Domain Ⅲ of Dengue Virus Serotype 2 Envelope: Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry

Obejective The domainⅢof dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the inhibited effects of recombinant protein on virus was detected. Methods In this study, the domainⅢ(DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was puriifed using enzymatic cleavage and afifnity puriifcation. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢprotein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a speciifc soluble 29 kD protein was obtained, and the expression product accounted for 68.87%of the total protein of the cell lysate. Western blot demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was puriifed using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢprotein. At a 1︰16 dilution, the antibodies produced at least 90%neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢantibody titer reached 1︰1 024. Conclusions DENV-2 EDⅢwas cloned and expressed successfully. DENV-2 EDⅢprotein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.

A multiple-target mRNA-LNP vaccine induces protective immunity against experimental multi-serotype DENV in mice

Dengue virus(DENV)is a mosquito-borne virus with a rapid spread to humans,causing mild to potentially fatal illness in hundreds of millions of people each year.Due to the large number of serotypes of the virus,there remains an unmet need to develop protective vaccines for a broad spectrum of the virus.Here,we constructed a modified mRNA vaccine containing envelope domain III(E-DIII)and non-structural protein 1(NS1)coated with lipid nanoparticles.This multi-target vaccine induced a robust antiviral immune response and increased neutralizing antibody titers that blocked all four types of DENV infection in vitro without significant antibodydependent enhancement(ADE).In addition,there was more bias for Th1 than Th2 in the exact E-DIII and NS1-specific T cell responses after a single injection.Importantly,intramuscular immunization limited DENV transmission in vivo and eliminated vascular leakage.Our findings highlight that chimeric allogeneic structural and non-structural proteins can be effective targets for DENV vaccine and that they can prevent the further development of congenital DENV syndrome.