A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

Abstract

Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4(+) and CD8(+) cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

The Chimeric Protein Domain III-Capsid of Dengue Virus Serotype 2 (DEN-2) Successfully Boosts Neutralizing Antibodies Generated in Monkeys upon Infection with DEN-2

“…rified in this expression system. When it was presented as a particulate aggregate with random ODNs, it induced antiviral and neutralizing antibodies and CMI and conferred a significant level of protection in mice (26). Based on these features, we selected this molecule, in a particulate form with ODNs, to combine it with infective DEN-2 in a heterologous prime-boost regime of immunization in monkeys.…”

“…The recombinant domain III-capsid protein comprises the domain III region of the envelope protein and the capsid protein, both from DEN-2. Previously, this protein in aggregated form was able to induce neutralizing antibodies, cell-mediated immunity (CMI), and protection against DEN-2 challenge in mice (26). In the present study, primed animals received one dose of the infective DEN-2 and were then vaccinated with the recombinant domain III-capsid protein.…”

“…To obtain the particulate form of the protein, the recombinant domain III-capsid molecule was purified as described previously (26). The purified preparation was incubated with a mixture of 50-base-long DNA oligonucleotides (ODNs) at a 3:1 protein/oligonucleotide molecular ratio basically according to the procedure described by Valdés et al in 2009 (26).…”

“…High levels of IFN-␥ were also obtained after in vitro stimulation with the chimeric protein, demonstrating that epitopes of the viral regions present in the protein are able to stimulate the PBMCs of the vaccinated monkeys. The capsid protein as well as the domain III-capsid protein were also able to induce high levels of IFN-␥ in mice after in vitro stimulation of the splenocytes with the infective virus (10,26).…”

Type I Interferon Signals in Macrophages and Dendritic Cells Control Dengue Virus Infection: Implications for a New Mouse Model To Test Dengue Vaccines

“…Intriguingly, mice lacking the type I IFN receptor (IFNAR) on either LysM ϩ or CD11c ϩ cells were susceptible to disease but frequently recovered by 15 days postinfection (p.i. ); furthermore, immunization of either of these mice with a dengue virus EDIII-capsid (EDIIIC-2) subunit vaccine (30,31) induced high titers of dengue-specific neutralizing antibodies. Therefore, we have developed a novel model which has revealed that type I IFN receptor signaling in CD11c-and LysM-expressing cells has important and nonredundant roles in determining susceptibility to dengue.…”