Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

BACKGROUND: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. METHODS: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. RESULTS: The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. CONCLUSIONS: The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine

Correlation of Serotype-Specific Dengue Virus Infection with Clinical Manifestations

Dengue virus (DENV) causes disease in millions of people annually and disproportionately affects those in the developing world. DENVs may be divided into four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) and a geographical region may be affected by one or more DENV serotypes simultaneously. Infection with DENV may cause life-threatening disease such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), but more often causes less severe manifestations affecting a wide range of organs. Although many previous reports have explored the role of the different DENV serotypes in the development of severe manifestations, little attention has focused on the relative role of each DENV serotype in the development of cutaneous, respiratory, gastrointestinal, musculoskeletal, and neurological manifestations. We recruited a large group of participants from four countries in South America to compare the prevalence of more than 30 manifestations among the four different DENV serotypes. We found that certain DENV serotypes were often associated with a higher prevalence of a certain manifestation (e.g., DENV-3 and diarrhea) or manifestation group (e.g., DENV-4 and cutaneous manifestations)

Dengue 1 Diversity and Microevolution, French Polynesia 2001–2006: Connection with Epidemiology and Clinics

The molecular characterization of 181 serotype 1 Dengue fever (DENV-1) viruses collected regularly during the 2001–2006 period in French Polynesia (FP) from patients experiencing various clinical presentations revealed that the virus responsible for the severe 2001 outbreak was introduced from South-East Asia, and evolved under an endemic mode until a new epidemic five years later. The dynamics of DENV-1 epidemics in FP did not follow the model of repeated virus introductions described in other South Pacific islands. They were characterized by a long sustained viral circulation and the absence of new viral introduction over a six-year period. Viral genetic variability was not observed only during outbreaks. In contrast with conventional thinking, a significant part of DENV-1 evolution may occur during endemic periods, and may reflect adaptation to the mosquito vector. However, DENV-1 evolution was globally characterized by strong purifying selection pressures leading to genome conservation, like other DENV serotypes and other arboviruses subject to constraints imposed by the host-vector alternating replication of viruses. Severe cases—dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS)—may be linked to both viral and host factors. For the first time, we report a significant correlation between intra-host viral genetic variability and clinical outcome. Severe cases were characterized by more homogeneous viral populations with lower intra-host genetic variability

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue

Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

BACKGROUND:Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. METHODOLOGY/PRINCIPAL FINDINGS:Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51-65, 71-90, 131-170, 196-210 and 246-260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51-65), 27 and 28 (aa131-150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51-65, 131-170, 196-210 and 246-260 elicited the highest antibody response and epitopes131-170, 196-210 and 246-260, elicited IFN-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. CONCLUSIONS/SIGNIFICANCE:Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine

A Non Mouse-Adapted Dengue Virus Strain as a New Model of Severe Dengue Infection in AG129 Mice

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing

STAT2 Mediates Innate Immunity to Dengue Virus in the Absence of STAT1 via the Type I Interferon Receptor

Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50–100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo

Phylogenetic reconstruction of dengue virus type 2 in Colombia

Background: Dengue fever is perhaps the most important viral re-emergent disease especially in tropical and subtropical countries, affecting about 50 million people around the world yearly. In Colombia, dengue virus was first detected in 1971 and still remains as a major public health issue. Although four viral serotypes have been recurrently identified, dengue virus type 2 (DENV-2) has been involved in the most important outbreaks during the last 20 years, including 2010 when the fatality rate highly increased. As there are no major studies reviewing virus origin and genotype distribution in this country, the present study attempts to reconstruct the phylogenetic history of DENV-2 using a sequence analysis from a 224 bp PCR-amplified product corresponding to the carboxyl terminus of the envelope (E) gene from 48 Colombian isolates. Results: As expected, the oldest isolates belonged to the American genotype (subtype V), but the strains collected since 1990 represent the American/Asian genotype (subtype IIIb) as previously reported in different American countries. Interestingly, the introduction of this genotype coincides with the first report of dengue hemorrhagic fever in Colombia at the end of 1989 and the increase of cases during the next years. Conclusion: After replacement of the American genotype, several lineages of American/Asian subtype have rapidly spread all over the country evolving in new clades. Nevertheless, the direct association of these new variants in the raise of lethality rate observed during the last outbreak has to be demonstrated

Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia

Background: Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70′s when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution. Results: We used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found. Conclusion: DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages

Mosquitoes Put the Brake on Arbovirus Evolution: Experimental Evolution Reveals Slower Mutation Accumulation in Mosquito Than Vertebrate Cells

Like other arthropod-borne viruses (arboviruses), mosquito-borne dengue virus (DENV) is maintained in an alternating cycle of replication in arthropod and vertebrate hosts. The trade-off hypothesis suggests that this alternation constrains DENV evolution because a fitness increase in one host usually diminishes fitness in the other. Moreover, the hypothesis predicts that releasing DENV from host alternation should facilitate adaptation. To test this prediction, DENV was serially passaged in either a single human cell line (Huh-7), a single mosquito cell line (C6/36), or in alternating passages between Huh-7 and C6/36 cells. After 10 passages, consensus mutations were identified and fitness was assayed by evaluating replication kinetics in both cell types as well as in a novel cell type (Vero) that was not utilized in any of the passage series. Viruses allowed to specialize in single host cell types exhibited fitness gains in the cell type in which they were passaged, but fitness losses in the bypassed cell type, and most alternating passages, exhibited fitness gains in both cell types. Interestingly, fitness gains were observed in the alternately passaged, cloned viruses, an observation that may be attributed to the acquisition of both host cell–specific and amphi-cell-specific adaptations or to recovery from the fitness losses due to the genetic bottleneck of biological cloning. Amino acid changes common to both passage series suggested convergent evolution to replication in cell culture via positive selection. However, intriguingly, mutations accumulated more rapidly in viruses passed in Huh-7 cells than in those passed in C6/36 cells or in alternation. These results support the hypothesis that releasing DENV from host alternation facilitates adaptation, but there is limited support for the hypothesis that such alternation necessitates a fitness trade-off. Moreover, these findings suggest that patterns of genetic evolution may differ between viruses replicating in mammalian and mosquito cells