To develop monovalent dengue virus-like particle for serotype 3 (DENV-LP/3), we prepared and expressed two structural polyprotein constructs using silkworm and Bm5 cells: DENV-3 Capsid-premembrane-envelope (DENV-3CprME) and premembrane-envelope (DENV-3prME). The expressed PA-tagged 3CprME and 3prME polypeptides were partially purified by PA-tag affinity chromatography and had molecular weights of 85 and 75 kDa, respectively. Expressed proteins were separately verified using the following primary antibodies: the anti-PA tag antibody, DENV premembrane polyclonal antibody, and DENV envelope polyclonal antibody. Transmission electron microscopy revealed that these DENV-3CprME and 3prME formed rough, spherical DENV-LPs (DENV-LP/3CprME and DENV-LP/3prME), respectively, with a diameter of 30-55 nm. The heparin-binding assay demonstrated that these DENV-LPs contained the envelope protein domain III on their surfaces. Both DENV-LPs showed an affinity to sera from human dengue patients and immunized mice. Immunization of mice with DENV-LP/3prME significantly induced the level of antibodies compared with DENV-LP/3CprME. These results indicate that DENV-LP/3prME is suitable as a vaccine candidate compared with DENV-LP/3CprME.

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SUMMARY:The development of a dengue virus vaccine is a major priority in efforts to control the diseases. Several researchers are currently using the Asian 1 and Asian 2 genotypes as vaccine candidates for dengue type 2 virus (DENV-2). However, in this study, we constructed a recombinant plasmid-based prM/E gene, from a DENV-2 Cosmopolitan genotype strain as a dengue DNA vaccine candidate. The protein expression of the recombinant plasmid in CHO cells was analyzed using an enzyme-linked immunosorbent assay, western blotting, and sucrose gradient sedimentation. After being used to immunize ddY mice three times at doses of 25 or 100 mg, the DNA vaccine induced humoral immune responses. There was no difference in the neutralizing antibody titer (focus reduction neutralization test 50z value) of mice immunized with 25 and 100 mg DNA vaccine doses. When challenged with 3 × 10 5 FFU DENV-2, immunized mice could raise anamnestic neutralizing antibody responses, which were observed at day 4 and day 8 post-challenge. Analysis of immunogenicity using BALB/c mice showed that their antibody neutralization titers were lower than those of ddY mice. In addition, the antibodies produced after immunization and challenge could also neutralize a DENV-2 Asian 2 genotype (New Guinea C) strain. Therefore, the DENV-2 Cosmopolitan genotype may be a DENV-2 vaccine candidate.

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) genebased expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.Key words Antibody-dependent enhancement, chimera, dengue, vaccine.Dengue virus, a member of the genus Flavivirus, is the causative agent of dengue fever and dengue hemorrhagic fever, an estimated 50 million infections occurring annually (1). Although DENV infection is a global public health concern, no approved vaccines or antiviral substances are currently available (2). Several research groups are currently trying to establish an effective DENV vaccine by using various strategies to induce anti-DENV neutralizing antibodies, which mediate the most effective anti-DENV immune response (3-6).The pathogenic mechanism of DENV infection has not been fully elucidated. Difference in disease severity upon secondary exposure to homotypic (protection) and heterotypic (deterioration) infection is the primary characteristic distinguishing dengue fever from dengue hemorrhagic fever (7). Antibody dependent enhancement (ADE) is the proposed mechanism for deterioration induced by secondary DENV infection; this is suggested by the observation that pre-existing neutralizing antibodies that have been induced by primary infection crossprotectively neutralize viruses during some post-infection periods only. In addition, the Fc portion of the antibody molecule may facilitate viral uptake to Fc receptorbearing cells, eventually enhancing viral propagation (8). Intriguingly, ADE activity has been detected in both polyclonal and monoclonal antibody states (9). List of Abbreviations: ADE, antibody-dependent enhancement; DENV, dengue virus; DENV-1, DENV type-1; DI, domain I; DII, domain II; DIII, domain III; E, envelope; JEV, Japanese encephalitis virus; pcD1ME, pcDNA3-based expression plasmid DENV1 Mochizuki; pcJEME, pcDNA3-based expression plasmid for prM and E proteins of JEV Nakayama; prM, premembrane.