Evaluation of plasma anti-CS3 and anti-LTB IgG avidity among Zambian children vaccinated with ETVAX

Background: Enterotoxigenic Escherichia coli (ETEC) remains a major cause of diarrheal disease in low- and middle-income countries (LMICs). To curb ETEC related diarrhoea, several candidate vaccines are in development, with ETVAX® being the most advanced. Although immunogenicity studies have primarily focused on measuring antibody titres, assessing antibody avidity offers additional valuable insight into antibody quality and immune maturity. This study assessed anti-CS3 and anti-LTB IgG avidity in Zambian children to better understand vaccine-induced antibody responses in an endemic setting. Methods: Children aged 6–23 months (n=60) received three quarter-doses of ETVAX® with dmLT adjuvant on days 0, 14, and 90. Plasma samples collected at baseline (V1), seven days after the second dose (V5), and seven days after the third dose (V7) were analysed by limiting antigen dilution ELISA to calculate avidity indices (AI). Naïve classification was performed using titre-based thresholds (20th percentile of baseline titres) and avidity-defined naivety (AI < 0.5). Receiver operating characteristic (ROC) analysis was used to evaluate the discriminatory performance of avidity indices against titre-defined naïve status. Results: Baseline avidity was detectable for both CS3 and LTB, consistent with prior natural exposure. Mean CS3 IgG avidity decreased from 0.7 at baseline to 0.6 after the third dose (p<0.001), while LTB IgG avidity showed transient decreases but no net gain. Naïve classification at baseline revealed that 9/60 children had titres but low avidity (functional naivety), and 6/60 had waned titres but high avidity. Only one child was naïve by both criteria for CS3, and none for LTB. ROC analysis demonstrated moderate discrimination for CS3 (AUC=0.65; optimal cut-off AI=0.36) but poor discrimination for LTB (AUC=0.30). Conclusion: In this endemic population, ETVAX® induced strong antibody titres but minimal changes in avidity over time with inter-individual differences, while ROC analysis highlighted the need for context-specific thresholds. These findings show the need for both antibody titre and avidity assessment in vaccine evaluations in endemic settings.