Structure-based vaccine design or structural vaccinology aided by computational modeling and atomistic simulation provides a new tool to overcome antigen heterogeneity challenge in vaccine development [18,34–40]. For ETEC vaccine development, heterogeneity of ETEC bacterial virulence factors remains the key challenge. Different ETEC bacteria produce immunologically heterogeneous adhesins and enterotoxins. ETEC bacteria expressing any one or two types of these adhesins (over 23 ETEC adhesins have been identified) and either toxin (heat-labile toxin-LT or heat-stable toxin-STa) can cause diarrhea. Therefore, only vaccines inducing broad immunity against these adhesins and/or toxins are expected effective against ETEC . Conventional vaccine candidates mixing together of a few live or killed strains induce immunity against homologous adhesins . Excessive somatic antigens particularly harmful LPS carried by these cocktail products, however, could link to side effects, lower immune responses, and unsatisfied protection against ETEC diarrhea . Instead of combining different bacteria strains, structure-based technology allows to include representative antigenic elements or epitopes from various ETEC virulence factors into a single MEFA immunogen for precision and broad immunogenicity. In silico structure data, however, are more of prediction at present; validation from empirical data is still considered essential for structural vaccinology . The current study demonstrated that structural vaccinology helped to characterize structure and immunogenicity of the ETEC MEFA immunogen, and showed congruence between computational data and the empirical mouse immunization data at MEFA immunogenicity. That suggests the feasibility of applying structural vaccinology to assist ETEC vaccine development.