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Outer membrane protein (Omp) K, OmpR and VAA have been identified with good immunogenicity from Vibrio anguillarum, and their recombinant proteins showed variable relative percent survival (RPS) in previous study. In order to develop effective bivalent vaccine candidates, recombinant (r) VAA + rOmpK (AK), rVAA + rOmpR (AR), rOmpK + rOmpR (KR) among VAA, OmpK and OmpR, or formalin-killed cells (FKC) of V. anguillarum were immunized in flounder, respectively. Results revealed that AK, AR, KR and FKC could induce the proliferation of surface membrane immunoglobulin-positive B lymphocytes or CD3+ T lymphocytes in peripheral blood lymphocytes, and significantly enhance the total antibodies, specific antibodies and immune-related gene than those of control group. AK, AR, KR or FKC showed RPS of 74.92%, 78.49%, 82.09% and 56.99%, respectively. These results indicated that three bivalent vaccines AK, AR and KR could induce strong cellular and humoral immunity, and had high protection against V. anguillarum infection in flounders.AK, AR, KR and FKC groups increased the percentages of CD3+ T and sIg+ B lymphocytes in PBLs.AK, AR, KR and FKC groups induced production of total and specific antibodies.AK, AR, KR and FKC groups induced up-regulation of the CD3, IgM, CD4-1, CD4-2, CD8α and CD8β genes.The RPS of AK, AR, KR and FKC groups were 74.92%, 78.49%, 82.09%, or 56.99%.