Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortality. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturally not susceptible to DENV and only become infected if highly immunocompromised. -cell response to viral infection, compared to a weak response in IFNAR؊/؊ mice. Furthermore, mice lacking IFNAR on either CD11c ؉ or LysM ؉ cells were also sufficiently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These data demonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study of DENV immunology and screening of vaccine candidates. IMPORTANCEDengue virus infects 400 million people every year worldwide, causing 100 million clinically apparent infections, which can be fatal if untreated. Despite many years of research, there are no effective vaccine and no antiviral treatment available for dengue. Development of vaccines has been hampered in particular by the lack of a suitable small animal model. Mouse models used to test dengue vaccine are deficient in interferon (IFN) type I signaling and severely immunocompromised and therefore likely not ideal for the testing of vaccines. In this study, we explored alternative models lacking the IFN receptor only on certain cell types. We show that mice lacking the IFN receptor on either CD11c-or LysM-expressing cells (conditional IFNAR mice) are susceptible to dengue virus infection. Importantly, we demonstrate that conditional IFN receptor knockout mice generate a better immune response to live virus and a candidate dengue vaccine compared to IFNAR mice and are resistant to subsequent challenge. D engue virus (DENV, a member of the Flaviviridae family, is a mosquito-borne pathogen that infects approximately 400 million people every year (1, 2). Each of the four DENV serotypes causes a spectrum of clinical symptoms ranging from mild fever to potentially fatal manifestations of dengue shock syndrome. DENV causes an acute infection with high fever, which usually resolves after 5 to 7 days. At this time, most patients have cleared the high virus load. Intriguingly, however, this is also the time point when some patients start to develop vascular leakage, which, if untreated, can lead to a collapse of the metabolism and organ failure. The frequency, severity, and geographical spread of cases has increased over the past decades (3, 4), and DENV infection is now considered a leading cause of morbidity in the tropics.There are no effective treatments for dengue fever, and the development of a vaccine has been hampered by the lack of suitable small animal models. Wild-type (wt) mice are not susceptible to infection with field strains of DENV, and while viral replication in these animals can be forced by intracranial injections of hightiter mouse-adapted DENV strains, the resulting clinical disease bears little resemblance to dengue fev...
The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.
Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum. Dengue virus (DENV) is a mosquito-borne flavivirus and the causative agent of dengue and severe dengue. It is estimated that nearly half of the world's population is at risk of infection, with up to 50 million people infected each year and frequent epidemic activity in Southeast Asia, South America and Western Pacific regions. 1,2 Infection with one serotype confers immunity to infection with the same dengue serotype, but does not prevent infection with the others. Viral and host factors have been proposed to have a role in the development of the severe disease, but epidemiological evidence has led to the realization that the majority of severe cases occur in individuals who suffer secondary or sequential DENV infections. 3,4 This implies that immunity to a heterotypic virus is not only ineffective in preventing secondary infection, but may also enhance the disease.Primary prevention through dengue vaccines is considered a research priority in the dengue agenda. Although a vaccine is feasible, its development faces several challenges: (a) a dengue vaccine must be able to protect against the four serotypes; (b) long-term protection is needed; (c) no suitable animal model exists for dengue; and (d) although the protective role of neutralizing antibodies is widely held, correlates of protection need to be defined.There are several dengue vaccine candidates at advanced preclinical and clinical stages, although no vaccine is licensed. The most advanced strategies (phase II-III) are based on live attenuated viruses and are led by the Sanofi Pasteur's ChimeriVax-dengue vaccine candidate (Paris, France). Despite the balanced reactogenicity and immunogenicity profile of the tetravalent ChimeriVax-dengue vaccine candidate, three doses are required during 1 year to induce high neutralizing antibody seroconversion...
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.
The role of cellular immune response in dengue virus infection is not yet fully understood. Only few studies in murine models propose that CD8(+) T-cells are associated with protection from infection and disease. At the light of recent reports about the protective role of CD8(+) T-cells in humans and the no correlation between neutralizing antibodies and protection observed in several studies, a vaccine based on cell-mediated immunity constitute an attractive approach. Our group has developed a capsid-based vaccine as nucleocpasid-like particles from dengue-2 virus, which induced a protective CD4(+) and CD8(+) cell-mediated immunity in mice, without the contribution of neutralizing antibodies. Herein we evaluated the immunogenicity and protective efficacy of this molecule in monkeys. Neither IgG antibodies against the whole virus nor neutralizing antibodies were elicited after the antigen inoculation. However, animals developed a cell-mediated immunity, measured by gamma interferon secretion and cytotoxic capacity. Although only one out of three vaccinated animals was fully protected against viral challenge, a viral load reduction was observed in this group compared with the placebo one, suggesting that capsid could be the base on an attractive vaccine against dengue.
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