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Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

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Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope ( Dengue virus (DENV) is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens, especially in tropical and subtropical countries (1, 2). It is transmitted to humans through the bite of Aedes mosquitoes. A recent study estimated that 390 million DENV infections occur annually worldwide, with 500,000 of these cases being severe and with 25,000 cases resulting in death, mostly among children (3). Despite the impact of this disease, neither a licensed vaccine nor a specific antiviral drug is available; vector control is the only control measure available (4, 5). There are four antigenically distinct serotypes of the virus (DENV serotype 1 [DENV-1] to DENV-4), and each can cause a wide spectrum of clinical manifestations, including asymptomatic infection, self-limited flu-like dengue fever (DF), and the severe life-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) (2, 6).

The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Pairedreceiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP-and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP-and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great utility in diagnostic and surveillance activities in which test accuracy is of utmost importance for effective disease intervention. Mosquito-borne flaviviruses in the family Flaviviridae are responsible for a number of globally significant diseases and are serologically divided into several complexes, including the Japanese encephalitis virus (JEV), dengue virus (DENV), and yellow fever virus (YFV) serocomplexes (1). JEV and West Nile virus (WNV) are two of the most important members of the JEV serocomplex that have emerged into new geographic ranges in the past years (2, 3). JEV occurs in East, South, and Southeast Asia, where DENV is also commonly distributed, but it has spread from the Indonesian archipelago to Papua New Guinea and the Torres Strait islands of northern Australia, and to new areas in western India and Pakistan (4). WNV is originally endemic in parts of Africa, Europe, the Middle East, West Asia, India, and Australia; it then unexpectedly emerged in New York City in 1999 and rapidly expanded over North America to Central America and fin...