Children (age 1-12 y) who were enrolled at the start of the study in 2004 [6], and who participated in regular follow-up and immunogenicity assessments in 2007 and 2010, were re-valuated in 2014 (Fig 1). Institutional ethics committee approval and informed consent were obtained. Participants were compensated for travel and loss of wages (where applicable). After routine clinical assessments, blood samples were collected and sent for estimation of total and IgM anti-HAV antibody analyses (Cobas anti-HAV electro-chemiluminescence immuno-assay, ECLIA, Roche Diagnostics Deutschland GmbH) [8] to an independent accredited laboratory (SRL Diagnostics, Mumbai, India). Seroprotection rate was defined as proportion of subjects with total anti-HAV antibody level ³20 mIU/mL. Geometric mean titers (GMTs) were calculated as per standard method.

Fig.1 Flow chart of 10-year study (2004-2014).

Of the original 143 children who received a single dose of live attenuated HAV vaccine in 2004, 121 subjects reported for 10 year follow-up assessment in 2014. Of these, four who had received two doses of licensed inactivated HAV vaccine (Havrix, GSK Biologicals) in 2004 (vaccine failures) and nine who had received a second dose of live HAV vaccine in 2007 for low titers (antibody levels <20 mIU/mL), were not evaluated in present analysis (Fig. 1). Therefore, 108 children who received a single dose of the live vaccine were analyzed.

The clinical examination of the participants did not reveal any abnormal findings and none had a history of hepatitis-like illness in the past. In the present analysis, 106 of the 108 included children had anti-HAV titres ³20 mIU/mL; seroprotection rate of 98.1% (95% CI 93.5%, 99.8%); 10 years after a single dose of the live attenuated vaccine. Only two participants had anti-HAV titres <20 mIU/mL (11.5 mIU/mL and 13.5 mIU/mL). Anti-HAV IgM was negative in all the children studied. The GMT of anti HAV antibodies of seroprotected children was 100.5 mIU/mL (95% CI 87.4 mIU/mL, 115.4 mIU/mL). On analysis of all 121 children (including 4 ‘vaccine failures’ and 9 with low titres at 30 months post-vaccination), the immunogenicity was 87.6%. The comparison of 10 year immunogenicity data with previous assessments (2004, 2007 and 2010) is presented in Fig. 2.

* GMT of 2004 not presented as anti HAV titres >100mIU/mL were not quantified in 2004.

Fig. 2 (a) Serial Seroprotection Rates (2004-2014); (b) Serial GMTs (2004-2014).

This long term follow-up of a cohort of children vaccinated with a single dose of live attenuated HAV vaccine in 2004 demonstrated a seroprotection rate of 98.1% (anti – HAV GMT of 100.46mIU/mL.), 10 years after vaccination, in those who showed adequate seroprotection in earlier studies. The seroprotection rate would be 87.6%, on inclusion of 13 participants who had to be given additional hepatitis A doses because of poor immunogenicity documented in earlier evaluations [6,7]. None reported hepatitis-like illness.

The strength of this study is that 85% of the cohort enrolled in 2004 reported regularly for follow-up. Differences in the kits used for evaluation of titers is one of the limitation of this study. In the first three follow-ups, immunogenicity was assessed using quantitative Axsym HAVB 2.0 ELISA from Abbott Laboratories (Abbott Park, Illinois, USA). These kits were not available in 2014, and hence different kits had to be used for the present analysis. AxSYM HAVB 2.0 uses microparticle enzyme immunoassay (MEIA) whereas present kits were based on ECLIA technology [8]. Some reports have shown higher sensitivity of ECLIA as compared to ELISA-based immunoassays [9], and this could be a reason for the higher titers of antibodies observed in this analysis. Cross validation of immunogenicity data between the two kits was not possible as sera from previous analyses were not available. Another limitation was the cohort contamination with second dose of the vaccine. Due to ethical reasons, children who remained seronegative after vaccination in 2004 were given 2 doses of inactivated (licensed) HAV vaccine; in 2007 those who remained seronegative received a second dose of now licensed live HAV vaccine, In 2010, however, in view of the encouraging reports of persisting immunological memory despite low titres [10,11], no revaccination was done in seronegative children. The contribution of asymptomatic infections towards immunogenicity also cannot be ruled out as there was no control group in our study.