Study On Multivalent Dengue Virus-like Particles Vaccine

Dengue viruses (DENV), existing as four antigenically distinct serotypes, belong to the family of Flaviviridae, genus Flavivirus. DENV are transmitted among humans by mosquitos, such as Aedes aegypti and Aedes albopictus and may cause a self-limited febrile illness known as dengue fever (DF), or result in a life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Dengue viruses are the most important arboviral pathogens in tropical and subtropical regions throughout the world. It has been estimated that 2.5 to 3 billion people are at risk of dengue infection and about 25000 deaths worldwide annually. Because of the widespread geographical distribution and the severe clinical symptoms, dengue vaccine is urgently needed. However, licensed vaccine is not currently available for prevention of DENV infection. One major reason is the phenomenon of antibody dependent-enhancement (ADE). DENV infection lacks cross-protection among serotypes. Furthermore, subsequent infection with an alternate serotype may enhance disease severity, which contributes to the onset of DHF or DSS.Virus-like particle (VLP) vaccine has shown considerable promise as vaccine candidate for some viral diseases. Similar to infectious virions in both structural and biochemical properties but are non-replicating and free of genome, VLPs preserve immunogenicity in native forms and are of better safety. Recombinant VLPs can be efficiently taken up, internalized and processed by antigen presenting cells (APCs), and capable to elicit strong humoral and cellular immune responses against viruses. Thus it opened a new avenue for dengue vaccine research. Dengue virion contains a positive-sense single-strand RNA genome of approximately 11 kb, with a long open reading frame coding for capsid (C), premembrane(prM), and envelope(E) structural proteins, as well as seven non-structural(NS) proteins:NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. Among the three structural proteins, envelope glycoprotein (E) is the major protective antigen of dengue virus which contains several B and T cells epitopes and involves in inducing neutralizing antibodies. prM, the precursor of membrane protein M, could induce neutralizing antibody and contribute to the correct folding and stability of E protein. A common feature in the replication of flaviviruses is to assemble prM and E proteins into subviral particles. Recombinant VLPs of flaviviruses have been shown to be produced efficiently by co-expressing the prM and E proteins in the absence of C protein. Thus, the prM and E proteins become the main targets for dengue vaccine research. In this study, prM-E was used as target gene and mammalian or insect cells were used as expression cells for the construction of recombinant dengue VLPs of four serotypes. The immune efficiency of monovalent and tetravalent dengue VLPs was evaluated.The entire prM-E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, strain SA14-14-2) was introduced using overlapping PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3'terminus and one chimeric gene, which was generated by replacing the 3'terminal 20% region of E gene with the corresponding sequence of JEV (strain SA14-14-2). The PCR segments were inserted into the corresponding sites of pcDNA5/FRT vector of mammalian cells expression system or into pAcUW51-M, pAcGP67-B and pAcSecG2T three vectors of insect cells expression system. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of dengue VLPs were detected by immunofluorescence assay (IFA) and Western blot analysis. After transected into 293T cells or Sf9 cells, all constructs expressed VLPs intracellularly indentified by IFA. In mammalian cells, VLPs of all four serotypes were secreted into cells supernatants while in insect cells only DENV-1 VLPs could be secreted into tissue culture using Western blot analysis. Based on these findings, we finally chose mammalian cells as the expression system of dengue virus-like particles and pJD1prME,pJD2-D4prMEâ–³20%JEV were identified as the recombinant expression plasmids of DENV 1-4 VLPs respectively.For DENV 1-4 VLPs preparation,293T cells in T75 flasks were transiently transfected with optimized plasmid of each serotype mentioned above. A large quantity of culture supernatants containing extracellular VLPs were harvested and purified by a two-step sucrose gradient ultracentrifugation. To compare the size and morphology of recombinant DENV 1-4 VLPs and their corresponding serotype of DENV virions, purified dengue VLPs and virions were observed under transmission electron microscopy (TEM). All four serotypes of recombinant VLPs exhibited as electron-dense spherical particles of 45-55nm size, which were similar to the morphology and size of authentic DENV virions.The immunogenicity of dengue VLPs was evaluated in BALB/c mice. The analysis of humoral immune responses revealed that dengue VLPs, in either monovalent or tetravalent formula, induced high levels of dengue rEIII-specific IgG antibodies. Both monovalent and tetravalent dengue VLPs could efficiently trigger in vivo development of neutralizing antibodies, the tetravalent formula was able to simultaneously induce balanced neutralizing antibodies against all four serotypes. Furthermore, cellular immune responses were assessed by IFN-y releasing ability of VLPs or virus-stimulated spleen cells using ELISPOT assay. Spleen cells from mice vaccinated with monovalent or tetravalent dengue VLPs and virions produced comparable levels of IFN-y.In summary, recombinant VLPs of four antigenically different DENV serotypes DENV 1-4 were produced and intraperitoneally applied to immunize BALB/c mice. The results showed that monovalent or tetravalent dengue VLPs produced by mammalian cells were capable to induce dengue-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection. Meanwhile, dengue VLPs could also be applied to serological diagnosis of dengue fever.