Immunologic hypo- or non-responder in natural dengue virus infection

Serologically defined primary dengue virus infection and/or subsequent homologous serotype infection is known to be associated with less severe disease as compared with secondary subsequent heterologous serotype infection. In geographical locales of high dengue endemicity, almost all individuals in the population are infected at some point in time and should therefore are at high risk of secondary infection. Interestingly, dengue viremia in healthy blood donors whose sera apparently lack detectable levels of specific antibody to dengue viral antigens has been reported. The incidence rate of potential immunologic hypo- or non-responders following natural primary dengue virus infection in dengue endemic regions, who do become immune responders only after repeated exposure, has not been described. These are the patients who may be diagnosed as primary infection in the subsequent infection, but actually are secondary infection. This concept has important implications with regards to the hypothesis of immunological enhancement of dengue pathogenesis, which has largely been advanced based on empirical observations and/or from in vitro experimental assays. The fact that dengue naïve travelers can suffer from severe dengue upon primary exposure while visiting dengue endemic countries underscores one of the major problems in explaining the role of immune enhancement in the pathogenesis of severe dengue virus infection. This evidence suggests that the mechanism(s) leading to severe dengue may not be associated with pre-existing enhancing antibody. Consequently, we propose a new paradigm for dengue virus infection classification. These include a) patients with naïve primary infection, b) those that are serologically defined primary in dengue endemic zones and c) those who are serologically defined secondary dengue virus infection. We submit that clarity with regards to such definitions may help facilitate the delineation of the potential mechanisms of severe dengue virus infection.     

登革病毒疫苗研究进展

登革病毒属黄病毒属,可通过蚊虫传播,感染人体后可引发一系列临床症状,从轻微发热到严重的并发症,称为登革热、登革出血热以及登革休克综合征。过去50年,全球登革热感染病例增加了约30倍。目前,全球热带、亚热带地区约占世界2/5的人口存在感染风险。由于缺乏有效的治疗药物,疫苗研究已成为登革热疾病防控的重心。然而,由于缺乏对病毒致病机理及病毒感染免疫应答深入的了解,候选疫苗的研发受到阻碍。但经过几十年的努力,疫苗研究取得了明显进展。目前正在研究的登革病毒疫苗依托各种技术平台,种类多样,对正处于临床前研究及临床试验阶段的不同类型疫苗进行阐述。     

Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexes after rIFN-gamma treatment. Pretreatment of U937 cells with rIFN-gamma resulted in a significant increase in the number of dengue virus-infected cells and in the yield of infectious virus. rIFN-gamma did not augment dengue virus infection when cells were infected with virus in the absence of anti-dengue antibodies. Gamma interferon (IFN-gamma) produced by peripheral blood lymphocytes from dengue-immune donors after in vitro stimulation with dengue antigens also augmented dengue virus infection of U937 cells. IFN-gamma did not augment dengue virus infections when cells were infected with virus in the presence of F(ab')2 prepared from anti-dengue immunoglobulin G. Human immunoglobulin inhibited IFN-gamma-induced augmentation. IFN-gamma increased the number of Fc gamma receptors on U937 cells. The increase in the percentage of dengue antigen-positive cells correlated with the increase in the number of Fc gamma receptors after rIFN-gamma treatment. These results indicate that IFN-gamma-induced augmentation of dengue virus infection is Fc gamma receptor mediated. Based on these results we conclude that IFN-gamma increases the number of Fc gamma receptors and that this leads to an augmented uptake of dengue virus in the form of dengue virus-antibody complexes, which results in augmented dengue virus infection.     

Dengue virus selectively induces human mast cell chemokine production

Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1alpha, and MIP-1beta, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P < 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1alpha, or MIP-1beta response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.     

Dengue epidemiology and pathogenesis:images of the future viewed through a mirror of the past

Every year, millions of individuals throughout the world are seriously affected by dengue virus. The unavailability of a vaccine and of anti-viral drugs has made this mosquito-borne disease a serious health concern. Not only does dengue cause fatalities but it also has a profoundly negative economic impact. In recent decades, extensive research has been performed on epidemiology, vector biology, life cycle, pathogenesis, vaccine development and prevention. Although dengue research is still not at a stage to suggest definite hopes of a cure, encouraging significant advances have provided remarkable progress in the fight against infection. Recent developments indicate that both anti-viral drug and vaccine research should be pursued, in parallel with vector control programs.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Dermal-type macrophages expressing CD209/DC-SIGN show inherent resistance to dengue virus growth

Background

An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection.

Methods and Findings

Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-α was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes.

Conclusions

Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito.     

Viremia and the Magnitude of the Immune Response upon Infection of Green Monkeys with Dengue Virus Type 2 Are Strain-Dependent

Testing in non-human primates is a generally accepted necessary step preceding the evaluation of dengue vaccine candidates in humans. A reduction of viremia in these animals after virus challenge is generally used as an indicator of vaccine efficacy. In this work, we compared the infectivity of three strains of dengue virus type 2 in a non-human primate model of dengue infection, with the aim of selecting a virus for vaccine protection studies. As a result, strain SB8553 produced the longest duration of viremia, with a mean of 3 days/animal. In addition, it induced the highest antiviral and neutralizing antibody titers. These results support the use of strain SB8553 in challenge assays in this model and demonstrate that infection of green monkeys with dengue virus type 2 is dependent on the strain of virus used.     

Dengue virus induces expression of CXC chemokine ligand 10/IFN-gamma-inducible protein 10, which competitively inhibits viral binding to cell surface heparan sulfate

Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-gamma-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.     

Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo

Previous studies have suggested that α-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P ≤ 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.     

Productive dengue virus infection of human endothelial cells is directed by heparan sulfate-containing proteoglycan receptors

Dengue virus causes leakage of the vascular endothelium, resulting in dengue hemorrhagic fever and dengue shock syndrome. The endothelial cell lining of the vasculature regulates capillary permeability and is altered by immune and chemokine responses which affect fluid barrier functions of the endothelium. Our findings indicate that human endothelial cells are highly susceptible to infection by dengue virus (type 4). We found that dengue virus productively infects ～80% of primary human endothelial cells, resulting in the rapid release of ～10(5) virions 1 day postinfection. Analysis of potential inhibitors of dengue virus entry demonstrated that antibodies and ligands to integrins and cellular receptors were unable to inhibit dengue virus infection of endothelial cells. In contrast, pretreating cells with heparin or heparan sulfate resulted in a 60 to 80% reduction in dengue virus-infected cells, and pretreatment of endothelial cells with heparinase III or protease reduced dengue infectivity by >80%. Dengue virus bound specifically to resin immobilized heparin, and binding was competitively inhibited by excess heparin but not other ligands. Collectively, these findings suggest that dengue virus specifically attaches to heparan sulfate-containing proteoglycan receptors on endothelial cells. Following attachment to human endothelial cell receptors, dengue virus causes a highly productive infection that has the potential to increase viral dissemination and viremia. This provides the potential for dengue virus-infected endothelial cells to directly alter barrier functions of the endothelium, contribute to enhancement of immune cell activation, and serve as potential targets of immune responses which play a central role in dengue pathogenesis.     

The monocyte-macrophage-mast cell axis in dengue pathogenesis

Dengue virus, the causative agent of dengue disease which may have hemorrhagic complications, poses a global health threat. Among the numerous target cells for dengue virus in humans are monocytes, macrophages and mast cells which are important regulators of vascular integrity and which undergo dramatic cellular responses after infection by dengue virus. The strategic locations of these three cell types, inside blood vessels (monocytes) or outside blood vessels (macrophages and mast cells) allow them to respond to dengue virus infection with the production of both intracellular and secretory factors which affect virus replication, vascular permeability and/or leukocyte extravasation. Moreover, the expression of Fc receptors on the surface of monocytes, macrophages and mast cells makes them important target cells for antibody-enhanced dengue virus infection which is a major risk factor for severe dengue disease, involving hemorrhage. Collectively, these features of monocytes, macrophages and mast cells contribute to both beneficial and harmful responses of importance to understanding and controlling dengue infection and disease.     

Molecular Mechanisms Involved in Antibody‐Dependent Enhancement of Dengue Virus Infection in Humans

Dengue is the most common arthropod‐borne viral infection in humans with ～50 million cases annually worldwide. In recent decades, a steady increase in the number of severe dengue cases has been seen. Severe dengue disease is most often observed in individuals that have pre‐existing immunity against heterotypic dengue subtypes and in infants with low levels of maternal dengue antibodies. The generally accepted hypothesis explaining the immunopathogenesis of severe dengue is called antibody‐dependent enhancement of dengue infection. Here, circulating antibodies bind to the newly infecting virus but do not neutralize infection. Rather, these antibodies increase the infected cell mass and virus production. Additionally, antiviral responses are diminished allowing massive virus particle production early in infection. The large infected cell mass and the high viral load are prelude for severe disease development. In this review, we discuss what is known about the trafficking of dengue virus in its human host cells, and the signalling pathways activated after virus detection, both in the absence and presence of antibodies against the virus. This review summarizes work that aims to better understand the complex immunopathogenesis of severe dengue disease.      

Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors.

It is known that antibodies to dengue viruses at subneutralizing concentrations enhance dengue virus infection of Fc gamma R+ cells. This phenomenon called antibody-dependent enhancement (ADE) occurs when virus-antibody complexes bind to the Fc gamma R via the Fc portion of the Ig. It has been hypothesized that ADE may be responsible for the pathogenesis of the severe manifestations of dengue virus infection including dengue hemorrhagic fever/dengue shock syndrome. To further analyze the mechanisms of ADE, we prepared bispecific antibodies by chemically cross-linking antidengue virus antibodies to antibodies specific for Fc gamma RI or Fc gamma RII and the non-Fc R molecules beta2 microglobulin, CD15 or CD33 and examined whether these bispecific antibodies could enhance infection. Bispecific antibodies targeting dengue virus to Fc gamma RI or Fc gamma RII enhanced dengue virus infection, consistent with previous reports using conventional antibodies. Furthermore, bispecific antibodies targeting dengue virus to beta2 microglobulin, CD15 or CD33 also enhanced dengue virus infection. Bispecific antibody mediated ADE was inhibited by pretreating the cells with the appropriate blocking mAb. These results indicate that cell surface molecules other than Fc gamma R can mediate ADE and suggest that the Fc gamma R does not provide a unique signal necessary for enhanced infection. We hypothesize that directing dengue virus to the cell surface by a bispecific antibody facilitates the interaction between dengue virus and its receptor, thereby increasing its infectivity.     

MCP-1 and MIP-1α expression in a model resembling early immune response to dengue

Dengue virus has become endemic in most tropical urban areas throughout the world, and DHF has appeared concomitantly with this expansion. The intensity of dengue virus replication during the early stages of infection could determine clinical outcomes; therefore, it is important to understand the impact of dengue virus infection on the earliest immune defense against microbial infection, which also strongly regulates the adaptive immune responses. This study was aimed at evaluating the expression of the CC-chemokines MIP-1α/CCL3 and MCP-1/CCL2 in peripheral blood leukocytes using an ex vivo model resembling dengue infection in vivo, in subjects with a well characterized dengue immune background, due to the exceptional Cuban epidemiological situation in dengue. The expression of IFNγ, TNFα and IL10 was also evaluated, giving insight about the role of MCP-1 and MIP-1α in the interplay between innate and adaptive immunity. From individuals with different dengue immune background after dengue virus challenge, increased and different expression of the chemokines and cytokines studied was verified in peripheral blood mononuclear cells, thus demonstrating that the previous immunity to a dengue virus serotype has a strong influence on the early immune response after dengue re-infection.     

The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection

Using flow cytometric assay and monoclonal anti-dengue Ab, we observed that both anti-E and anti-prM Abs could enhance dengue virus infection in a concentration-dependent but serotype-independent manner. Increases were found in both the percentage of dengue-infected cells and the expression of dengue E and NS1 protein per cell. Dengue virion binding and infection were enhanced on FcR-bearing cells via the Fc-FcgammaRII pathway. Furthermore, anti-prM Ab also enhanced dengue virion binding and infection on cells lacking FcR, such as BHK-21 or A549 cells, by the mechanism of peptide (CPFLKQNEPEDIDCW)-specific binding. Anti-prM Ab cross-reacted with BHK-21 or A549 cells and recognized self-Ags such as heat shock protein 60. In summary, a novel mechanism of anti-prM Ab-mediated enhancement on dengue virus infection was found to be mediated by dual specific binding to dengue virion and to target cells, in addition to the traditional enhancement on FcR-bearing cells.     

A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan

ABSTRACT: Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue.     

Endothelial cells elicit immune-enhancing responses to dengue virus infection

Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Preexisting antibodies to dengue virus disposes patients to immune-enhanced edema (DSS) or hemorrhagic (DHF) disease following infection by a discrete dengue virus serotype. Although the endothelium is the primary vascular fluid barrier, direct effects of dengue virus on endothelial cells (ECs) have not been considered primary factors in pathogenesis. Here, we show that dengue virus infection of human ECs elicits immune-enhancing EC responses. Our results suggest that rapid early dengue virus proliferation within ECs is permitted by dengue virus regulation of early, but not late, beta interferon (IFN-β) responses. The analysis of EC responses following synchronous dengue virus infection revealed the high-level induction and secretion of immune cells (T cells, B cells, and mast cells) as well as activating and recruiting cytokines BAFF (119-fold), IL-6/8 (4- to 7-fold), CXCL9/10/11 (45- to 338-fold), RANTES (724-fold), and interleukin-7 (IL-7; 128-fold). Moreover, we found that properdin factor B, an alternative pathway complement activator that directs chemotactic anaphylatoxin C3a and C5a production, was induced 34-fold. Thus, dengue virus-infected ECs evoke key inflammatory responses observed in dengue virus patients which are linked to DHF and DSS. Our findings suggest that dengue virus-infected ECs directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These data implicate EC responses in dengue virus pathogenesis and further rationalize therapeutic targeting of the endothelium as a means of reducing the severity of dengue virus disease.