Cross-reactive memory CD8(+) T cells alter the immune response to heterologous secondary dengue virus infections in mice in a sequence-specific manner

Dengue virus is the causative agent of dengue fever and the more-severe dengue hemorrhagic fever (DHF). Human studies suggest that the increased risk of DHF during secondary infection is due to immunopathology partially mediated by cross-reactive memory T cells from the primary infection. To model T cell responses to sequential infections, we immunized mice with different sequences of dengue virus serotypes and measured the frequency of peptide-specific T cells after infection. The acute response after heterologous secondary infections was enhanced compared with the acute or memory response after primary infection. Also, the hierarchy of epitope-specific responses was influenced by the specific sequence of infection. Adoptive-transfer experiments showed that memory T cells responded preferentially to the secondary infection. These findings demonstrate that cross-reactive T cells from a primary infection alter the immune response during a heterologous secondary infection.     

The human antibody response to dengue virus infection

Dengue viruses (DENV) are the causative agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). Here we review the current state of knowledge about the human antibody response to dengue and identify important knowledge gaps. A large body of work has demonstrated that antibodies can neutralize or enhance DENV infection. Investigators have mainly used mouse monoclonal antibodies (MAbs) to study interactions between DENV and antibodies. These studies indicate that antibody neutralization of DENVs is a "multi-hit" phenomenon that requires the binding of multiple antibodies to neutralize a virion. The most potently neutralizing mouse MAbs bind to surface exposed epitopes on domain III of the dengue envelope (E) protein. One challenge facing the dengue field now is to extend these studies with mouse MAbs to better understand the human antibody response. The human antibody response is complex as it involves a polyclonal response to primary and secondary infections with 4 different DENV serotypes. Here we review studies conducted with immune sera and MAbs isolated from people exposed to dengue infections. Most dengue-specific antibodies in human immune sera are weakly neutralizing and bind to multiple DENV serotypes. The human antibodies that potently and type specifically neutralize DENV represent a small fraction of the total DENV-specific antibody response. Moreover, these neutralizing antibodies appear to bind to novel epitopes including complex, quaternary epitopes that are only preserved on the intact virion. These studies establish that human and mouse antibodies recognize distinct epitopes on the dengue virion. The leading theory proposed to explain the increased risk of severe disease in secondary cases is antibody dependent enhancement (ADE), which postulates that weakly neutralizing antibodies from the first infection bind to the second serotype and enhance infection of FcγR bearing myeloid cells such as monocytes and macrophages. Here we review results from human, animal and cell culture studies relevant to the ADE hypothesis. By understanding how human antibodies neutralize or enhance DENV, it will be possible to better evaluate existing vaccines and develop the next generation of novel vaccines.     

Severe dengue virus infection in travelers: risk factors and laboratory indicators

BACKGROUND: Dengue fever is the most common arboviral disease in travelers. In countries where dengue virus is endemic, sequential (secondary) infections with different dengue virus serotypes are associated with disease severity. Data on severity and secondary infection rates in a population of travelers are lacking. METHODS: Intensified surveillance of dengue fever in travelers was performed within the European Network on Surveillance of Imported Infectious Diseases. Data were collected at 14 European clinical referral centers between 2003 and 2005. RESULTS: A total of 219 dengue virus infections imported from various regions of endemicity were reported. Serological analysis revealed a secondary immune response in 17%. Spontaneous bleeding was observed in 17 (8%) patients and was associated with increased serum alanine and aspartate aminotransferase levels and lower median platelet counts. Two (0.9%) patients fulfilled the World Health Organization (WHO) case definition for dengue hemorrhagic fever. However, 23 (11%) travelers had severe clinical manifestations (internal hemorrhage, plasma leakage, shock, or marked thrombocytopenia). A secondary immune response was significantly associated with both spontaneous bleeding and other severe clinical manifestations. CONCLUSIONS: In travelers, severe dengue virus infections are not uncommon but may be missed if the WHO classification is strictly applied. High liver enzyme levels and low platelet counts could serve as indicators of disease severity.     

Dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles

A prospective study of clinical and cytokine profiles of 107 infants with dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) was conducted. Fever, petechiae on the skin, and hepatomegaly were the most common clinical findings associated with DHF/DSS in infants. DSS occurred in 20.5% of the patients. Hemoconcentration and thrombocytopenia were observed in 91.5% and 92.5% of the patients, respectively. Serologic testing revealed that almost all of the patients (95.3%) had primary dengue virus infections. These data demonstrate that clinical and laboratory findings of DHF/DSS in infants are compatible with the World Health Organization's clinical diagnostic criteria for pediatric DHF. The present study is the first to report evidence of production of cytokines in infants with DHF/DSS and to describe the difference between the cytokine profile of infants with primary dengue virus infections and children with secondary infections. Overproduction of both proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) and anti-inflammatory cytokines (interleukin-10 and -6) may play a role in the pathogenesis of DHF/DSS in infants.     

A prospective seroepidemiologic study on dengue in children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995-1996.

A prospective study on dengue (DEN) viruses was initiated in October 1995 in Gondokusuman kecamatan, Yogyakarta, Indonesia. This report presents data from the first year of the study. The studied cohort included all children 4-9 years of age living in the kecamatan. Blood samples for serology were collected from 1,837 children in October 1995 and again in October 1996. Blood samples for virus isolation and serology were collected from cohort children who were seen in municipal health clinics with febrile syndromes or admitted to hospitals with a provisional diagnosis of dengue hemorrhagic fever. Dengue serotype antibody prevalence and 1995-1996 infection rates were calculated using a single dilution (1:60) 70% plaque reduction endpoint neutralization test. Prevalence of dengue antibody at the beginning of the study was DEN 1 = 12%, DEN 2 = 16%, DEN 3 = 2%, DEN 4 = 4%, and two or more dengue infections = 22%. Total dengue antibody prevalence increased from 38% in 4-year-old children to 69% in 9-year-old children. During the observation period, primary dengue infection rates were DEN 1 = 4.8%, DEN 2 = 7.7%, DEN 3 = 4.2%, and DEN 4 = 3.4%, while two or more dengue infections occurred in 6.7% of the study population. The secondary dengue infection rate was 19.0%. From febrile cases, all four dengue viruses were isolated with DEN 3 predominating. Seven children were hospitalized, including one fatal case with a hospital diagnosis of dengue shock syndrome. Based upon presence of antibody in the initial cohort bleeding and the serologic response both weeks and several months following illness, all had secondary dengue infections. Neutralizing antibody patterns in the initial cohort bleeding and in late convalescent serum samples permitted recognition of dengue infection sequence in five patients: DEN 2-DEN 1 (3), DEN 2-DEN 4 (1), DEN 1-DEN 3 (1), and none in the sequence DEN 1-DEN 2. In the total cohort 6.5% of the observed secondary infections were of the sequence DEN 2-DEN 1, while 4.9% were DEN 1-DEN 2, a highly pathogenic sequence in previous studies. Reduced pathogenic expression of secondary DEN 2 with enhanced pathogenic expression of secondary DEN 1 infections was an unexpected finding. Further studies will be required to understand the respective contributions to pathogenicity of antibody from initial dengue infections versus the biological attributes of the second infecting dengue viruses.     

Infections with two dengue viruses in Greece in the 20th century. Did dengue hemorrhagic fever occur in the 1928 epidemic?

From contemporary clinical accounts we hypothesized that the 1928 dengue epidemic in Greece may have been an earlier occurrence of dengue hemorrhagic fiver/dengue shock syndrome (DHF/DSS). To study the possibility that two different dengue viruses may have been involved, serums from 62 Athenians alive during the epidemic were examined for dengue antibodies; 73 per cent showed evidence of prior dengue infection. Monotypic neutralizing antibodies were found to two different dengue viruses, types 1 and 2. A large proportion of the sampled population had evidence of two or more past dengue infections. Since there is no evidence that dengue viruses have been transmitted in Greece since 1928, during the epidemic a very large number of persons immune to one dengue type must have acquired infections with a secon type. The virological criteria for secondary infection DHF/DSS are thus satisfied. Although DHF/DSS is currently restricted to Asia and the Pacific, the Greek oubreak suggests a biological potential for fatal consequences of dengue infections in Caucasians, particularly the elderly.     

Immunopathological mechanisms in dengue and dengue hemorrhagic fever

PURPOSE OF REVIEW: The continued emergence of dengue virus infection and its severe disease manifestation, dengue hemorrhagic fever, is a growing public health problem. The majority of severe infections occur upon secondary encounters with heterologous dengue virus serotypes, suggesting an immune-mediated process. RECENT FINDINGS: Significant findings in the past year include a greater understanding of dengue virus interactions with target cells such as dendritic cells, hepatocytes and endothelial cells. Infection of these cells results in the production of immune mediators that then shape the adaptive humoral and cellular immune response. The circulation of high levels of secreted NS1 in the presence of pre-existing heterologous non-neutralizing antibody may mediate complement activation and trigger plasma leakage. The role of enhancing antibodies in disease pathogenesis remains unclear. Recent studies demonstrate low avidity crossreactive T cells, which may produce an altered profile of cytokines leading to plasma leakage. Ongoing prospective studies that include epidemiological, virological and immunological risk factors are crucial to our understanding of the mechanisms of immunopathogenesis of dengue hemorrhagic fever. SUMMARY: The immune mechanisms that lead to dengue hemorrhagic fever are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious dengue vaccines.     

Immunodominant T-cell responses to dengue virus NS3 are associated with DHF

Dengue infections are increasing at an alarming rate in many tropical and subtropical countries, where epidemics can put health care systems under extreme pressure. The more severe infections lead to dengue hemorrhagic fever (DHF), which can be life threatening. A variety of viral and host factors have been associated with the severity of dengue infections. Because secondary dengue infection is more commonly associated with DHF than primary infections, the acquired immune response to dengue, both B cells and T cells have been implicated. In this study, we set out to study T-cell responses across the entire dengue virus proteome and to see whether these were related to disease severity in a cohort of dengue-infected children from Thailand. Robust responses were observed in most infected individuals against most viral proteins. Responses to NS3 were the most frequent, and there was a very strong association between the magnitude of the response and disease severity. Furthermore, in DHF, cytokine-high CD107a-negative cells predominated.     

Intracellular cytokine production by dengue virus-specific T cells correlates with subclinical secondary infection

The pathophysiology of dengue virus infection remains poorly understood, although secondary infection is strongly associated with more severe disease. In the present study, we performed a nested, case-control study comparing the responses of pre-illness peripheral blood mononuclear cells between children who would subsequently develop either subclinical or symptomatic secondary infection 6-11 months after the baseline blood samples were obtained and frozen. We analyzed intracellular cytokine production by CD4(+) and CD8(+) cells in response to stimulation with dengue antigen. We found higher frequencies of dengue virus-specific TNFα, IFNγ-, and IL-2-producing T cells among schoolchildren who subsequently developed subclinical infection, compared with those who developed symptomatic secondary dengue virus infection. Although other studies have correlated immune responses during secondary infection with severity of disease, to our knowledge this is the first study to demonstrate a pre-infection dengue-specific immune response that correlates specifically with a subclinical secondary infection.     

Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a prospective cohort study of DV infection in Thailand

BACKGROUND: Infection with any 1 of the 4 dengue viruses (DVs) can produce several illnesses, ranging from a mild febrile illness to classic dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. Most DHF cases occur after sequential heterotypic DV infections. The role of preexisting humoral immunity in modifying severity of dengue disease is not well understood. METHODS: We conducted a prospective cohort study of children in a region where dengue disease is hyperendemic and examined the role of preexisting neutralizing anti-DV antibodies (Abs) in modifying secondary dengue-3 virus (D3V), dengue-2 virus (D2V), and dengue-1 virus (D1V) infections. RESULTS: In secondary D3V infection, higher levels of preexisting neutralizing Ab directed against D3V (reference virus strain and patient's virus isolate) were associated with lower viremia levels and milder disease. Preexisting neutralizing Ab levels against D2V were not associated with severity of secondary D2V infection. The levels of preexisting neutralizing Ab against the infecting virus isolates were not associated with viremia levels in secondary D2V or D1V infections. CONCLUSIONS: Cross-reactive memory humoral immune responses appear to be beneficial in symptomatic secondary D3V infection, but not in secondary D2V or D1V infection. These results may have important implications for the development of live attenuated tetravalent dengue vaccines.     

Influence of the dengue serotype, previous dengue infection, and plasma viral load on clinical presentation and outcome during a dengue-2 and dengue-4 co-epidemic

Martinique experienced a dengue outbreak with co-circulation of DENV-2 and DENV-4. In an emergency department-based study, we analyzed whether the clinical presentation and outcome of adult patients were related to serotype, immune status, or plasma viral load. Of the 146 adult patients who had confirmed dengue infection, 91 (62.3%) were classified as having classic dengue fever, 11 (7.5%) fulfilled World Health Organization criteria for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), 21 other patients (14.4%) presented with at least one typical feature of DHF/DSS [i.e., internal hemorrhage, plasma leakage, marked thrombocytopenia (platelet count < or = 50,000 platelets/mm(3)) and/or shock], and 23 further patients (15.8%) had unusual manifestations. Four patients died. Severe illness was more frequent in patients with secondary dengue infection (odds ratio, 7.18; 95% confidence interval, 3.1-16.7; P < 0.001). Multivariate regression analysis showed that gastrointestinal symptoms and other unusual manifestations were independently associated with DENV-2 infection, whereas cough and DHF/DSS features were independently associated with secondary immune response. A high plasma viral load was associated with DENV-2 infection, increased serum liver enzymes, and with DHF/DSS features in patients presenting after the third day of illness. The most severe cases of dengue resulted from the combined effects of DENV-2 and secondary infection.     

Capillary leakage in travelers with dengue infection: implications for pathogenesis

Dengue hemorrhagic fever is characterized by the presence of a capillary leak syndrome. Its pathogenesis is presumed to differ from that of classical dengue fever (DF) and to be associated with secondary dengue infection. Returning travelers given a diagnosis of DF were evaluated for capillary leakage with abdominal sonography. Data were compared between travelers with primary/secondary infection defined by epidemiologic and serologic parameters. A total of 12 (34.3%) of 35 patients had sonographic signs of capillary leakage. Most (85%) patients with capillary leakage had classical DF. Capillary leak was diagnosed in 32% of primary dengue cases and in 40% of secondary dengue cases (P = 0.69). The two patients given a diagnosis of dengue hemorrhagic fever had primary infections. The high prevalence of capillary leakage among travelers, most of them with primary exposure to dengue, calls into question the importance of secondary infection in causing capillary leakage in dengue infection.     

Innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4

Dengue infection is an important public health issue worldwide. The ChimeriVax-Dengue (CYD) vaccine uses yellow fever (YF) 17D vaccine as a live vector. Dendritic cells (DCs) play a key role in initiating immune responses and could be an important primary target of dengue infection. We investigated in vitro the consequences of CYD infection of DCs on their activation/maturation and cytokine production. In CYD-infected DCs, we observed an up-regulation of HLA-DR, CD80, CD86, and CD83. Cells exposed to CYD secreted type I interferons, monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2), interleukin-6 (IL-6), and low amounts of tumor necrosis factor-alpha (TNF-alpha), but no IL-10, IL-12, or IL-1alpha. Parental dengue viruses induced a similar array of cytokines, but more TNF-alpha, less IL-6, and less MCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation and a controlled response accompanied by limited inflammatory cytokine production and consistent expression of anti-viral interferons, in agreement with clinical observations of safety and immunogenicity.     

Serum levels of cytokines in two ethnic groups with dengue virus infection

This study compared the serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon gamma (IFN-gamma) in 78 Colombian patients, from two ethnic groups, with dengue virus infection. TNF-alpha levels were significantly higher in Afro-Colombians than in Mestizos and IL-6 levels were significantly higher in Mestizos than in Afro-Colombians, during the acute phase. IFN-gamma levels were similar in both ethnic groups. Significantly higher TNF-alpha levels were found in Afro-Colombians than in Mestizos in both dengue fever (DF) and dengue hemorrhagic fever (DHF). The IL-6 levels were higher in Mestizos than in Afro-Colombians among patients with DF, but levels of this cytokine were higher in Afro-Colombians than in Mestizos among patients with DHF. Levels of IFN-gamma were higher in patients with DHF than DF. Higher levels of these cytokines were observed in secondary infection. These results suggest that ethnicity may contribute to differences in immune responses to dengue infections.     

Differences in dengue severity in infants, children, and adults in a 3-year hospital-based study in Nicaragua

To investigate age-related differences in dengue severity, 114 infants, 1,211 children, and 346 adults with laboratory-confirmed dengue virus (DEN) infections presenting to three hospitals in major urban centers in Nicaragua were recruited from 1999 to 2001. The age distribution of dengue cases and the circulating serotype (predominantly DEN2) were representative of national data. Similar results were obtained when either dengue hemorrhagic fever/dengue shock syndrome or its principal manifestations (vascular permeability, internal hemorrhage, marked thrombocytopenia, and/or shock) were analyzed in relation to age and immune status. The burden of disease and of severe dengue was found predominantly in infants 4-9 months of age and in children 5-9 years old, and secondary DEN infection was a risk factor for severity in children. Age-related differences were identified in the prevalence of specific clinical manifestations as well as in their association with a confirmed DEN diagnosis. This represents one of the few comprehensive studies to analyze characteristics of dengue in infants, children, and adults in the same population and highlights age-related differences in dengue severity.     

The use of intravenous gammaglobulin in dengue fever, a case report

We managed a serologically documented case of dengue fever with thrombocytopenia with IV IgG. Acute and convalescent ELISA and HI titers indicated an acute dengue infection with a secondary response that clinically, and by laboratory parameters dramatically improved after IV IgG administration. The use of IV IgG in cases of thrombocytopenia associated with dengue has both theoretical advantages and disadvantages. IV IgG may have a role in the management of DHF/DSS because of the significant morbidity and mortality associated with DHF/DSS. A prospective randomized study should be developed to determine if IV IgG does indeed effect the course of thrombocytopenia in dengue infections.     

Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections

Dengue hemorrhagic fever (DHF), the most severe form of illness following infection with a dengue virus, is characterized by plasma leakage, thrombocytopenia, and hepatic inflammation. The interrelationships among virus burden, immune activation, and development of DHF were examined in 54 children with secondary dengue-3 virus infections participating in a prospective, hospital-based study. DHF was associated with higher mean plasma viremia early in illness and earlier peak plasma interferon-gamma levels. Maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia. Maximum plasma levels of interleukin (IL)-10 and soluble tumor necrosis factor receptor-II correlated with the degree of thrombocytopenia, independently of viremia levels. Hepatic transaminase elevation correlated with plasma soluble IL-2 receptor levels and not with viremia levels. Quantitative differences in virus burden and host immune responses, and the timing of type 1 cytokine responses, have differing influences on the severity of disease manifestations during secondary dengue-3 virus infections.     

Fulminant liver failure secondary to haemorrhagic dengue in an international traveller.

Dengue infections are caused by a single-stranded RNA virus, which has four serotypes (DEN 1-4); mosquitoes of the genus Aedes serve as vectors of transmission. Risk factors for dengue infection are related to both the host and virus. Age, gender, immune status, and genetic background of the host all contribute to the severity of dengue infection. Recently, international travel to endemic areas has also been identified as a major risk factor for both primary and secondary dengue infection. Dengue remains a diagnostic challenge, given its protean nature, ranging from mild febrile illness to profound shock. The most severe manifestation of dengue infection is dengue shock syndrome, which has an estimated mortality rate close to 50%. Dengue shock syndrome typically presents with increased anion gap metabolic acidosis, disseminated intravascular coagulation, severe hypotension, and jaundice. Liver involvement appears to occur more frequently when infections involve DEN-3 and DEN-4 serotypes. While hepatocellular damage has been reported previously in dengue infection, acute liver failure is an extremely rare occurrence in adults. We report a patient with dengue shock syndrome who presented with acute liver failure and hepatic encephalopathy after recent travel to an endemic area.     

Cytokine Factors Present in Dengue Patient Sera Induces Alterations of Junctional Proteins in Human Endothelial Cells

Plasma leakage in severe dengue has been postulated to be associated with skewed cytokine immune responses. In this study, the association of cytokines with vascular permeability in dengue patients was investigated. Human serum samples collected from 48 persons (13 with dengue fever, 29 with dengue hemorrhagic fever, and 6 healthy) were subjected to cytokines analysis by using Luminex Multiplex Technology. Selected serum samples from patients with dengue hemorrhagic fever sera and recombinant human cytokines were then tested for roles on inducing vascular permeability by treatment of human umbilical vein endothelial cells. Confocal immunofluorescence staining indicated morphologic alteration of human umbilical vein endothelial cells treated with serum samples from patients with dengue hemorrhagic fever compared with serum samples from healthy persons. The findings suggest that cytokines produced during dengue hemorrhagic infections could induce alterations in the vascular endothelium, which may play a fundamental role in the pathophysiology of dengue.