Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus

Abstract

Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.

Tetravalent dengue DIIIC protein together with alum and ODN elicits a Th1 response and neutralizing antibodies in mice

“…1B). A higher amount of DENV-4 was chosen based on previous results showing that the antibody titers to DENV-4 were lower compared to the other serotypes [30]. As a control, mice were infected with live DENV-1, -2, -3 or -4 at day 0.…”

“…The formulation of the Tetra DIIIC vaccine candidate tested in this study is essential for its immunogenicity [14,30] and likely shapes the immune response generated against it. The adjuvant Fig.…”

“…The design, cloning, expression and purification of the recombinant proteins DIIIC-1, DIIIC-2, DIIIC-3 and DIIIC-4, were described previously [14,30]. Briefly, the sequence coding for the domain III fragment (amino acids 286-426) or capsid protein (C) were amplified from the viral genome of the following dengue strains: DENV-1 Jamaica (AF425621); DENV-2 Jamaica (M20558.1), DENV-3 Nicaragua (FJ882576), and DENV-4 Dominica 814669 (AF326573).…”

Dengue vaccine: an update on recombinant subunit strategies

“…Tetravalent candidate evaluated in mice; monovalent DV1 and DV2 candidates evaluated in NHPs Hermida et al, 2006;Lazo et al, 2014;Valdés et al, 2009a] EDIII-capsid fusion protein (DIIIC) expressed in E. coli Monovalent DV2 candidate evaluated in NHPs [Gil et al, 2015;Suzarte et al, 2014;Valdés et al, 2009b;Zuest et al, 2015] Tetravalent EDIII-STF2 fusion proteins expressed in E. coli Tetravalent candidate evaluated in mice and NHPs [Liu et al, 2015] EDIII-HBcAg fusion protein expressed in P. pastoris Monovalent DV2 candidate evaluated in mice Recombinant EDII-EDIII-NS1 fusion protein expressed in Drosophila S2 Tetravalent lipidated consensus EDIII (LcED III) expressed in E. coli Tetravalent candidate evaluated in mice [Chen et al, 2013;Chiang et al, 2011;Leng et al, 2009] Lipidated EDIII (LED III) expressed in E. coli Monovalent candidates (DV1,2,4) evaluated in mice [Chiang et al, 2011[Chiang et al, , 2013a MixBiEDIII: bivalent EDIIIs (DV1-2/ DV3-4) expressed in E. coli Tetravalent candidate evaluated in mice [Zhao et al, 2014] Bivalent EDIII (DV1-2) expressed in E. coli Bivalent candidate (DV1-2) evaluated in mice [Zhang et al, 2015] Tetravalent chimeric EDIII expressed in P. pastoris Tetravalent candidate evaluated in mice [Etemad et al, 2008] Non-structural proteins Recombinant DV2 NS1 protein expressed in E. coli Monovalent DV2 candidate evaluated in mice [Amorim et al, 2012] Recombinant DV2 NS1-DEC205 fusion protein expressed in E. coli Monovalent DV2 candidate evaluated in mice [Henriques et al, 2013] Full-length recombinant DV2 NS3 protein expressed in E. coli Monovalent DV2 candidate evaluated in mice [Ramírez et al, 2014] Capsid protein Recombinant capsid protein expressed in E. coli Monovalent DV2 candidate evaluated in mice and NHPs …”

“…Th e sequences coding for the envelope domain III fragments of the four DV serotypes (aa 286-426) were fused to the N-terminus of the homologous capsid gene (Valdés et al, 2009b;Suzarte et al, 2014). Soluble recombinant EDIIIC proteins were expressed in E. coli.…”