High ELISA titres were a hall mark of properly folded immunogens. Indeed, titres observed in mice immunised with the reduced alkylated antigens were several log units lower than those elicited by the non-reduced antigens. ELISA titres were poorly related with biological activity such as surface reactivity, differing from some studies on Var2CSA-derived immunogens. Thus, although ELISA titres were poorly informative with regard to antibody functionality, they provided a robust profile characteristic of the immunogen. All recombinant PfEMP1-VarO domains elicited strong immunoblot reactivity with the parasite-derived PfEMP1-VarO protein and, apart from eDBL3 and eDBL5, all constructs elicited surface-reactive antibodies. Strong MFI values were observed by FACS and here as well, there was no major influence of the expression system, contrasting with reported data for PfEMP1-Var2CSA.The titres of iRBC surface reactivity of anti-DBL1 antisera compare well with titres reported for various PfEMP1 domains of rosette-forming parasites. Titres were lower for the downstream domains, yet higher than the surface reactivity reported for various PfEMP1-derived domains. The reason for the failure of eDBL3 and eDBL5 domains to elicit surface-reactive antibodies is unclear. It is possible that these domains are buried within the iRBC surface-displayed molecule. An alternative is they were not expressed with the native fold. The eDBL3and eDBL5CD spectra did not depart from the CD spectra of the other domains indicating absence of major biophysical PF-03814735 differences. Moreover, both were readily recognized by sera from humans living in endemic areas. This indicates that they display epitopes exposed to the immune system during natural parasite infection, but does not preclude that they do not display some surface-exposed epitopes. As we could not assign any specific binding characteristics to eDBL3and eDBL5, we cannot ascertain that the recombinant antigens are functional. More work is PF-00562271 needed to understand surface display of each individual PfEMP1-VarO domain.