Designing a vaccination strategy against dengue

In this work we propose a mathematical approach to estimate the dengue force of infection, the average age of dengue first infection, the optimum age to vaccinate children against dengue in a routine fashion and the optimum age interval to introduce the dengue vaccine in a mass vaccination campaign. The model is based on previously published models for vaccination against other childhood infections, which resulted in actual vaccination programmes in Brazil. The model was applied for three areas of distinct levels of endemicity of the city of Recife in Northeastern State of Pernambuco, Brazil. Our results point to an optimal age to introduce the dengue vaccine in the routine immunization programme at two years of age and an age interval to introduce a mass vaccination between three and 14 years of age.

The burden of dengue: Jundiaí, Brazil - January 2010

OBJECTIVE: To study the antibody prevalence against dengue in the municipality of Jundiaí, São Paulo, Brazil, due to the low number of official confirmed autochthonous cases. METHODS: A serological study on dengue infection was conducted during January 2010 and previous reports on dengue and entomological surveillance during that period were reviewed. RESULTS: A prevalence of 7.8% IgG positive (68:876) was found. Furthermore, based on the detection of IgM antibodies in five samples, it was observed that the incidence of dengue in the city at the time of the survey contrasts with the absence of notifications by local health authorities over the same period of time. CONCLUSION: These results highlight the discrepancies between the actual and the detected number of dengue infections, possibly due to significant numbers of asymptomatic infections aggravated by difficulties with dengue clinical diagnosis.

Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background: Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods: Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of Sa˜o Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results: Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions: The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

Dengue virus type 3 adaptive changes during epidemics in São Jose de Rio Preto, Brazil, 2006–2007

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

Worldwide spread of Dengue virus type 1

BACKGROUND: DENV-1 is one of the four viral serotypes that causes Dengue, the most common mosquito-borne viral disease of humans. The prevalence of these viruses has grown in recent decades and is now present in more than 100 countries. Limited studies document the spread of DENV-1 over the world despite its importance for human health. METHODOLOGY/PRINCIPAL FINDINGS: We used representative DENV-1 envelope gene sequences to unravel the dynamics of viral diffusion under a Bayesian phylogeographic approach. Data included strains from 45 distinct geographic locations isolated from 1944 to 2009. The estimated mean rate of nucleotide substitution was 6.56 × 10⁻⁴ substitutions/site/year. The larger genotypes (I, IV and V) had a distinctive phylogenetic structure and since 1990 they experienced effective population size oscillations. Thailand and Indonesia represented the main sources of strains for neighboring countries. Besides, Asia broadcast lineages into the Americas and the Pacific region that diverged in isolation. Also, a transmission network analysis revealed the pivotal role of Indochina in the global diffusion of DENV-1 and of the Caribbean in the diffusion over the Americas. CONCLUSIONS/SIGNIFICANCE: The study summarizes the spatiotemporal DENV-1 worldwide spread that may help disease control.

Risk factors associated with death in Brazilian children with severe dengue: a case-control study

Objective: The purpose of this case-control study was to evaluate risk factors associated with death in children with severe dengue. Methods: The clinical condition of hospitalized patients with severe dengue who died (cases, n = 18) was compared with that of hospitalized patients with severe dengue who survived (controls, n = 77). The inclusion criteria for this study were age under 13 years; hospital admission in São Luis, northeastern Brazil; and laboratory-confirmed diagnosis of dengue. Results: Severe bleeding (hemoptysis), a defining criterion for dengue severity, was the factor most strongly associated with death in our study. We also found that epistaxis and persistent vomiting, both included as warning signs in the World Health Organization (WHO) classification of dengue, were strongly associated with death. No significant association was observed between any of the laboratory findings and death. Conclusions: The finding that epistaxis and persistent vomiting were also associated with death in children with severe dengue was unexpected and deserves to be explored in future studies. Because intensive care units are often limited in resource-poor settings, any information that can help to distinguish patients with severe dengue with a higher risk to progress to death may be crucial.

Genomic mosaicism in two strains of Dengue virus type 3

Recombination is a significant factor driving genomic evolution, but it is not well understood in Dengue virus. We used phylogenetic methods to search for recombination in 636 Dengue virus type 3 (DENV-3) genomes and unveiled complex recombination patterns in two strains, which appear to be the outcome of recombination between genotype II and genotype I parental DENV-3 lineages. Our findings of genomic mosaic structures suggest that strand switching during RNA synthesis may be involved in the generation of genetic diversity in dengue viruses.

A comparative analysis of the relative efficacy of vector-control strategies against dengue fever

Dengue is considered one of the most important vector-borne infection, affecting almost half of the world population with 50 to 100 million cases every year. In this paper, we present one of the simplest models that can encapsulate all the important variables related to vector control of dengue fever. The model considers the human population, the adult mosquito population and the population of immature stages, which includes eggs, larvae and pupae. The model also considers the vertical transmission of dengue in the mosquitoes and the seasonal variation in the mosquito population. From this basic model describing the dynamics of dengue infection, we deduce thresholds for avoiding the introduction of the disease and for the elimination of the disease. In particular, we deduce a Basic Reproduction Number for dengue that includes parameters related to the immature stages of the mosquito. By neglecting seasonal variation, we calculate the equilibrium values of the model’s variables. We also present a sensitivity analysis of the impact of four vector-control strategies on the Basic Reproduction Number, on the Force of Infection and on the human prevalence of dengue. Each of the strategies was studied separately from the others. The analysis presented allows us to conclude that of the available vector control strategies, adulticide application is the most effective, followed by the reduction of the exposure to mosquito bites, locating and destroying breeding places and, finally, larvicides. Current vector-control methods are concentrated on mechanical destruction of mosquitoes’ breeding places. Our results suggest that reducing the contact between vector and hosts (biting rates) is as efficient as the logistically difficult but very efficient adult mosquito’s control.

The dengue virus non-structural 1 protein: risks and benefits

The dengue virus (DENV) non-structural 1 (NS1) protein plays a critical role in viral RNA replication and has a central position in DENV pathogenesis. DENV NS1 is a glycoprotein expressed in infected mammalian cells as soluble monomers that dimerize in the lumen of the endoplasmic reticulum; NS1 is subsequently transported to the cell surface, where it remains membrane associated or is secreted into the extracellular milieu as a hexameric complex. During the last three decades, the DENV NS1 protein has also been intensively investigated as a potential target for vaccines and antiviral drugs. In addition, NS1 is the major diagnostic marker for dengue infection. This review highlights some important issues regarding the role of NS1 in DENV pathogenesis and its biotechnological applications, both as a target for the development of safe and effective vaccines and antiviral drugs and as a tool for the generation of accurate diagnostic methods

Synthese, Charakterisierung und Testung von Phenylacrylsäure-Derivaten, Benzothiazolen und Diarylthioethern als potentielle Inhibitoren der Dengue-Virus-Typ-2-NS2B-NS3-Protease

Dengue-Fieber ist eine durch Stechmücken der Gattungen Aedes aegypti und Aedes albopticus übertragene, virale Infektionskrankheit des Menschen, welche eine zunehmende Bedrohung für die Weltbevölkerung darstellt; das Infektionsrisiko betrifft vorwiegend Menschen, die in tropischen und subtropischen Gebieten der Erde (Asien, Afrika, Amerika) leben. Bei dem Erreger handelt es sich um ein Flavivirus, bestehend aus einer positiv polarisierten Einzelstrang-RNA, welches in vier verschiedenen Serotypen existiert. Eine Infektion mit Dengue-Viren zeigt sich durch drei mögliche Krankheitsbilder: Klassisches Dengue-Fieber (DF), hämorrhagisches Dengue-Fieber (DHF) oder Dengue-Schock-Syndrom (DSS). Das Dengue-Virus-Genom codiert eine Serin-Protease mit einer klassischen katalytischen Triade, bestehend aus den Aminosäuren His51, Asp75 und Ser135. Die Funktion der Dengue-Virus-Protease besteht in der post-translationalen, proteolytischen Prozessierung des viralen Polyprotein-Vorläufers, womit sie essentiell für die Virus-Replikation ist und damit einen wichtigen therapeutischen Ansatz für die Entwicklung neuer Wirkstoffe gegen Dengue-Fieber darstellt. Die Ziele der vorliegenden Arbeit bestanden darin, neue potentielle Inhibitoren der Dengue-Virus Typ 2 NS2B-NS3 Protease (DEN-2 NS2B-NS3pro) zu synthetisieren, deren Hemmwirkung sowie den Inhibitionstyp mithilfe fluorimetrischer Enzym-Assays zu bestimmen, Struktur-Wirkungs-Beziehungen (u.a. mithilfe von Molecular Docking-Rechnungen) zu analysieren und die erhaltenen Leitstrukturen zu optimieren. In der vorliegenden Arbeit wurden zwei Substanzklassen und damit zwei Teilprojekte behandelt: Phenylacrylsäureamide im ersten Teilprojekt, Benzothiazole und Diarylthioether zusammen im zweiten Teilprojekt. Im ersten Teilprojekt zeigten einige Phenylacrylsäureamide eine schwache Hemmung der DEN-2 NS2B-NS3pro zwischen ca. 50 und 61 % bei einer Inhibitorkonzentration von 50 µM sowie eine nicht-kompetitive Hemmung, welche jedoch durch vielfältige Derivatisierung kaum verändert oder verbessert werden konnte. Darüber hinaus wurden die endogenen Serin-Proteasen alpha-Chymotrypsin und Trypsin durch einige Phenylacrylsäureamide erheblich stärker gehemmt als die DEN-2 NS2B-NS3pro. Das zweite Teilprojekt befasste sich mit der Synthese und Testung von Diarylthioethern mit hydroxy-substituierten Benzothiazol-Bausteinen sowie der Testung einiger methoxy-substituierter Synthese-Vorstufen der Endverbindungen, um die Relevanz und den Einfluss der einzelnen Bausteine auf die Hemmung der DEN-2 NS2B-NS3pro zu untersuchen. Der in der vorliegenden Arbeit synthetisierte, potenteste Inhibitor der DEN-2 NS2B-NS3pro (Hemmung: 90 % [50 µM]; IC50 = 3.6 +/- 0.11 µM) und der DEN-3 NS2B-NS3pro (Hemmung: &amp;gt;99 % [100 µM]; IC50 = 9.1 +/- 1.02 µM), SH65, ein Diarylthioether-Benzothiazol-Derivat, entstand aufgrund der Vorhersage zweier möglicher Bindungsmodi (kompetitiv und nicht-kompetitiv) mithilfe von Molecular Docking-Experimenten an der Röntgen-Kristall-struktur der DEN-3 NS2B-NS3pro (PDB-Code: 3U1I). Nach experimenteller Bestimmung der IC50-Werte bei unterschiedlichen Substratkonzentrationen erwies sich SH65 jedoch als nicht-kompetitiver Inhibitor der DEN-2 NS2B-NS3pro. Trypsin wurde von SH65 vergleichbar stark gehemmt (96% [50 µM]; IC50 = 6.27 +/- 0.68 µM) wie die beiden getesteten Dengue-Virus-Proteasen, nicht jedoch alpha-Chymotrypsin (nur 21% Hemmung bei 50 µM), wodurch diesem Inhibitor zumindest eine relative Selektivität gegenüber Serin-Proteasen zugeschrieben werden kann. SH65 zeigte lediglich Protease-Hemmung in den Enzym-Assays, jedoch keine antivirale Aktivität bei der Testung an Dengue-Virus-infizierten Zellen, was aber wiederum bei der synthetisierten Vorstufe von SH65, welche anstelle der beiden Hydroxy-Gruppen über Methoxy-Gruppen verfügt, der Fall war. Diarylthioether mit mehrfach hydroxy-substituiertem Benzothiazol-Baustein stellen hiermit eine neue, vielversprechende Wirkstoffgruppe zur Hemmung sowohl der Dengue-Virus Typ 2- als auch der Dengue-Virus Typ 3 NS2B-NS3 Protease dar.

Dengue risk factor distribution in Harris County, Texas

As an important emerging arboviral disease in Texas and throughout the world, dengue fever has the potential to make a re-emergence in the Harris County/Houston metropolitan area. Harris County has seen dengue epidemics in the past. The area has a competent vector, Aedes aegypti, capable of transmission of the virus should it be introduced. It is important to examine areas of highest risk for dengue emergence and transmission in Harris County so that surveillance and educational programs can be properly implemented. This study uses mapping software to visually represent risk factor information with areas of known Ae. aegypti populations. This study focused on known demographic risk factors such as race/ethnicity, place of birth, gender as well as socioeconomic status represented by educational attainment and income. This study found that there are several areas, particularly in central Harris County that are at particular risk for dengue transmission. The findings support the hypothesis that in areas of lower socioeconomic status there were increased populations of foreign born populations, Hispanic populations, and identified locations of a competent vector present. These findings suggest that more specific surveillance of Ae. aegypti, testing of the mosquitoes for dengue virus, and active surveillance for human cases should be implemented in these areas. ^

Evidence of secondary dengue infection and potential for association with clinical depression in a subset of a randomly selected cohort in South Texas

Globally, dengue is an emerging disease resulting in an estimated 50 million new cases and 22, 000 deaths each year. Anecdotally, depression has been reported as a possible sequelae of dengue virus infection. To test the association, we performed a cross-sectional analysis in a selected sub-set of participants from the Cameron County Hispanic Cohort (CCHC) in South Texas. All study subjects in the analysis had Center for Epidemiological Studies Depression scale (CES-D) scores and were tested for dengue antibodies using stored plasma. We found that 5.0% of participants tested either positive or equivocal for anti-dengue IgG antibodies using the capture antibody test, which detects acute secondary infections. Logistic regression identified that evidence of acute secondary dengue infection was not associated with depression (Odds Ratio [OR] = 0.97, 95%Confidence Interval [CI] 0.47-1.98); however, both being female (OR = 1.53, 95%CI 1.09-2.15) and obese body mass index (BMI > 30) (OR = 1.84, 95%CI 1.19-2.84) were associated with depression. ^