3. Pathogenesis of Dengue virus infection

movement of people (and consequently viruses), international travel and breakdown in public health infrastructure, and vector control programs. The actual numbers of dengue cases are underreported and many cases are misclassified. The prevalence of dengue is estimated at 3.9 billion people who are at risk of infection in 128 tropical and subtropical countries, mainly southeast and south Asia, Central and South America, and the Caribbean. A recent estimate indicates 390 million dengue infections per year (95% credible interval 284–528 million), of which 96 million (67–136 million) manifest clinically (with any severity of disease), with an estimated 500,000 cases each year of life-threatening disease in the form of severe dengue—including dengue hemorrhagic fever and dengue shock syndrome mostly in the pediatric population, with about 20,000 succumbing to it, and is the leading cause of childhood death in many countries [1– 3]. Dengue is associated with considerable social, economic, and political consequences caused by urban epidemics, such as those seen in Delhi (1996), Cuba (1977–1979 and 1997), Taiwan (2002), and Brazil (2008). Furthermore, Dengue is currently also a major cause of morbidity in American and European travelers and military personnel [4]. This disease places a high economic burden on both governments and individuals; for instance, in America, Dengue illness costs US\$2.1 billion per year on average, excluding vector control, exceeding costs of other viral illnesses. In addition, in Southeast Asia, there is an estimate of 2.9 million dengue episodes and

5906 deaths annually, with an annual economic burden of \$950 million [3].

Dengue virus infections encompass a range of well-described clinical illnesses ranging from an asymptomatic infection to a self-limiting febrile illness, dengue fever, to severe dengue (shock and death), a clinical syndrome that typically presents with capillary permeability and can lead to dengue shock syndrome and dengue hemorrhagic fever. Among less common presentations of severe dengue are encephalitis, hepatitis, and renal dysfunction [4]. Infection by any dengue virus requires a 4- to 8-day incubation period and can produce a wide spectrum of illnesses, the majority of these being asymptomatic or subclinical. Although most patients are able to recover after a self-limiting (yet debilitating) illness, a small proportion develops a severe form of the disease, which is mainly characterized by plasma leakage with or without bleeding [3]. The acute illness is usually benign and self-limiting. Moreover, a secondary infection, corresponding to a subsequent infection with a different serotype is also characterized by acute fever and several other nonspecific signs and symptoms, usually indistinguishable from a range of other illnesses. However, in 2–3% of secondary infections with another serotype there is a higher risk of increased disease severity, causing life-threatening Dengue with Warning Signs (DWS+) and Severe dengue (SD), according to the revised WHO dengue case classification (DENCO) [2, 5]. Serotype-cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE) [6, 7]. Dengue without Warning Signs (DWS) is more often observed in adults and adolescents and can manifest with only a mild fever only or a more disabling disease. This latter form is characterized by symptoms occurring mainly in the early febrile stage, such as the sudden onset of high fever, severe headache, retroorbital pain, myalgia, arthralgia, and rash. In the critical phase, the skin is flushed with the appearance of a petechial rash, occurring predominantly around the time of defervescence,

2. Natural clinical evolution

146 Dengue Fever - a Resilient Threat in the Face of Innovation

The pathophysiological basis for severe dengue is multifactorial, resulting from a complex interaction between the virus, the host, and, at least in part, immune-mediated mechanisms. Individuals with primary Dengue virus infection induce a lifelong protective immunity to the infecting serotype, accompanied by a short-term cross-immunity against other serotypes and development only leads to self-limited dengue fever characterized by high fever and debilitating joint pain, recovery from infection by one provides lifelong immunity against that serotype. However, subsequent infection with a different dengue serotype is more likely to cause DWS+/SD, and cross-immunity to the other serotypes after recovery is only partial and temporary. Epidemiological data suggest an increased risk of DWS+/SD in people with preexisting heterotypic dengue virus antibodies, and this has led to research in dengue pathogenesis focusing on the subsequent infections by other serotypes and an increased risk of developing severe dengue. Accordingly, antibodies against a given serotype can cross-react with, but not cross-protect against, the remaining three virus serotypes [4] during secondary infections. Immunopathological mechanisms have been proposed, such as the immune system improvement phenomenon (immunopotentiation), which contribute to the increased risk of DWS+ or SD. It is widely accepted that these cross-reactive antibodies can promote enhanced uptake of the heterologous virus into host cells, precipitating a hyperimmune reaction resulting in blood vessel leakage and potentially fatal hypovolemic shock [1, 2]. This increase occurs when nonneutralizing antibodies resulting from the primary infection favor the invasion of the second virus with a different serotype into the target cells, a phenomenon known as antibody-dependent amplification or antibody-dependent enhancement (ADE) [8, 9]. Subsequent infection with a different dengue serotype is more likely to cause DWS+/SD [8, 9]. According to the ADE hypothesis, a primary infection with DENV produces an insufficient concentration of antibodies or avidity to neutralize a secondary infection by DENV of a different serotype that differs in its amino acid sequence by 30–35%; and these sub-neutralizing antibody concentrations can promote infection in such cells by facilitating Fc-receptor-mediated entry [10]. These concentrations could be enough to opsonize the secondary virus and target it for Fc-receptormediated endocytosis into myeloid cells such as monocytes and macrophages (which constitute the main site of DENV replication) and in this manner promote higher viral loads. ADE can be observed in vitro and has also been proven to drive higher viral loads of DENV in animal models [11]. The following findings support the hypothesis of the antibody-dependent enhancement (ADE): undiluted sera obtained early from patients with secondary infection was shown to enhance dengue virus infection in vitro, infants born from dengue-immune mothers displayed a higher viral burden than infants born to dengue nonimmune mothers and further demonstrated immune activation associated with disease severity, and lethal antibodydependent enhancement has been shown in dengue mouse models, and virus-antibody complexes bind to Fcγ receptor-bearing cells, resulting in increased infected cell mass and a rise in viremia. The cardinal feature of DWS+ is plasma leakage believed to arise from proinflammatory cytokine-inflicted damage to the vascular endothelium. Although the cause of severe form of dengue infection is not clear, it has been revealed that secondary infection with a different serotype of DENV, or even a homotypic reinfection, are major risk factors for SD probably due to antibody-dependent enhancement. The phenomena of original antigenic sin, as well as immune evasion that inhibits interferon (IFN)-α and IFN-β signaling by suppressing Jak-Stat activation, a cytokine storm, and autoimmune responses are thought to contribute to the pathogenesis of severe form of dengue disease [12, 13], see Figure 1. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The proteolytic activity of the NS2B3 protease complex of DENV allows it to function as an antagonist of type I IFN production. Other DENV proteins that antagonize type I IFN signaling include NS2A, NS4A, NS4B, and NS5 by targeting different components of this signaling pathway, such as STATs [15]. During a primary infection, serotype-specific as well as cross-reactive memory T-cell responses are produced. On the other hand, during a secondary dengue virus infection, viral epitopes expressed on infected cells trigger activation of serotype-cross-reactive memory T-cells, with the production of pro-inflammatory cytokines. The latter ultimately lead to plasma leakage in the vascular endothelium. The specific cellular

response against DENV begins with the activation of CD4+ T-cell during viremia and subsequently the activation of CD8+ T-cell. In individuals with DHF or DWS+ due to secondary infections, the presence of CD4+/CD8+ memory T-cell and cytotoxic CD4+/CD8+ T-cell has been demonstrated [16], and the activation of T-cell and the production of cytokines are important factors in the pathogenesis of DWS+ [17]. Likewise, the cellular immune response, in the case of DWS+, exacerbates the activation and release of cytokines, which is related to the greater severity of the clinical picture. The activation of the complement system has also been demonstrated in DHF, and high concentrations of C3 and C1q proteins can be detected in severe cases. It is suggested that complex virus-circular antibodies could activate the cascade reaction of the complement [18], see Figure 2. Regulatory immune pattern in homologous versus a pro-inflammatory pattern in heterologous dengue virus secondary infection has been reported. Several soluble factors produced by T cells, monocytes, macrophages, and mast cells have been proposed to increase vascular permeability in primary endothelial cells. These factors include TNFα, interleukin 6, interleukin 8, interleukin 10, interleukin 12, macrophage migration inhibitory factor, HMGB1, MCP-1, and matrix metalloproteinases. Endothelial permeability can also be influenced by the maturation state of NS4B, which modulates the cytokine response in monocytic cell lines. In addition, secreted NS1 protein, along with anti-NS1 antibodies and complement activation, might be involved in dengue virus-induced vascular leakage. Moreover, around defervescence, when plasma leakage is apparent, high levels of complement activation products C3a and C5a are detected in plasma, followed by accelerated consumption and large reduction of complement components in patients with dengue shock syndrome. Activation of the complement system can stimulate the production of inflammatory cytokines associated with DWS+/SD, and trigger local and systemic effects implicated

Current Status of Vaccines against Dengue Virus http://dx.doi.org/10.5772/intechopen.80820 149

Figure 2. Schematic representation of the immunopathogenesis of severe dengue disease. Adapted from Webster et al.

[4].

Figure 1. A hypothetical model of dengue pathogenesis. Viral and immunological factors contribute to clinical manifestations, including severe hemorrhage, thrombocytopenia, plasma leakage, hepatomegaly, and neurological compromise. DV: dengue virus. Adapted from Wan et al. [14].

response against DENV begins with the activation of CD4+ T-cell during viremia and subsequently the activation of CD8+ T-cell. In individuals with DHF or DWS+ due to secondary infections, the presence of CD4+/CD8+ memory T-cell and cytotoxic CD4+/CD8+ T-cell has been demonstrated [16], and the activation of T-cell and the production of cytokines are important factors in the pathogenesis of DWS+ [17]. Likewise, the cellular immune response, in the case of DWS+, exacerbates the activation and release of cytokines, which is related to the greater severity of the clinical picture. The activation of the complement system has also been demonstrated in DHF, and high concentrations of C3 and C1q proteins can be detected in severe cases. It is suggested that complex virus-circular antibodies could activate the cascade reaction of the complement [18], see Figure 2. Regulatory immune pattern in homologous versus a pro-inflammatory pattern in heterologous dengue virus secondary infection has been reported. Several soluble factors produced by T cells, monocytes, macrophages, and mast cells have been proposed to increase vascular permeability in primary endothelial cells. These factors include TNFα, interleukin 6, interleukin 8, interleukin 10, interleukin 12, macrophage migration inhibitory factor, HMGB1, MCP-1, and matrix metalloproteinases. Endothelial permeability can also be influenced by the maturation state of NS4B, which modulates the cytokine response in monocytic cell lines. In addition, secreted NS1 protein, along with anti-NS1 antibodies and complement activation, might be involved in dengue virus-induced vascular leakage. Moreover, around defervescence, when plasma leakage is apparent, high levels of complement activation products C3a and C5a are detected in plasma, followed by accelerated consumption and large reduction of complement components in patients with dengue shock syndrome. Activation of the complement system can stimulate the production of inflammatory cytokines associated with DWS+/SD, and trigger local and systemic effects implicated

enhancement (ADE): undiluted sera obtained early from patients with secondary infection was shown to enhance dengue virus infection in vitro, infants born from dengue-immune mothers displayed a higher viral burden than infants born to dengue nonimmune mothers and further demonstrated immune activation associated with disease severity, and lethal antibodydependent enhancement has been shown in dengue mouse models, and virus-antibody complexes bind to Fcγ receptor-bearing cells, resulting in increased infected cell mass and a rise in viremia. The cardinal feature of DWS+ is plasma leakage believed to arise from proinflammatory cytokine-inflicted damage to the vascular endothelium. Although the cause of severe form of dengue infection is not clear, it has been revealed that secondary infection with a different serotype of DENV, or even a homotypic reinfection, are major risk factors for SD probably due to antibody-dependent enhancement. The phenomena of original antigenic sin, as well as immune evasion that inhibits interferon (IFN)-α and IFN-β signaling by suppressing Jak-Stat activation, a cytokine storm, and autoimmune responses are thought to contribute to the pathogenesis of severe form of dengue disease [12, 13], see Figure 1. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The proteolytic activity of the NS2B3 protease complex of DENV allows it to function as an antagonist of type I IFN production. Other DENV proteins that antagonize type I IFN signaling include NS2A, NS4A, NS4B, and NS5 by targeting different components of this signaling pathway, such as STATs [15]. During a primary infection, serotype-specific as well as cross-reactive memory T-cell responses are produced. On the other hand, during a secondary dengue virus infection, viral epitopes expressed on infected cells trigger activation of serotype-cross-reactive memory T-cells, with the production of pro-inflammatory cytokines. The latter ultimately lead to plasma leakage in the vascular endothelium. The specific cellular

148 Dengue Fever - a Resilient Threat in the Face of Innovation

Figure 1. A hypothetical model of dengue pathogenesis. Viral and immunological factors contribute to clinical manifestations, including severe hemorrhage, thrombocytopenia, plasma leakage, hepatomegaly, and neurological compromise.

DV: dengue virus. Adapted from Wan et al. [14].

Figure 2. Schematic representation of the immunopathogenesis of severe dengue disease. Adapted from Webster et al. [4].

in intravascular coagulation. Finally, although controversial, the role of autoimmunity in the pathogenesis of dengue is mentioned, as autoantibodies resulting in platelet and endothelial cell dysfunction might be involved in severe dengue pathogenesis. Not all cases of DWS+ occur in people who experience a secondary infection, since in some cases the virus' own virulence, added to the characteristics of the host, leads to the complication of the disease [19], which may be due to the presence of antibodies against viral proteins that have crossreactivity with platelets and coagulation factors [18]. Certain nonstructural proteins such as NS1, NS2, and NS3 appear to have a certain structural homology with coagulation factors, platelets, integrins, and adhesins of human endothelial cells, allowing the activation of autoreactive T lymphocytes that participate in the pathology of dengue [16, 20]. Anti-NS1 antibodies correlate with disease severity, and cross-reaction of anti-NS1 antibodies with liver and endothelial cells are also implicated in affecting the integrity of the vascular endothelium and platelets has been proposed to trigger these cells to express nitric oxide and undergo apoptosis [2, 3, 14]. Certain antibodies to some E protein epitopes can bind to human plasminogen and inhibit plasmin activity (see Figure 3). Recently, it has been reported that Tropomyosin (TPM)-1 may play an important role in the pathogenesis of SD. It is plausible that the elevation of TPM-1 in the plasma of SD patients can be due to excessive cell death, thereby releasing TPM into the circulation as DAMPs, and leading to mast cell activation. Moreover, the insulin pathway may play a role in the pathogenesis of SD, hence, regulating the insulin signaling pathway may be a key intervention to reduce plasma leakage in patients with SD

Current Status of Vaccines against Dengue Virus http://dx.doi.org/10.5772/intechopen.80820 151

A protective versus pathological outcome depends on the balance between the host's genetic and immunological background and viral factors. Vaccine development has been slowed by fears that immunization might predispose individuals to the severe form of dengue infection [3, 4]. There are four distinct, but closely related, serotypes of the virus that cause dengue

No DENV-specific therapies are available, while a DENV vaccine that elicits protection in people with prior DENV exposure but not in naive individuals and that is not equally protective against all four serotypes has recently begun to be licensed on a country-by-country basis. This is mostly due to an incomplete understanding of the interplay between viral and host factors that contribute to DENV pathogenesis. On the virus side, some DENV lineages are more virologically and epidemiologically fit than others and are thus associated with DWS+/ SD manifestations. On the host side, DENV infection history is the primary determinant associated with the development of more severe dengue disease, with potential contributions

Several studies have demonstrated that DENV-specific antibodies can protect against infection and, under certain conditions, enhance infection and disease severity, whereas the role of T cells remains unclear. Thus, to avoid the risk of enhancement, a safe vaccine against dengue virus will need to confer protective immunity against all four serotypes [10]. Consequently, the adaptive immune response to dengue can be both protective and pathogenic, which compli-

Dengue virus is widespread throughout the tropics, representing an important, rapidly growing public health problem with an estimated 2.5–3.9 billion people at risk of dengue fever and the life-threatening severe dengue disease. Therefore, the need for a safe and effective vaccine for dengue is immediate. Vaccine development has been slowed by fears that immunization might predispose individuals to the severe form of dengue infection [4]. The characteristics and challenges that the ideal vaccine for the dengue virus must have are described in the following.

• Rapid immunization regime requiring a single vaccine or two that fit in with established

4.1. Characteristics of an ideal dengue vaccine and challenges to its development

• Avoids ADE (antibody-dependent enhancement) and pathogenesis

[12], see Figures 1–3.

4. Dengue vaccines

4.1.1. Characteristics

vaccine programs

• Safe in children and adults [3, 4]

(DEN-1, DEN-2, DEN-3, and DEN-4).

from other factors such as genetic variation, age, and sex.

cates vaccine development, as discussed in this chapter.

Figure 3. A schematic model of autoantibody-mediated immunopathogenesis in DENV infection. Molecular mimicry between platelets, endothelial cells, and coagulatory molecules with NS1, prM, E, and C proteins underlies the crossreactivity of anti-NS1, anti-prM, anti-E, and anti-C Abs, respectively, to host proteins. Abs Z antibodies; C Z capsid protein; DENV Z dengue virus; E Z envelope protein; NS Z nonstructural protein; prM Z precursor membrane protein. Adapted from Wan et al. [14].

signaling pathway may be a key intervention to reduce plasma leakage in patients with SD [12], see Figures 1–3.

A protective versus pathological outcome depends on the balance between the host's genetic and immunological background and viral factors. Vaccine development has been slowed by fears that immunization might predispose individuals to the severe form of dengue infection [3, 4]. There are four distinct, but closely related, serotypes of the virus that cause dengue (DEN-1, DEN-2, DEN-3, and DEN-4).

No DENV-specific therapies are available, while a DENV vaccine that elicits protection in people with prior DENV exposure but not in naive individuals and that is not equally protective against all four serotypes has recently begun to be licensed on a country-by-country basis. This is mostly due to an incomplete understanding of the interplay between viral and host factors that contribute to DENV pathogenesis. On the virus side, some DENV lineages are more virologically and epidemiologically fit than others and are thus associated with DWS+/ SD manifestations. On the host side, DENV infection history is the primary determinant associated with the development of more severe dengue disease, with potential contributions from other factors such as genetic variation, age, and sex.

Several studies have demonstrated that DENV-specific antibodies can protect against infection and, under certain conditions, enhance infection and disease severity, whereas the role of T cells remains unclear. Thus, to avoid the risk of enhancement, a safe vaccine against dengue virus will need to confer protective immunity against all four serotypes [10]. Consequently, the adaptive immune response to dengue can be both protective and pathogenic, which complicates vaccine development, as discussed in this chapter.
