27 Keywords: Dengue virus, DENV-2 vaccine, epitope modification, cross-reactivity reduced, 28 neutralizing antibodies, prime-boost immunization 29 30 Word count: Abstract 191 words; Main text 2,652 words 31 32 33 Abstract 34The four serotypes of dengue virus (DENV) cause the most important rapidly 35 emerging arthropod-borne disease globally. The humoral immune response to DENV 36 infection is predominantly directed against the immunodominant cross-reactive weakly 37 neutralizing epitopes located in the highly conserved fusion peptide of ectodomain II of 38 envelope (E) protein (EDII FP ). Antibodies recognizing EDII FP have been shown to associate 39 with immune enhancement in an ex vivo animal model. In this study, we explored how prime-40 boost strategies influence the immunogenicity of a cross-reactivity reduced (CRR) DENV-2 41 vaccine with substitutions in EDII FP residues (DENV-2 RD) and found that mice in various 42 DENV-2 RD prime-boost immunizations had significantly reduced levels of EDII FP 43antibodies. In addition, heterologous DENV-2 RD DNA-VLP prime-boost immunization 44 induced higher and broader levels of total IgG and neutralizing antibodies (NtAbs) although 45IgG titers to DENV-2 and 3 were statistically significant. Consistently, mice from DENV-2 46 RD DNA-VLP prime-boost immunization were fully protected from homologous DENV-2 47 lethal challenge and partially protected (60% survival rate) from heterologous lethal DENV-3 48 challenge. Our results conclude that the CRR DENV-2 RD vaccine requires a multivalent 49 format to effectively elicit a balanced and protective immunity across all four DENV 50 serotypes. 51 52 53 54 Importance 55The low vaccine efficacy of the live-attenuated chimeric yellow fever virus-DENV 56 tetravalent dengue vaccine (CYD-TDV) is unexpected and there is an urgent need to develop 57 a next generation of dengue vaccine. Antibodies against the fusion peptide in envelope 58 protein (E) ectodomain II (EDII FP ) can potentially induce a severe disease via antibody-59 dependent enhancement (ADE) of infection. This study evaluated different formats of an 60 EDII FP -modified DENV-2 vaccine (DENV-2 RD) in its capability of inducing a reduced 61 EDII FP antibodies, and sculpting the immune response towards an increased DENV complex-62 reactive neutralizing antibodies (CR NtAb). The results from this study confirmed the poor 63 correlate of neutralizing assay with protection as suggested by the results of CYD-TDV 64 clinical trials. There is a urgent need to develop a biological correlate with protection while 65 evaluating the efficacy of the next generation dengue vaccine. 66 67