAC (n = 227) no. (%) | JEEV–JEEV (n = 228) no. (%) | |
| Age (in months) | ||||
| Mean (SD) | 9.5 (0.77) | 9.5 (0.73) | 9.4 (0.69) | 9.5 (0.77) |
| Sex | ||||
| Male | 142 (62.3) | 113 (49.6) | 125 (55.1) | 135 (59.2) |
| Female | 86 (37.7) | 115 (50.4) | 102 (44.9) | 93 (40.8) |
| Anthropometry | ||||
| Mean weight (kg) (SD) | 7.9 (1.1) | 7.9 (1.0) | 7.9 (0.9) | 8.0 (0.9) |
| Mean height (cm) (SD) | 70.9 (3.2) | 70.7 (2.8) | 70.7 (3.1) | 71.2 (3.0) |
Of the 911 randomly assigned participants, 909 (99.8%) received the first assigned vaccine dose, and 867 (95.1%) received the second dose. The concomitant vaccines administered with first and second dose of JE vaccine are summarized in Appendix p 4.
Baseline blood samples were collected from 902 (99.2%) of the 909 vaccine recipients. Evaluable post dose-2 serum samples were available from 841 participants and comprised the mITT population (JENVAC–JEEV: 216, JENVAC–JENVAC: 204, JEEV–JENVAC: 208, JEEV–JEEV: 213) (Fig. 1, Appendix pp. 5–9). Of these, 775 samples were collected within the prespecified post-dose-2 visit window and constituted the PP population (Appendix pp. 5–9). The median interval between dose-1 and dose-2 was comparable among the four vaccine groups with an overall median (IQR) of 7 months (IQR: 6.7–7.4) (Appendix p 10). Overall, 77 (8.5%) of the 902 participants had PRNT50 antibody titer against JEV of 1:10 or more at the baseline (Appendix pp 11 and 12).
In the mITT analysis, the proportion of participants who seroconverted 28 days after the first vaccine dose ranged from 48.8% to 56.3% across different vaccine schedules. Before administration of the second dose, 34.5%–41.3% remained seroconverted (Table 2). In the PP analysis, the corresponding proportions were 47.1%–56.1% after the first dose and 33.2%–42.9% before the second dose (Table 2).
Table 2.
Seroconversion (%) at different timepoints by vaccine schedules.
| Vaccine schedules | No. (%) with PRNT50 titers of ≥1:10 at baseline | Seroconversiona (%) |
Difference in seroconversion (%) after second dose (mixed product—same product schedule; 95% CI) | ||
|---|---|---|---|---|---|
| 28 days after first dose n/N (%) | Before second dose n/N (%) | 28 days after second doseb n/N (%) | |||
| Modified intention-to-treat analysis set (n = 841) | |||||
| JENVAC–JEEV | 22/216 (10.2) | 101/207 (48.8) | 67/194 (34.5) | 194/216 (89.8) | −0.38 (−6.1 to 5.4) |
| JENVAC–JENVAC | 15/204 (7.4) | 100/197 (50.8) | 78/189 (41.3) | 184/204 (90.2) | Reference |
| JEEV–JENVAC | 12/208 (5.8) | 113/203 (55.7) | 69/197 (35.0) | 189/208 (90.9) | 1.66 (−4.1 to 7.4) |
| JEEV–JEEV | 20/213 (9.4) | 116/206 (56.3) | 69/198 (34.8) | 190/213 (89.2) | Reference |
| Per-protocol analysis set (within window) (n = 775) | |||||
| JENVAC–JEEV | 20/198 (10.1) | 90/191 (47.1) | 64/184 (34.8) | 180/198 (90.9) | −1.11 (−6.7 to 4.5) |
| JENVAC–JENVAC | 13/188 (6.9) | 89/183 (48.6) | 76/177 (42.9) | 173/188 (92.0) | Reference |
| JEEV–JENVAC | 12/193 (6.2) | 106/189 (56.1) | 62/187 (33.2) | 176/193 (91.2) | 0.89 (−4.9 to 6.6) |
| JEEV–JEEV | 18/196 (9.2) | 107/191 (56.0) | 66/185 (35.7) | 177/196 (90.3) | Reference |
Denominators vary across visits because serum samples were not available from all participants at each timepoint.
Seroconversion was defined as a PRNT50 antibody titer against JEV of <1:10 at baseline and ≥1:10 after vaccination, or a ≥4-fold rise in PRNT50 titer against JEV among participants with a baseline titer ≥1:10.
Primary endpoint.
In the mITT analysis, the proportion of participants who seroconverted 28 days after the second vaccine dose was 89.8% (194/216) in the JENVAC–JEEV group, 90.2% (184/204) in the JENVAC–JENVAC group, 90.9% (189/208) in the JEEV–JENVAC group, and 89.2% (190/213) in the JEEV–JEEV group (Table 2). The difference in seroconversion between the mixed-product and same-product vaccine schedule was −0.38% (95% CI −6.1 to 5.4) among JENVAC-primed participants and 1.66% (95% CI −4.1 to 7.4) among JEEV-primed participants, meeting the prespecified criterion for non-inferiority (Table 2). For both comparisons, the lower bound of the 95% CI was greater than the prespecified non-inferiority margin of −10 percentage points. The corresponding estimates in the PP analysis were −1.11% (95% CI −6.7 to 4.5) and 0.89% (95% CI −4.9 to 6.6) respectively (Table 2). The seroprotection 28 days after the second dose of vaccine ranged between 93.6% and 96.4% in the PP analysis and between 92.2% and 95.3% in the mITT analysis (Appendix p 13).
Among the 772 participants who were seronegative at baseline, the proportions who seroconverted 28 days after the second dose in the mITT analysis ranged from 92.6% to 94.8% across the four vaccine regimens [JENVAC–JEEV: 93.3% (181/194), JENVAC–JENVAC: 92.6% (175/189), JEEV–JENVAC: 92.9% (182/196), and JEEV–JEEV: 94.8% (183/193)]. Among the 69 participants who were seropositive at baseline, the proportions who seroconverted were 35.0%–60.0% across the four vaccine schedules [JENVAC–JEEV: 59.1% (13/22), JENVAC–JENVAC: 60.0% (9/15), JEEV–JENVAC: 58.3% (7/12) and JEEV–JEEV: 35.0% (7/20)]. A similar pattern of seroconversion was also found in the PP analysis set (Appendix pp 14 and 15).
The geometric mean PRNT50 antibody titers against JEV increased after the second vaccine dose in all vaccine groups (Table 3). In the mITT analysis, GMTs after the second dose were 74.3 (95% CI 61.5–89.8) in the JENVAC–JEEV group, 73.3 (95% CI 59.7–90.1) in the JENVAC–JENVAC group, 69.9 (95% CI 57.3–85.1) in the JEEV–JENVAC group, and 67.6 (95% CI 56.8–80.4) in the JEEV–JEEV group. The corresponding GMTs in the PP analysis were 79.8 (95% CI 65.5–97.2), 75.0 (95% CI 61.6–91.2), 73.8 (95% CI 59.7–91.2), and 71.2 (95% CI 59.6–85.1) respectively (Table 3).
Table 3.
Geometric mean of neutralizing antibody titers (95% CI) at different timepoints by vaccine schedules.
| Vaccine regimen | Geometric mean of neutralizing antibody titers (95% CI) |
28 days after second dose | ||
|---|---|---|---|---|
| Before first dose | 28 days after first dose | Before second dose | ||
| Modified intention-to-treat analysis set | ||||
| JENVAC–JEEV | 2.2 (1.9–2.5) | 9.4 (7.8–11.3) | 5.6 (4.7–6.8) | 74.3 (61.5–89.8) |
| JENVAC–JENVAC | 2.0 (1.8–2.3) | 10.2 (8.5–12.4) | 5.8 (4.8–6.9) | 73.3 (59.7–90.1) |
| JEEV–JENVAC | 2.0 (1.8–2.2) | 8.9 (7.4–10.6) | 5.7 (4.8–6.8) | 69.9 (57.3–85.1) |
| JEEV–JEEV (n = 228) | 2.2 (2.0–2.5) | 10.3 (8.5–12.4) | 5.3 (4.5–6.3) | 67.6 (56.8–80.4) |
| Per-protocol analysis set | ||||
| JENVAC–JEEV | 2.1 (1.9–2.4) | 9.2 (7.6–11.1) | 5.6 (4.6–6.8) | 79.8 (65.5–97.2) |
| JENVAC–JENVAC | 1.9 (1.7–2.1) | 8.8 (7.3–10.6) | 5.8 (4.8–7.0) | 75.0 (61.6–91.2) |
| JEEV–JENVAC | 2.1 (1.8–2.3) | 10.4 (8.6–12.7) | 5.7 (4.7–6.9) | 73.8 (59.7–91.2) |
| JEEV–JEEV | 2.2 (1.9–2.5) | 10.0 (8.2–12.1) | 5.4 (4.6–6.5) | 71.2 (59.6–85.1) |
Among the 114 children whose sera were tested against the Kolar-821564XY and SA-14-14-2 strains, the proportions who seroconverted and the neutralizing antibody titers were generally higher against vaccine homologous strains than the wild type JEV strain, particularly after the first dose and before the second dose. Post-dose-2 responses were high across all three tested strains (Fig. 2, Appendix p 16).
Fig. 2.
Seroconversion against three JE virus strains at four timepoints by vaccine schedules.
In the safety population, 333 participants reported at least one solicited adverse event within 7 days of vaccination, with the incidence ranging between 35.5% and 37.9% across the four vaccine schedules (Tables 4 and 5). Fever was the most frequently reported solicited adverse event, followed by rhinorrhea and cough. A total of 721 solicited AEs and 793 unsolicited AEs were reported. SAEs occurred in 20 participants with 23 events reported. This included eight events in the JENVAC–JEEV group, six in the JENVAC–JENVAC group, five in the JEEV–JENVAC and four in the JEEV–JEEV group. No serious adverse event was assessed as related to vaccination (Appendix pp 17–19). The majority (92.5%) of the AEs were mild and resolved without any sequelae (Appendix pp 20 and 21).
Table 4.
Distribution of solicited and unsolicited adverse events (AEs) following JE vaccination in JENVAC primed schedules.
| Adverse events | JENVAC–JEEV (n = 228) |
JENVAC–JENVAC (n = 228) |
||||
|---|---|---|---|---|---|---|
| No. of participants | Incidence (95% CI) | Total AEs | No. of participants | Incidence (95% CI) | Total AEs | |
| Solicited eventsa | 81 | 35.5 (29.3, 42.1) | 197 | 81 | 35.5 (29.3, 42.1) | 169 |
| Fever | 66 | 28.9 (23.2, 35.3) | 75 | 68 | 29.8 (24, 36.2) | 72 |
| Runny nose | 20 | 8.8 (5.4, 13.2) | 23 | 22 | 9.6 (6.1, 14.2) | 24 |
| Cough | 14 | 6.1 (3.4, 10.1) | 19 | 8 | 3.5 (1.5, 6.8) | 8 |
| Vomiting | 12 | 5.3 (2.7, 9) | 19 | 11 | 4.8 (2.4, 8.5) | 12 |
| Loose stools | 14 | 6.1 (3.4, 10.1) | 16 | 5 | 2.2 (0.7, 5) | 5 |
| Irritability | 12 | 5.3 (2.7, 9) | 15 | 12 | 5.3 (2.7, 9) | 13 |
| Refusal to feed | 8 | 3.5 (1.5, 6.8) | 9 | 10 | 4.4 (2.1, 7.9) | 11 |
| Pain/tenderness in the injection site | 5 | 2.2 (0.7, 5) | 6 | 7 | 3.1 (1.2, 6.2) | 7 |
| Swelling in the injection site | 4 | 1.8 (0.5, 4.4) | 4 | 4 | 1.8 (0.5, 4.4) | 4 |
| Hardness in the injection site | 4 | 1.8 (0.5, 4.4) | 4 | 4 | 1.8 (0.5, 4.4) | 4 |
| Excessive sleepiness | 2 | 0.9 (0.1, 3.1) | 2 | 3 | 1.3 (0.3, 3.8) | 3 |
| Redness in the injection site | 2 | 0.9 (0.1, 3.1) | 2 | 3 | 1.3 (0.3, 3.8) | 3 |
| Activity level | 1 | 0.4 (0, 2.4) | 1 | 3 | 1.3 (0.3, 3.8) | 3 |
| Others (not listed) | 1 | 0.4 (0, 2.4) | 2 | – | – | – |
| Unsolicited events | 86 | 37.7 (31.4, 44.4) | 229 | 81 | 35.5 (29.3, 42.1) | 195 |
| Serious adverse events | 8 | 3.5 (1.5, 6.8) | 8 | 5 | 2.2 (0.7, 5) | 6 |
Solicited AEs were recorded for 7 days after each dose and unsolicited AEs were collected throughout the follow-up. Incidence represents the cumulative proportion of participants who experienced at least one event following either Dose 1 and Dose 2.
Table 5.
Distribution of solicited and unsolicited adverse events (AEs) following JE vaccination in JEEV primed schedules.
| Adverse events | JEEV–JENVAC (n = 227) |
JEEV–JEEV (n = 228) |
||||
|---|---|---|---|---|---|---|
| No. of participants | Incidence (95% CI) | Total AEs | No. of participants | Incidence (95% CI) | Total AEs | |
| Solicited eventsa | 86 | 37.9 (31.6, 44.5) | 165 | 85 | 37.3 (31, 43.9) | 190 |
| Fever | 62 | 27.3 (21.6, 33.6) | 63 | 61 | 26.8 (21.1, 33) | 68 |
| Runny nose | 20 | 8.8 (5.5, 13.3) | 24 | 15 | 6.6 (3.7, 10.6) | 16 |
| Cough | 18 | 7.9 (4.8, 12.2) | 18 | 8 | 3.5 (1.5, 6.8) | 8 |
| Vomiting | 6 | 2.6 (1, 5.7) | 6 | 12 | 5.3 (2.7, 9) | 15 |
| Loose stools | 10 | 4.4 (2.1, 8) | 10 | 11 | 4.8 (2.4, 8.5) | 12 |
| Irritability | 8 | 3.5 (1.5, 6.8) | 8 | 11 | 4.8 (2.4, 8.5) | 16 |
| Refusal to feed | 5 | 2.2 (0.7, 5.1) | 5 | 9 | 3.9 (1.8, 7.4) | 14 |
| Pain/tenderness in the injection site | 6 | 2.6 (1, 5.7) | 6 | 11 | 4.8 (2.4, 8.5) | 12 |
| Swelling in the injection site | 4 | 1.8 (0.5, 4.5) | 4 | 8 | 3.5 (1.5, 6.8) | 8 |
| Hardness in the injection site | 7 | 3.1 (1.2, 6.3) | 7 | 5 | 2.2 (0.7, 5) | 6 |
| Excessive sleepiness | 3 | 1.3 (0.3, 3.8) | 3 | 4 | 1.8 (0.5, 4.4) | 6 |
| Redness in the injection site | 5 | 2.2 (0.7, 5.1) | 5 | 3 | 1.3 (0.3, 3.8) | 3 |
| Activity level | 5 | 2.2 (0.7, 5.1) | 5 | 4 | 1.8 (0.5, 4.4) | 4 |
| Others (not listed) | 1 | 0.4 (0, 2.4) | 1 | 2 | 0.9 (0.1, 3.1) | 2 |
| Unsolicited events | 82 | 36.1 (29.9, 42.7) | 179 | 80 | 35.1 (28.9, 41.7) | 190 |
| Serious adverse events | 4 | 1.8 (0.5, 4.5) | 5 | 3 | 1.3 (0.3, 3.8) | 4 |
Solicited AEs were recorded for 7 days after each dose and unsolicited AEs were collected throughout the follow-up. Incidence represents the cumulative proportion of participants who experienced at least one event following either Dose 1 and Dose 2.
Discussion
In this multicentre, randomized, non-inferiority trial among Indian children vaccinated according to the national immunization schedule, mixed-product schedules of JENVAC and JEEV were non-inferior to same-product vaccine schedules with respect to seroconversion 28 days after the second dose. These findings were consistent for both JENVAC-primed and JEEV-primed comparisons and in both mITT and PP analyses. The mixed-product vaccine schedules were well tolerated, with no safety signals identified. No SAEs were assessed as related to vaccination. These findings support the interchangeable use of JENVAC and JEEV in routine immunization programme when children cannot complete two-dose JE vaccination schedule with the same-product because of vaccine supply, procurement changes, or population mobility.
Previous studies of inactivated JE vaccines have generally evaluated same-product vaccination schedules using shorter intervals between the doses. For example, in the earlier trial in which JENVAC was administered as a two-dose regimen one-month apart, 95.9% (95% CI: 92.9–98.9) participants seroconverted at 28 days after the first dose and 98.8% (97.2–100.0) seroconverted 28 days after the second dose.22 Similarly, high seroconversion rates (range: 99–100%) have been reported following two doses of inactivated SA 14-14-2–derived vaccines (for e.g., IXIARO/IC51) given one month apart.23,24 In the present trial, seroconversion after completion of the two-dose schedule were slightly lower (89–91%) across both mixed-product and same-product vaccine schedules.
An important methodological difference that might explain the lower seroconversion in our study was the choice of viral strain used for neutralization assays. Earlier studies assessed the immunogenicity using homologous strains of JE vaccine. For example, the JENVAC trial used the Kolar strain, whereas trials with IXIARO/IC51 used SA 14-14-2 strains.22, 23, 24 In contrast, our primary immunogenicity assessment used an Indian wild-type JEV strain isolated during a large outbreak in 2005 in Uttar Pradesh, India.19,20 This strain is heterologous to both JENVAC and JEEV vaccines. This approach is programmatically relevant because it assessed neutralizing antibody responses against a field strain, but it might have resulted in lower measured seroconversion and GMTs than assays using vaccine-homologous strains. In the subset tested against Kolar-821564XY and SA 14-14-2, neutralizing antibody responses were generally higher against vaccine homologous strains, particularly after the first dose and before the second dose. After the second dose, seroconversion and seroprotection were high across all vaccination schedules, irrespective of a type of the strain used for PRNT50.
Among the participants seronegative at baseline, the proportions who seroconverted after the two-dose schedule ranged from 92% to 96% across four vaccine schedules in both mITT or PP analyses. However, among participants who were seropositive at baseline, the proportion who met the seroconversion criteria was lower, ranging from 35% to 60%. This was primarily because many participants who were seropositive at baseline did not achieve a four-fold or greater increase in PRNT50 antibody titers against JEV after vaccination. Nevertheless, the majority (97.1%) of them were seroprotected.
Both JENVAC and JEEV were originally licensed as two-dose vaccines administered one month apart. However, India's UIP uses a two-dose schedule with a longer interval between doses, with the first dose given at 9–12 months of age and the second at 16–24 months. Consequently, the interval between doses in programmatic settings could range from 4 to 15 months. In our study, the longer interval two-dose schedule resulted in 90% seroconversion 28 days after the second dose, supporting its immunogenicity in the programmatic setting. This vaccination schedule aligns with other routine childhood vaccinations, including the first dose of MRCV, third dose of PCV and third dose of fIPV at 9–12 months, and the second dose of MRCV and first booster of the DPT vaccine at 16–24 months. This alignment simplifies vaccine delivery by health-care workers.
In our study, only about one-third of children (34–41%) were seroconverted before the receipt of the second dose. This finding suggests a possible period of lower antibody-mediated protection between the first and second doses and suggest a potential vulnerability to JE infection/disease. Although, our trial was not designed to assess JE disease incidence during this interval, analysis of surveillance data from India's Virus Research and Diagnostic Laboratory Network (VRDLN) showed that children aged 12–24 months accounted for 3.4%–6.5% of all JE cases diagnosed through the network each year during 2014–2025 (Appendix p 22) with no apparent increasing trend. However, age-specific JE incidence data are needed to determine whether the longer interval between doses increases the risk of JE in this age group. Nevertheless, these findings underscore the importance of timely administration of both doses to maximize protection during early childhood.
The public-health relevance of our study findings extends beyond immunogenicity. In large immunization programmes, children may not always receive the same vaccine product for both doses because of migration, stock availability, procurement cycles, or changes in vaccine supply. In such situations, lack of evidence on immunogenicity of mixed-product schedules could lead to missed opportunities for vaccination and unnecessary deferral of the second dose. Evidence that JENVAC and JEEV can be used interchangeably without compromising short-term immunogenicity or safety provides practical reassurance for immunization programmes. This flexibility could be particularly important in endemic regions, where maintaining high two-dose coverage is essential for JE control.
Our study has certain limitations. First, immunogenicity of JE vaccines was assessed using neutralizing antibody titers, which are an accepted correlate of protection against JE. However, we did not assess cell-mediated immune responses, which might contribute to protection, particularly in individuals with lower antibody titers. Second, the trial was open-label. However, the laboratory staff performing PRNT50 assays were masked to group allocation, reducing the potential for bias in the assessment of primary immunogenicity outcomes. The open label nature of the trial, however, could influence reporting of adverse events. Third, the number of participants evaluable for immunogenicity in the PP analysis was slightly lower than the planned sample size. Fourth, the immunogenicity was assessed only one month after the second dose, limiting the assessment of long term durability of immune responses after completion of two dose schedule.
In conclusion, mixed-product schedules of JENVAC and JEEV elicited non-inferior short-term neutralizing antibody responses compared to same-product schedules and showed no safety signals during the follow-up period. The findings support interchangeable use of these two inactivated Vero cell-derived JE vaccines in routine programme settings. The lower proportion of children remaining seroconverted before the second dose highlights the importance of timely completion of the two-dose schedule and the need for further studies on durability of immune responses after vaccination.
Contributors
MVM, SKV, JK, JWT, MAJ, SK, RK and BD conceptualized and designed the study; JWT, JK, KK, HR, AS and VK did protocol training and monitoring of trial; MAJ, BD, SK, RK, SP, CM, SPP, AM, MR, ARL, SS, JS, SKP did the data collection, KK, JWT, HR, AS and VK did the data management; GNS, GRD, VPB, RS and AS did sample testing and laboratory analysis; SKV, JWT, JK and MVM did the data analysis; JWT, HR, AS, VK, LSG and NG did the project management; SKV, JWT, JK and MVM did the data interpretation. MVM, JWT, SKV, JK and HR wrote the first draft of the manuscript. MVM and JWT had accessed and verified the underlying data. All authors had full access to the datasets; they reviewed and approved the final draft of the manuscript.
Data sharing statement
All key anonymized individual participant data pertaining to the results reported in the article, along with a data dictionary and study proposal, are available upon request to the corresponding author after approval of a proposal and a signed data access agreement.
Declaration of interests
Vaccines for the trial were provided by Bharat Biotech International Ltd, Hyderabad and Biological E. Ltd, Hyderabad free of cost. The manufacturers had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. All other authors declare no competing interests.
Acknowledgements
We sincerely thank Late Mrs Anita D, for the support provided for the study. We acknowledge the support of study site project staff, local health staff at the vaccination centres, ASHAS and community health volunteers in implementing the study. During the preparation of this work the authors used ChatGPT to assist with grammar, spelling, and language refinement. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.
Footnotes
Supplementary data related to this article can be found at https://doi.org/10.1016/j.eclinm.2026.104235.
Appendix A. Supplementary data
References
- 1.Dwivedi G.R., Mishra A., Singh A., Singh H., Kant R., Mishra N. Gorakhpur, India experience of Japanese encephalitis management: a successful one health approach. J Vector Borne Dis. 2025;62:287–294. doi: 10.4103/jvbd.jvbd_171_24. [DOI] [PubMed] [Google Scholar]
- 2.Campbell G.L., Hills S.L., Fischer M., et al. Estimated global incidence of Japanese encephalitis: a systematic review. Bull World Health Organ. 2011;89:766–774E. doi: 10.2471/BLT.10.085233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Yun S.-I., Lee Y.-M. Japanese encephalitis: the virus and vaccines. Hum Vaccin Immunother. 2014;10:263–279. doi: 10.4161/hv.26902. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Quan T.M., Thao T.T.N., Duy N.M., Nhat T.M., Clapham H. Estimates of the global burden of Japanese encephalitis and the impact of vaccination from 2000-2015. eLife. 2020;9 doi: 10.7554/eLife.51027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Fischer M., Hills S.L., Staples E. Emerging infections. Wiley; 2008. Japanese encephalitis prevention and control: advances, challenges, and new initiatives; pp. 93–124. [Google Scholar]
- 6.Halstead SB, Jacobson J, Dubischar-Kastner K, et al. In: Vaccines. 6th ed. Plotkin SA, Ornestein WA, Offit PA, editors. Elseiver/Saunders; 2013. Japanese encephalitis vaccines; pp. 312–351. [Google Scholar]
- 7.Srivastava K.S., Jeswani V., Pal N., et al. Japanese encephalitis virus: an update on the potential antivirals and vaccines. Vaccines. 2023;11:742. doi: 10.3390/vaccines11040742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.WHO Immunization Data portal Vaccination schedule for Japanese Encephalitis. https://immunizationdata.who.int/global/wiise-detail-page/vaccination-schedule-for-japanese-encephalitis?ISO_3_CODE=&TARGETPOP_GENERAL=
- 9.JE Cases and Deaths in the Country Since 2018 National Center for Vector Borne Diseases Control (NCVBDC). State wise number of JE cases and deaths from 2018-2026. https://ncvbdc.mohfw.gov.in/index1.php?lang=1&level=2&lid=4001&sublinkid=5917
- 10.Hegde N.R., Gore M.M. Japanese encephalitis vaccines: immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease. Hum Vaccin Immunother. 2017;13:1–18. doi: 10.1080/21645515.2017.1285472. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Balasubramanian S, Chatterjee P, Shah AK, et al. IAP Guidebook on Immunization 2018-2019. Jaypee Brothers Medical Publishers; 2019. [Google Scholar]
- 12.World Health Organization . World Health Organization; 2016. Public Assessment Summary report Update– Japanese encephalitis vaccine (Human) (Purified inactivated Vaccine-Adsorbed) JEEV®.https://extranet.who.int/prequal/sites/default/files/vwa_vaccine/pq_266_308_JEEV_1dose_BiolE_updated_Aug16_vpsar.pdf [Google Scholar]
- 13.Bharat Biotech International Limited . Bharat Biotech International Limited; 2023. Summary of Product Characteristics (SmPC)https://www.cdsco.gov.in/opencms/resources/UploadCDSCOWeb/2018/UploadSmPC/114.%20JE%20Vaccine%20PFS%20Bharat%20Biotech.pdf [Google Scholar]
- 14.Ministry of Health and Family Welfare . 2024. Routine Immunization Manual for Medical Officers Routine Immunization Manual for Medical Officers.https://nccmis.mohfw.gov.in/document/RI_Manual_For_Medical_Officers.pdf [Google Scholar]
- 15.Islam N., Xu C., Lau C.L., et al. Persistence of antibodies, boostability, and interchangeability of Japanese encephalitis vaccines: a systematic review and dose-response meta-analysis. Vaccine. 2022;40:3546–3555. doi: 10.1016/j.vaccine.2022.04.079. [DOI] [PubMed] [Google Scholar]
- 16.Bharat Biotech International Limited JENVAC 0.5 mL PFS PI. Bharat Biotech International Limited. https://www.bharatbiotech.com/images/PackageInserts/JENVAC%200.5%20mL%20PFS%20PI.pdf
- 17.Division of AIDS (DAIDS) table for grading the severity of adult and pediatric adverse events, v. 1.0 | DAIDS Regulatory Support Center (RSC) https://rsc-prod.niaid.nih.gov/clinical-research-sites/table-grading-severity-adult-pediatric-adverse-events-version-one
- 18.Causality Assessment of an Adverse Event Following Immunization (AEFI): User Manual for the Revised WHO Classification. 2nd ed. 2019. https://www.who.int/publications/i/item/9789241516990 update: [Google Scholar]
- 19.Parida M., Dash P.K., Tripathi N.K., et al. Japanese encephalitis outbreak, India. Emerg Infect Dis. 2006;12:1427–1430. doi: 10.3201/eid1209.060200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Japanese encephalitis virus isolate 057434, complete genome. 2011. http://www.ncbi.nlm.nih.gov/nuccore/EF623988.1
- 21.Hombach J., Solomon T., Kurane I., Jacobson J., Wood D. Report on a WHO consultation on immunological endpoints for evaluation of new Japanese encephalitis vaccines, WHO, Geneva, 2-3 September, 2004. Vaccine. 2005;23:5205–5211. doi: 10.1016/j.vaccine.2005.07.002. [DOI] [PubMed] [Google Scholar]
- 22.Singh A., Mitra M., Sampath G., et al. A Japanese encephalitis vaccine from India induces durable and cross-protective immunity against temporally and spatially wide-ranging global field strains. J Infect Dis. 2015;212:715–725. doi: 10.1093/infdis/jiv023. [DOI] [PubMed] [Google Scholar]
- 23.Jelinek T., Cromer M.A., Cramer J.P., et al. Safety and immunogenicity of an inactivated Vero cell derived - Japanese encephalitis vaccine (IXIARO®, JESPECT®) in a pediatric population in JE non-endemic countries: an uncontrolled, open-label phase 3 study. Travel Med Infect Dis. 2018;22:18–24. doi: 10.1016/j.tmaid.2018.03.003. [DOI] [PubMed] [Google Scholar]
- 24.Dubischar K.L., Kadlecek V., Sablan B., et al. Immunogenicity of the inactivated Japanese encephalitis virus vaccine IXIARO in children from a Japanese encephalitis virus-endemic region. Pediatr Infect Dis J. 2017;36:898–904. doi: 10.1097/INF.0000000000001615. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.