BACKGROUND: Universal two-dose hepatitis A virus (HAV) vaccination of toddlers effectively controls hepatitis A. High vaccine costs impede, however, implementation in endemic countries. To test single-dose vaccination as a possible alternative, we initiated an observational, longitudinal study in Nicaragua, to assess protective effectiveness and - through challenge vaccination - humoral immune memory response. METHODS: Following a 2003 serosurvey, 130 originally seronegative children received 2005 one dose virosomal HAV vaccine, followed by yearly serological and clinical assessments until 2012. After 7.5 years, a vaccine booster was administered. Concurrent antibody screening of patients presenting with hepatitis symptoms documented persistent HAV circulation in the communities studied. RESULTS: Between serosurvey and vaccination, 25 children contracted hepatitis A subclinically (>8,000 mIU/mL anti-HAV). In the remaining 105 children, immunisation resulted in 17-572 mIU/mL anti-HAV. Under the ≥15% annual infection risk, an estimated 60% of children were exposed to HAV encounters during follow-up. No child presented with hepatitis symptomatology. Serological breakthrough infection (7106 mIU/mL) was documented in one child, representing an estimated 98.3% (95%CI, 87.9-99.8) protective effectiveness. Boosting elicited an average 29.7-fold increase of anti-HAV levels. CONCLUSIONS: In children living in hyperendemic settings, one dose of virosomal HAV vaccine is sufficient to activate immune memory and may provide long-term protection.