The role of the unfolded protein response in dengue virus pathogenesis

Symptomatic dengue virus (DENV) infections range from mild fever to severe haemorrhagic disease and death. Host‐viral interactions play a significant role in deciding the fate of the infection. The unfolded protein response (UPR) is a prosurvival cellular reaction induced in response to DENV‐mediated endoplasmic reticulum stress. The UPR has complex interactions with the cellular autophagy machinery, apoptosis, and innate immunity. DENV has evolved to manipulate the UPR to facilitate its replication and to evade host immunity. Our knowledge of this intertwined network of events is continuously developing. A better understanding of the UPR mediated antiviral and proviral effects will shed light on dengue disease pathogenesis and may help development of anti‐DENV therapeutics. This review summarizes the role of the UPR in viral replication, autophagy, and DENV‐induced inflammation to describe how a host response contributes to DENV pathogenesis.     

An emerging role for the anti-inflammatory cytokine interleukin-10 in dengue virus infection

Infection with dengue virus (DENV) causes both mild dengue fever and severe dengue diseases, such as dengue hemorrhagic fever and dengue shock syndrome. The pathogenic mechanisms for DENV are complicated, involving viral cytotoxicity, immunopathogenesis, autoimmunity, and underlying host diseases. Viral load correlates with disease severity, while the antibody-dependent enhancement of infection largely determines the secondary effects of DENV infection. Epidemiological and experimental studies have revealed an association between the plasma levels of interleukin (IL)-10, which is the master anti-inflammatory cytokine, and disease severity in patients with DENV infection. Based on current knowledge of IL-10-mediated immune regulation during infection, researchers speculate an emerging role for IL-10 in clinical disease prognosis and dengue pathogenesis. However, the regulation of dengue pathogenesis has not been fully elucidated. This review article discusses the regulation and implications of IL-10 in DENV infection. For future strategies against DENV infection, manipulating IL-10 may be an effective antiviral treatment in addition to the development of a safe dengue vaccine.     

Dominant cross-reactive B cell response during secondary acute dengue virus infection in humans

The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Severe disease has been associated with heterotypic secondary DENV infection, mediated by cross-reactive antibodies (Abs) and/or cross-reactive T cells. The role of cross-reactive immunity in mediating enhanced disease versus cross-protection against secondary heterotypic DENV infection is not well defined. A better understanding of the cross-reactive immune response in natural infections is critical for development of safe and effective tetravalent vaccines. We studied the B cell phenotype of circulating B cells in the blood of pediatric patients suspected of dengue during the 2010-2011 dengue season in Managua, Nicaragua (n = 216), which was dominated by the DENV-3 serotype. We found a markedly larger percentage of plasmablast/plasma cells (PB/PCs) circulating in DENV-positive patients as compared to patients with Other Febrile Illnesses (OFIs). The percentage of DENV-specific PB/PCs against DENV-3 represented 10% of the circulating antibody-producing cells (ASCs) in secondary DENV-3 infections. Importantly, the cross-reactive DENV-specific B cell response was higher against a heterotypic serotype, with 46% of circulating PB/PCs specific to DENV-2 and 10% specific to DENV-3 during acute infection. We also observed a higher cross-reactive DENV-specific IgG serum avidity directed against DENV-2 as compared to DENV-3 during acute infection. The neutralization capacity of the serum was broadly cross-reactive against the four DENV serotypes both during the acute phase and at 3 months post-onset of symptoms. Overall, the cross-reactive B cell immune response dominates during secondary DENV infections in humans. These results reflect our recent findings in a mouse model of DENV cross-protection. In addition, this study enabled the development of increased technical and research capacity of Nicaraguan scientists and the implementation of several new immunological assays in the field.     

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection‐enhancing antibody response

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection‐enhancing activities at sub‐neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM‐E) gene‐based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM‐E gene backbone showed high neutralizing antibody titers with less DENV infection‐enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.     

Protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive B and T cells

The four dengue virus (DENV) serotypes cause dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Although severe disease has been associated with heterotypic secondary DENV infection, most secondary DENV infections are asymptomatic or result in classic DF. The role of cross-reactive immunity in mediating cross-protection against secondary heterotypic DENV infection is not well understood. DENV infection of IFN-α/β and IFN-γ receptor-deficient (AG129) mice reproduces key features of human disease. We previously demonstrated a role in cross-protection for pre-existing cross-reactive Abs, maintained by long-lived plasma cells. In this study, we use a sequential infection model, infecting AG129 mice with DENV-1, followed by DENV-2 6-8 wk later. We find that increased DENV-specific avidity during acute secondary heterotypic infection is mediated by cross-reactive memory B cells, as evidenced by increased numbers of DENV-1-specific cells by ELISPOT and higher avidity against DENV-1 of supernatants from polyclonally stimulated splenocytes isolated from mice experiencing secondary DENV-2 infection. However, increased DENV-specific avidity is not associated with increased DENV-specific neutralization, which appears to be mediated by naive B cells. Adoptive transfer of DENV-1-immune B and T cells into naive mice prior to secondary DENV-2 infection delayed mortality. Mice depleted of T cells developed signs of disease, but recovered after secondary DENV infection. Overall, we found that protective cross-reactive Abs are secreted by both long-lived plasma cells and memory B cells and that both cross-reactive B cells and T cells provide protection against a secondary heterotypic DENV infection. Understanding the protective immunity that develops naturally against DENV infection may help design future vaccines.     

A tetravalent dengue nanoparticle stimulates antibody production in mice

Background

Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progresses to the severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome. Currently, there are no licensed vaccines or antiviral drugs to prevent or treat dengue infections. Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines.

Findings

Here, we used a murine model to evaluate the IgG production after administration of inactivated DENV corresponding to all four serotypes adsorbed to bovine serum albumin nanoparticles. This formulation induced a production of anti-DENV IgG antibodies (p < 0.001). However, plaque reduction neutralization assays with the four DENV serotypes revealed that these antibodies have no neutralizing activity in the dilutions tested.

Conclusions

Our results show that while the nanoparticle system induces humoral responses against DENV, further investigation with different DENV antigens will be required to improve immunogenicity, epitope specicity, and functional activity to make this platform a viable option for DENV vaccines.     

A synthetic peptide analog of in silico-predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits

Development of a serotyping-capable dengue detection test is hampered by the absence of an identified unique marker that can detect specific dengue virus (DENV) serotype. In the current commercially available antibody-capture diagnostic methods, immobilized nonstructural 1 (NS1) antigen indiscriminately binds and detects immunoglobulin M or immunoglobulin G against any serotype, thus limiting its capability to distinguish existing serotypes of dengue. Identification of dengue serotype is important because certain serotypes are associated with severe forms of dengue as well as dengue hemorrhagic fever. In this study, we aimed to identify an immunogenic epitope unique to DENV2 NS1 antigen and determine the binding specificity of its synthetic peptide mimotope to antibodies raised in animal models. Selection of a putative B-cell epitope from the reported DENV2 NS1 antigen was done using Kolaskar and Tongaonkar Antigenicity prediction, Emini surface accessibility prediction, and Parker hydrophilicity prediction available at the immune epitope database and analysis resource. Uniqueness of the B-cell epitope to DENV2 was analyzed by BLASTp. Immunogenicity of the synthetic peptide analog of the predicted immunogenic epitope was tested in rabbits. The binding specificity of the antibodies raised in animals and the synthetic peptide mimotope was tested by indirect ELISA. A synthetic peptide analog comprising the unique epitope of DENV2 located at the 170th–183rd position of DENV2 NS1 was found to be immunogenic in animal models. The antipeptide antibody produced in rabbits showed specific binding to the synthetic peptide mimotope of the predicted unique DENV2 NS1 immunogenic epitope.     

Current progress in dengue vaccines

Dengue is one of the most important emerging vector-borne viral diseases. There are four serotypes of dengue viruses (DENV), each of which is capable of causing self-limited dengue fever (DF) or even life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The major clinical manifestations of severe DENV disease are vascular leakage, thrombocytopenia, and hemorrhage, yet the detailed mechanisms are not fully resolved. Besides the direct effects of the virus, immunopathological aspects are also involved in the development of dengue symptoms. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development, including live attenuated virus vaccines, live chimeric virus vaccines, inactivated virus vaccines, and live recombinant, DNA and subunit vaccines. The live attenuated virus vaccines and live chimeric virus vaccines are undergoing clinical evaluation. The other vaccine candidates have been evaluated in preclinical animal models or are being prepared for clinical trials. For the safety and efficacy of dengue vaccines, the immunopathogenic complications such as antibody-mediated enhancement and autoimmunity of dengue disease need to be considered.     

Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi,India

Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co‐circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT‐PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co‐infection with DENV and CHIKV in 10% of the samples by RT‐PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV‐1 strains, six DENV‐2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV‐1 strains grouped in the American African genotype, DENV‐2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV‐1 and DENV‐2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.     

Dengue infection in children in ratchaburi, Thailand: a cohort study. I. Epidemiology of symptomatic acute dengue infection in children, 2006-2009

Background

There is an urgent need to field test dengue vaccines to determine their role in the control of the disease. Our aims were to study dengue epidemiology and prepare the site for a dengue vaccine efficacy trial.

Methods and Findings

We performed a prospective cohort study of children in primary schools in central Thailand from 2006 through 2009. We assessed the epidemiology of dengue by active fever surveillance for acute febrile illness as detected by school absenteeism and telephone contact of parents, and dengue diagnostic testing. Dengue accounted for 394 (6.74%) of the 5,842 febrile cases identified in 2882, 3104, 2717 and 2312 student person-years over the four years, respectively. Dengue incidence was 1.77% in 2006, 3.58% in 2007, 5.74% in 2008 and 3.29% in 2009. Mean dengue incidence over the 4 years was 3.6%. Dengue virus (DENV) types were determined in 333 (84.5%) of positive specimens; DENV serotype 1 (DENV-1) was the most common (43%), followed by DENV-2 (29%), DENV-3 (20%) and DENV-4 (8%). Disease severity ranged from dengue hemorrhagic fever (DHF) in 42 (10.5%) cases, dengue fever (DF) in 142 (35.5%) cases and undifferentiated fever (UF) in 210 (52.5%) cases. All four DENV serotypes were involved in all disease severity. A majority of cases had secondary DENV infection, 95% in DHF, 88.7% in DF and 81.9% in UF. Two DHF (0.5%) cases had primary DENV-3 infection.

Conclusion

The results illustrate the high incidence of dengue with all four DENV serotypes in primary school children, with approximately 50% of disease manifesting as mild clinical symptoms of UF, not meeting the 1997 WHO criteria for dengue. Severe disease (DHF) occurred in one tenth of cases. Data of this type are required for clinical trials to evaluate the efficacy of dengue vaccines in large scale clinical trials.     

Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

BackgroundThere are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication.Conclusions/SignificanceThe method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.     

登革病毒疫苗研究现状与展望

登革病毒是属于黄病毒科的小型包膜病毒,在热带和亚热带地区通过蚊媒传播。其感染可引起临床症状轻微的登革热,甚至危及生命的登革出血热和登革休克综合征。登革病毒包含4种血清型,有效的登革病毒疫苗需对4种血清型的登革病毒均具有抗病毒保护作用。目前,尚未有针对登革病毒的特效药和成熟的疫苗产品。各类登革病毒疫苗均在研发中,其中一些已进入临床试验阶段。本文就登革病毒疫苗研究进展作一综述并对未来发展进行展望。     

Investigation of the First Case of Dengue Virus Infection Acquired in Western Australia in Seven Decades: Evidence of Importation of Infected Mosquitoes?

In October 2013, a locally-acquired case of dengue virus (DENV) infection was reported in Western Australia (WA) where local dengue transmission has not occurred for over 70 years. Laboratory testing confirmed recent DENV infection and the case demonstrated a clinically compatible illness. The infection was most likely acquired in the Pilbara region in the northwest of WA. Follow up investigations did not detect any other locally-acquired dengue cases or any known dengue vector species in the local region, despite intensive adult and larval mosquito surveillance, both immediately after the case was notified in October 2013 and after the start of the wet season in January 2014. The mechanism of infection with DENV in this case cannot be confirmed. However, it most likely followed a bite from a single infected mosquito vector that was transiently introduced into the Pilbara region but failed to establish a local breeding population. This case highlights the public health importance of maintaining surveillance efforts to ensure that any incursions of dengue vectors into WA are promptly identified and do not become established, particularly given the large numbers of viraemic dengue fever cases imported into WA by travellers returning from dengue-endemic regions.     

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.     

Changes in hematological and serum biochemical parameters in common marmosets (Callithrix jacchus) after inoculation with dengue virus

Background Marmosets are susceptible to dengue virus (DENV) infection. However, blood parameter data and clinical signs of DENV-infected marmosets are limited. Methods Blood hematological and serum biochemical values were obtained from twelve DENV-inoculated and four mock-infected marmosets. Additionally, body temperature and activity level were determined. Results Five DENV-inoculated marmosets demonstrated thrombocytopenia, nine demonstrated leucopenia, and five demonstrated an increase in the levels of AST, ALT, LDH, and BUN. Additionally, seven DENV-inoculated marmosets demonstrated clinical signs including fever and decreases in activity. None of the four mock-inoculated marmosets demonstrated changes in either hematological or biochemical parameters. Conclusions Marmosets inoculated with DENV exhibited clinical signs and changes in hematological and biochemical parameters. The results suggest that blood parameter data and clinical signs could potentially be useful markers for understanding the progress of DENV infection in studies using marmosets.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

登革四价疫苗研究进展

登革病毒感染引起的登革热和登革出血热是重要的蚊媒病毒病。近20年来,在全球范围内登革出血热的发病率和病死率急剧上升,但目前仍缺乏安全有效的疫苗。着重介绍目前已经或正在进入临床研究阶段的四价登革减毒活疫苗,以及灭活疫苗、亚单位疫苗、病毒载体疫苗和DNA疫苗的研究进展。