Dengue has been recognized as one of the most important vector-borne human diseases. The disease is induced by dengue virus infection resulting from the bite of an infected Aedes spp. mosquito after imbibing the tainted blood from animals or patients. Dynamic clinical spectrums ranging from asymptomatic, undifferentiated fever, typical dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) have been well documented. Initially, the disease was mainly restricted in tropical and subtropical zones. However, with factors such as ineffective vector control, frequency of human migration, unplanned urbanization, and changing climate temperature, the disease has been spotted at almost every territory of the earth. Dengue has been associated with human disease for more than two centuries. Although classic DF is viewed as a self-limited illness, subjects normally resolve within two weeks and recover without any noticeable complications or sequelae, some of these infected individuals may progress to life-threatening DHF/DSS, characterized with plasma leakage due to an increase in capillary permeability. The significantly increased public health threat and the burden of morbidity and mortality of dengue globally has caught the attention of public officials and prompted an action to find a way to contain and prevent the disease. The lack of specific dengue therapeutics has led to an emphasis on vaccine development, one of the best and effective strategies to reduce and prevent the illness. Making a dengue vaccine has been attempted more than six decades; although some of these products are in clinical trials, vaccine development for the prevention of dengue disease is still at its infancy. So far no dengue vaccine is available for the public. Dengue vaccine development may be hindered by the complexity of the clinical presentations, which implicates that multiple pathogenic mechanisms are involved in dengue disease. Some of these elements will be discussed in the current review. Opening up discussion on these pros and cons and engaging in more research to understand these features would not only improve the understanding of the pathogenesis of the dengue virus infection but also pave a new tactic to develop a safer and effective dengue vaccine.
Dengue is induced by infection with dengue virus and is one of the most important arthropod-borne human diseases. Although human beings are the natural host for the virus, it is transmitted by the bite of an Aedes spp. mosquito carrying infectious virus. Initially, the distribution of the disease was mainly in the tropical and subtropical regions. But, over time numerous factors such as global warming and increased frequency of human migration, and ineffective vector control have resulted in the expansion of dengue virus to almost every corner of the earth [
Dengue is introduced into the host during the blood meal taken by an infected mosquito. Epidemiological studies suggest that about 2.5 billion or 2/5 of the world’s population are at risk, of which approximately 1 billion live in tropical and subtropical regions. It is also estimated that about 50 million infections occur annually, roughly causing 500,000 hospitalizations, mainly in children. The case fatality rate is 1% - 2% in most dengue hyperendemic countries, but the rate of more than 5% has been reported in some areas [
Dengue virus is introduced to the human host by the bite of infected mosquitoes. Mosquitoes first become infected with dengue virus by feeding on the blood of a dengue-infected person. Several factors influence the rate of successful transmission, ranging from environmental temperature, density of Aedes spp. mosquitoes, competence of the mosquitoes, and levels of viremia in the host, interruption in exploration, deposition of the virus, and the pulse of the host [
Dengue has a significant impact on the economy and society and burdens the public health sectors in endemic areas. Globally, the societal burden has been estimated to be approximately 528 to 1300 disability-adjusted life years (DALY) per million to populations in endemic regions [12-15]. Children appear to be the most vulnerable group in many highly endemic countries and hence contribute the greatest number of DALYs attributable to dengue in the Torrid Zone. With more than a half-million cases reported annually, the average aggregate annual economic cost of dengue for eight study countries in the Americas and Asia is at approximate $587 million [
Dengue virus infections cause a spectrum of illnesses ranging from asymptomatic, mild undifferentiated fever to classical dengue fever (DF), and dengue hemorrhagic fever (DHF). DHF is classified as grades 1 to 4 depending on the severity [
ment [
The principle management of dengue cases is supportive or palliative care with close monitoring for shock and bleeding. The prompt and appropriate intravenous fluid therapy is the key to obtaining successful outcomes. Severe disease progresses rapidly but generally ceases by 48 hours after the critical stage. There has been no specific treatment or antiviral approved for therapy.
Dengue virus is in the family of Flaviviridae and under the genus Flavivirus, which includes Yellow Fever and West Nile Viruses. Though all these viruses are transmitted by mosquitoes, they differ in their primary host reservoir. Dengue primarily resides in humans, while animals and birds are the principal hosts for the Yellow Fever Virus and the West Nile Virus, respectively. The genome of dengue virus is single-stranded, positive-sense RNA with approximately 11,000 nucleotides. The RNA is infectious since it is capable of functioning as mRNA even though only equipped with a 5’ terminus type I cap (m7GpppAmp) but lack of 3’ terminus poly-A tail [20-22]. The genome encodes a single large open reading frame protein that is proteolytically cleaved into 10 known proteins consisting of 3 structural proteins, capsid (C), membrane (prM) and envelope (E), and 7 nonstructural proteins (NS) [23,24]. The arrangement and the order of the gene products is C-prM-E-NS1-NS2ANS2B-NS3-NS4A-NS4B-NS5 [25,26]. The functions of the proteins encoded by the virus and its importance in the life cycle of the virus in tissue culture system have been investigated very well and are not the focus of the current review. The readers are directed to these recent comprehensive review articles on the subject [2,5,27].
It has been known for a while that there are two variations of the dengue virion derived from different hosts. The lipid-associated viral particles, originally described in infected mouse brains, sediment slower through sucrose while having the same buoyant density as mousederived classical virions. The classical/conventional viral particles typically observed in fibroblast, lymphoblast or mosquito cell cultures, have a slightly heavier density [28, 29]. The earliest documentation on the diversity of the infectious dengue virus was addressed by Paul et al. [
Dengue viruses can be classified into four different antigenically distinct serotypes, a unique feature that distinguishes them from other flaviviruses. Each of the serotype is capable of inducing the spectrum of dengue diseases. Multiple dengue serotypes can co-circulate in endemic areas [
Despite its importance in human morbidity and mortality and decades of intensive efforts to learn and understand dengue, its pathogenesis remains a major enigma. Samples or specimens obtained from dengue patients are always at a late stage of the illness (
In tissue culture, various cells have been demonstrated to be permissive for dengue virus infection [
The severity of dengue diseases are observed not at the time when the viral burden is at its highest in vivo, but rather when the virus is being rapidly cleared from host tissues by the innate and adaptive immune responses. It is critical to bear in mind that evidence suggests that dengue viral antigen in leukocytes are most likely seen
after the cessation of viremia, based upon studies in rhesus monkeys [
Immune viral antigen-antibody complexes have been consistently identified in the plasma of patients with the hemagglutination-inhibition method [
The timing and dynamic spectrum of dengue disease indicates that many factors whether they are sequential, synergistic, or simultaneous occurring events contribute to the clinical outcomes. Intrinsic factors, such as individual’s genetic background, age, race, and sex and extrinsic elements, such as nutritional status, underlying illness, viral virulence, pre-existing antibodies, interval between first and subsequent infection, and the sequence of infections may influence pathogenesis [
Upon discovery that dengue virus is transmitted by the mosquitoes that imbibe blood from dengue infected individuals, there were attempts to reproduce the disease in small animals [
An animal model that can recapitulate the cardinal features of human dengue currently does not exist and is urgently needed. Nonetheless, laboratory animals, such as mice and other rodents, are somewhat susceptible to dengue virus infection and currently utilized for experimental infections; under certain settings, various disease models have been described in mice [84-88]. Although these models display some features of human dengue disease, they each have significant limitations [85,89]. For example, these models have mostly relied upon mouse-adapted viruses that appear to be attenuated with respect to human infection [81,83], and often present with a distinct paralysis phenotype, uncharacteristic of human disease [
Systemic research suggests that the nonhuman primate immune system and its responses, including profiles to dengue virus infection, are very similar to that of human beings. Chimpanzees and monkeys have been found to be highly susceptible for dengue virus infection, though the cardinal features present in human disease are hardly seen in these infected animals [30,46]. Therefore, humans, monkeys and chimpanzees are viewed to be the natural hosts for dengue virus. Subsequent investigations have revealed that the viremia levels in the circulation of monkeys and chimps are significantly lowered than that of infected humans [
Development of dengue vaccine has been attempted for more than 6 decades. A chronicle milestone on the activities is given in
The earliest literature describing the modulation of dengue virus virulence was by Cleland et al. [
The quest for a dengue vaccine was not stagnated during and after WWII. Although dengue has been shown to be caused by a filterable agent [47,99], the first dengue virus was not isolated until 1940, during WWII, by Dr. Hotta, S. in Japan and by Dr. Sabin, A.B. in the US [80-83]. During this period several innovative techniques were invented and developed. This offered a good opportunity to study dengue vaccine in much more specific manner. Several attempts to alter the virulence of the isolated dengue virus were made as well. These included studies of the immune response in mice using formalin-inactivated and attenuated dengue vaccines derived from suckling mice [80,100-102]. Sabin also observed that immunity elicited in volunteers who received the live
attenuated dengue vaccine could sustain the re-infection with the same dengue serotype at least for 18 months [
The isolation of the virus had significantly contributed to the development of biological and functional assays allowing for the study of immune responses to dengue virus. These new tools and techniques included the neutralization assay [
The major milestone from this era was that multiple serotypes of dengue virus were firmly established and designated as DENV1, DENV2, DENV3, and DENV4 [
This era brought about many golden milestones for dengue vaccine researchers. The numerous techniques and strategies employed to generate a variety of dengue vaccines have recently been reviewed [2-5,109] . These candidates include mutant, chimera, subunit, and DNA vaccines. Even though overor under-attenuated dengue vaccines developed under sponsorship of the U.S. Army proved not to be ideal for use in humans [110,111], some of them moved rapidly (at the speed of light) through safety, reactogenicity, and immunogenicity clinical trials. The safety and immunogenicity data obtained from these phase 1 clinical trials with monovalent vaccines in nonimmune volunteers revealed mixed results [112,113]. However, as a whole, the live attenuated vaccine did not show severe adverse reactions, the seroconversion rate was remarkable with very low virus doses, and neutralizing antibodies were still detectable one year later with just one dose [
A series of bivalent, trivalent, and tetravalent clinical trials were carried out for about a decade in both immune and non-immune young adults and in children residing in the endemic country of Thailand [113,115,116]. As a whole, the results were acceptable and efforts were shifted to find the best formulation for the four attenuated vaccine viruses to provide the best immunogenicity for all four serotypes. However, problems with antibody production due to interference among the different serotypes in the formulation are the major concern.
This era focused on bringing forward these established vaccines into clinical trials as soon as possible. A few set backs were encountered; for instance, one of the tetravalent live attenuated vaccines were shelved due to the imbalance in the antibody response to each serotype and consistent presence of high reactogenicity [
The dynamic nature of dengue clinical presentations makes the disease one of the most difficult vector-borne human diseases to be accurately diagnosed by doctors. Dengue is transmitted by the bite of mosquitoes carrying infectious dengue virus and is an acute disease. Patients do not normally recall when they were bitten but are often aware of how many days of fever he/she has experienced before seeking professional help from the clinics or hospitals. At that time, samples are taken and parameters are analyzed. Consequently, the conclusions drawn from these reports often conflict with each other, leading to debates over the clinical findings [50,55,122]. As a whole, reports from these clinical settings suggest that multiple factors are associated with disease development. These factors have been reviewed extensively [2,5,37] and will not be discussed in the current article. Instead, some of the underappreciated parameters are further discussed.
It is a well-established fact that antibodies play an important role in controlling infections from various pathogens. Hence, the humoral immune response is one of the critical indexes for the evaluation of vaccine efficacy. Dengue vaccine is not an exception. Unfortunately, due to its complicated viral biology in conjunction with the potential interference in antibody production between different serotypes, a consensus on the value of a neutralizing antibody response in dengue virus infections has not been reached [64,81,123]. The discrepancy may result from the fact that dengue is a timing disease; the lapse in time from the mosquito bite to the onset of the symptoms and the clinical presentation varies among infected individuals may dictate the outcomes of the disease and the analytic profiles [
account for this phenomenon? Nevertheless, the mystery of the low antibody levels observed in dengue patients suffering re-infection has not been addressed and investigated well. In order to improve accuracy in predicting and evaluating the performance of dengue vaccine in vaccinees, this critical area warrants further study. Answers to these questions would definitely improve dengue vaccine design and efficacy.
Despite the well-known fact on the importance of antibody in control of viral infections, very little information is available on the plasma cells, also called effector B cells, the antibody producing cells, in dengue patients. A review of the literature reveals conflicting information on the interaction of dengue virus with plasma cells or B cells in dengue patients and in dengue pathogenesis. Some reports advocate that B cells or plasma cells are permissive for dengue virus infection in vitro [29,48, 128-132], while others do not observe this feature [133, 134]. A few reports with samples taken from acute dengue patients suggest that viral antigens are associated with B-lymphocytes and that virus may replicate in these cells, a phenomenon that could explain the presence of atypical lymphocytes [135-137]. Another report indicates that a suppressor or inhibitor derived from dengue virus infection may contribute to the dysfunction of the B cells [
A properly functioning immune response to invaders or non-self substances is the result of the orchestration of a sophisticated network of signaling events coordinated by the immune system, which must balance these elements to prevent harm to the host. However, under certain conditions, the immune system can go amiss and attack its own components. Generally speaking, when an intruder’s components display molecular or structural similarity or are identical to its host, antibodies raised against these antigens can induce harmful consequences to the host. The immune system is constantly being challenged by infection. The intimate relationships between infectious agents and autoimmunity have been well-established. Thousands of infectious agents have been identified and numerous components within these pathogens mimic their hosts [71,72,141-146]. The components of dengue virus are not an exception; broadly cross-reactive antibodies to the viral antigens have been shown to react with a variety of host proteins [71,72,144-146]. However, exactly how these self reactive antibodies are generated in dengue virus infection remains to be further elaborated.
In the event of dengue virus infection, molecular mimicry has been implicated to contribute to dengue pathogenesis [144,145]. Conformational mimicry between proteins from platelets, endothelial cells, and coagulatory factors with those from dengue virus may explain the cross-reactive properties of anti-NS1, anti-prM, or anti-E antibody, which may participate in disease development [145,147]. Although the wide spectrum of dengue virus epitopes that exploit molecular mimicry have been investigated in great detail [144,146], the direct evidence that these peptides elicit cross-reactive T or B cell responses has not been provided. Thus more research needs to be done especially on this topic. Many questions abound, such as do the dengue virus-induced memory B or T cells provide agonists, antagonists or altered peptide ligands that can activate, block, or modulate effector functions? Do the avidities to the host protein and the viral mimicking epitope differ? Do pre-existing subclinical conditions make the activated memory B or T cells much easier to be aggravated upon re-infection with dengue virus? The actual incidence of autoimmune complications due to molecular mimicry in dengue patient is unknown and warrants further investigation. Nevertheless, the molecular mimicry observed in dengue virus infection should be considered an integral component to evaluating dengue vaccine safety.
Macrophages are one of the most important players in innate defense within the network of the immune system. They are bestowed with a dynamic set of mechanisms that are powerful at combating many types of pathogens under activated conditions. There are many subtypes of activated macrophages and each has its unique effector functions in dealing with infectious agents [148-157]. Considering the complexity of the operational immune system, all classes of activated macrophages are likely to be present in samples taken from infected individuals. However, the differential and proportional distribution of these subpopulations of activated macrophages during dengue virus infection has not yet been well-documented.
Dengue virus-induced disease has its unique features. One of the hallmarks of DHF/DSS is an acute vascular permeability syndrome accompanied by abnormalities in hemostasis. Although the underlying mechanisms in the development of this disease are unresolved, immunopathogenesis is thought to be a major factor [5,37,60]. However, DHF/DSS develops very rapidly, usually over a period of hours, and resolves within 1 to 2 days in patients who receive appropriate fluid resuscitation. No discernible sequelae are usually found. Therefore, an alternative scenario must exist. We have reported that dengue virus infection causes intense immune activation. Aberrant immune responses such as cytokine overproduction and generation of autoantibody acting against platelets and endothelial cells occur after dengue virus infection [71,145-147,158-160]. In addition, we have proposed that dengue virus can cause severe hemophagocytic syndrome [161,162]. In dengue patients, monocytes or macrophages could be activated by cytokines including IFNγ and TNF, dengue virus-immune complexes, and other stimuli. One of the likely functions of the activated macrophages is to implement the phagocytosis of the autoantibody-coated platelets and endothelial cells and thus contribute to the destruction of these uninfected cells. Anti-dengue antibodies become pathogenic and can enhance the dengue disease severity. The anti-NS1, anti-prM, and anti-E cross-reactive antibody action against platelets, endothelial cells, and coagulatory molecules provides an explanation for the targeted specificity and unique features of thrombocytopenia and plasma leakage observed during the development of DHF/DSS. We believe that this theory can explain the mechanistic steps of immunopathogenesis, and account for the specific characteristics of clinical, pathologic, and epidemiological observations that are unique in dengue virus-induced disease.
The autoantibody-associated macrophage activation has participated in many viral diseases [
The best known classical role of platelets is its importance as primary mediators of hemostasis, mainly in cooperation with other inflammatory processes. Cumulative information from the research has revealed an expansion of roles for platelets in unexpected areas, including a significant contribution in shaping and sharpening immune responses [164-168]. Recently, platelet expression of immunologically-related molecules that functionally modulate innate and adaptive immunity has been demonstrated [169-175]. One of the key aspects of platelets is their capability to bind leukocytes, especially monocytes, upon activation [176-178]. For example, platelet adhesive interactions with leukocytes and endothelial cells via P-selectin can lead to several pro-inflammatory events, including leukocyte rolling and activation, production of cytokine cascades, and recruitment of leukocytes to sites of tissue damage. Upon activation, platelets express on their surface CD154 protein that can induce the maturation of dendritic cells and regulate the adaptive immune response by stimulating Tand B-cell growth [
Obviously, other releasates from activated platelets are also present in the circulation of peripheral blood of dengue patients. Some of these factors may also be harmful to the host immune cells as well. One such molecule is polyphosphate (polyP), an inorganic, linear polymer of 60 - 100 phosphate residues linked by phosphoanhydride bonds [
Although multiple and diversified approaches to dengue vaccine development are in the pipeline at different stages of clinical trials, others are still under preclinical development. Challenges that have to be faced include but are not limited to reducing dynamic reactogenicity with a balanced and lasting immune response against all four dengue virus serotypes (the type of immunity that confers protection in natural infections), mitigating the potential immune-enhanced and autoimmune-mediated diseases, and development of a suitable animal model to evaluate candidate vaccines. Nevertheless, further understanding the factors that contribute to the dengue pathogenesis could diffuse some of the challenges and the concerns. Some essential points on future dengue vaccine developments are suggested.
The double-edged relationship between reactogenicity and immunity is a very intriguing topic in dengue virus infection [
Furthermore, the assays currently available to measure neutralizing antibodies are not suitable to identify the true protective homotypic antibody and cannot differentiate between type specific, heterotypic and cross-reactive antibodies [44,64,191,192]. This may be critically important in dengue endemic regions where the majority of populations already have pre-existing dengue antibodies to at least one dengue viral serotype, but the levels of neutralizing antibodies to each serotype are unknown. Therefore, one could envision that the enhanced vaccine reactogenicity may be a health threat when live attenuated tetravalent vaccine is administered to individuals from endemic countries with partial immunity. The results obtained from endemic regions demonstrate that the levels of pre-existing neutralizing antibodies against the infecting virus are not associated with viremia levels in secondary infection [
As aforementioned, why the majority of antibodies to dengue viral antigens are cross-reactive in nature, including to host proteins, remains a mystery. These antibodies may be beneficial, cross protecting the host from other flaviviruses, but are also likely harmful to the affected individual [194,195]. These unwanted cross-reactive antibodies could lead to some very serious situations-one dire consequence being death [63,196]. Some devastating effects have been documented, including autoantibody conjugating with circulating materials or inducing the release of deleterious substances from cells lined-up on the surface of capillaries; these events have the capacity to trigger hemorrhage and/or plasma leakage in dengue patients [72,73,145,147,197,198]. Currently, the magnitude of the potential side effects resulting from these autoantibodies, formed due to mimicry, has not been centered in the agenda of the current dengue vaccine developers. Hence, these potential adverse reactions have not been integrated as part of the efficacy evaluation index in tetravalent attenuated dengue vaccine clinical trials. Although investigations on the epitopes that can cause cross-reactive T or B cell responses has not been initiated, the molecular details of these protein sequences from viral antigens that elicit autoantibody production have been investigated very well [144,146, 199,200]. A strategy can be applied, such as modifying the coding sequence of the dengue viral genomes and altering these epitopes in the integrated virus strains to mitigate the potential side effects and enhance the safety profile of the dengue vaccines.
The importance of B cells, especially those constitutively producing antibody that control invading pathogens upon re-infection, has been well-established in vaccine regimens. However, antibody titers are low in patients who suffer from acute dengue in spite of high levels of pre-existing dengue specific antibodies. Also a paucity of detectable memory B cells in these patients during acute illness have been observed [
Dengue has become one of the most important mosquito-borne human diseases globally. Several measures have been applied and utilized to attempt to slow down or reduce the spread and the burden of the disease, but to no avail. Dengue seems to be a preventable disease considering the success achieved in vaccine design with other flaviviruses, such as yellow fever. Hence, a dengue vaccine appears to be an attainable effective method to control and prevent the spread of the dengue virus. With this notion, numerous dengue vaccines have been designed with a variety of approaches. Several of these dengue vaccines are currently going through clinical trials and some are still in pre-clinical development. Importantly, some of these dengue vaccines could be licensed within the next few years if all goes as planned [
We would like to thank the clinical staffs at the Division of Infectious Diseases in the Department of Pediatrics at the Siriraj Hospital, Mahidol University, Bangkok, Thailand. This study was supported by Emory SOM start-up fund (GCP) and Grants # NSC99-2321-B006-008 (HYL and YSL) from the National Science Council, Taiwan.