OBJECTIVES: Typhoid conjugate vaccines (TCVs) are essential for controlling endemic typhoid fever in low- and middle-income countries. However, evidence suggests that TCV-induced protection wanes 3-5 years post-vaccination, particularly among children vaccinated before two years of age, raising the question of whether a booster dose is needed. This study leverages a unique context in Nepal, where the nationwide rollout of the Vi-CRM197 TCV occurred four years after a large-scale TCV efficacy trial using Vi-TT, allowing the study to assess the immunogenicity of a heterologous prime-boost schedule. METHODS: We conducted a prospective observational study in Nepal, comparing antibody titres in participants who received: (1) a single dose of Vi-CRM197 (single-dose group); (2) a dose of Vi polysaccharide-tetanus toxoid (Vi-TT) vaccine followed by a Vi-CRM197 boost after a median interval of 4.2 years (4-year interval group); and (3) Vi-TT followed by Vi-CRM197 after a median interval of 2.1 years (2-year interval group). Anti-Vi IgG and IgA titres were measured 10-20 months after Vi-CRM197 vaccination. The primary objective was to show that the single-dose group was non-inferior to the 4-year interval group using a non-inferiority margin of 0.67 for the adjusted geometric mean ratio (aGMRs). Pairwise GMRs were obtained by exponentiating differences in mean log10-transformed titres. We also compared anti-Vi antibody responses following a single-dose Vi-CRM197 with those among Vi-TT-primed participants who had received a single dose of Vi-TT in the original trial, with antibody measurement taken one to two years post-vaccination, including analyses stratified by Vi-TT vaccination time to account for differences in post-vaccination follow-up time. RESULTS: The single-dose Vi-CRM197 group exhibited significantly lower anti-Vi IgG and IgA geometric mean concentrations (GMCs) compared with the 4-year interval prime-boost group (reference), with aGMRs of 0.24 (95% CI: 0.18, 0.32) for IgG and 0.40 (0.30, 0.52) for IgA, leading to rejection of non-inferiority. Antibody responses in the 2-year interval prime-boost group were intermediate between those in the single-dose and 4-year interval groups, with aGMRs of 0.65 (95% CI: 0.41, 1.03) for anti-Vi IgG and 0.83 (0.54, 1.26) for anti-Vi IgA, all relative to the 4-year interval group. Compared with the immunogenicity among Vi-TT-primed participants, a single dose of Vi-CRM197 elicited lower anti-Vi IgG levels (aGMR: 0.56 [95% CI 0.47, 0.66]). When Vi-TT-primed participants were stratified by vaccination time (2017-2018 and 2020-2021), a single-dose of Vi-CRM197 elicited lower anti-Vi IgG levels than those observed among participants primed with Vi-TT in both cohorts (aGMR: 0.52 [0.43, 0.61] and 0.68 [0.54, 0.85], respectively). CONCLUSIONS: A single dose of Vi-CRM197 is immunologically inferior to a heterologous prime-boost schedule, with the 2-year interval boost eliciting intermediate antibody responses and the 4-year interval boost inducing the highest immunogenicity. These findings provide critical real-world evidence supporting the implementation of a TCV booster dose with a longer interval, particularly in endemic settings, to maintain robust immunity against typhoid fever.