Responses were monitored using several readout assays: ELISA on recombinant domains, immunoblotting of reduced and non-reduced Palo Alto 89F5VarO protein extracts, the capacity to react with the native, surface-exposed PfEMP1-VarO as assessed by surface immunofluorescence and the capacity to prevent formation or disrupt PaloAl to 89F5VarO rosettes. We studied the influence of protein folding on the production of surface-reacting antibodies and explored inter domain cross-reactivity. These results underline the high immunogenicity of each individual domain and provide a strong basis for a rational vaccination strategy. Vaccination with recombinant ZM 323881 hydrochloride antigens necessitates optimising a large number of parameters, including expression system, amount of protein injected, immunisation schedule, number of doses, adjuvant and delivery route. Only some of these parameters were investigated here, as we wanted to explore inter-individual variation in outbred and inbred mice using five readout assays. The scatter of antibody titres observed after the second dose, was substantially reduced after the third dose, resulting in homogeneous profiles both on the immunising antigen and on the various parasite-dependent assays. Importantly, outbred OF1 mice responded by a consistent production of high ELISA titres and good titres of antibodies reacting with the parasite PfEMP1-VarO protein in the parasite iRBC context. This shows good prospects for an effective vaccination strategy against rosetting, as an important prerequisite is to induce a potent, functionally active immune response targeting the iRBC surface in a large proportion if not all, of vaccinees. Endpoint titres for all domains were higher than those usually reported for Nilutamide PfEMP1-Var2CSA individual domains injected in Freund’s adjuvant, including studies that reported improved immunogenicity and higher ELISA titres. Of note, immunization with different batches of eDBL1, bDBL1 or bDBL2 induced quite similar ELISA titres and surface IFA reactivity, indicating reproducible immunogenicity in outbred animals.