An affordable vaccine against all serogroups of Neisseria meningitidis causing reoccurring epidemics in sub-Saharan Africa is needed. Native outer membrane vesicles (NOMV) with over-expressed factor H binding protein (fHbp) represent a promising vaccine approach. We investigated the contribution of fHbp variant 1 amino acidic sequence to the cross-reactivity of the antibody response generated by NOMV OE fHbp. Coupling structure-sequence analysis of fHbp with epidemiological data of meningococcal prevalence in Africa, we selected four fHbp v.1 IDs (1, 5, 9, 74), to generate isogenic mutants of a serogroup W African isolate, over-expressing each of the them. NOMV from the mutants were purified, characterized, and the antibody response generated in mice was investigated, and compared to the corresponding recombinant fHbp. This is the first study indicating that the amino acid sequence of fHbp influences the specificity of the antibody response generated, not only as recombinant protein, but also when over-expressed on NOMV. In mice NOMV OE fHbp induced a fast, long-lasting antibody response, with high IgM and IgG antibody levels 7 days after immunization, and germinal centers induced. LipidA modifications do not impact primary antibody response, while precipitation of the antigen on alum attenuated the early antibody response, but enhance its longevity.