Abstract

Dengue virus is one of the most prevalent arborviruses that emerges fast around the world and imposes a substantial disease burden globally. The co-circulation of four serotypes of dengue viruses causes a large number of dengue fever and dengue hemorrhagic fever/dengue shock syndrome cases. As there is no specific antiviral treatment, an effective dengue vaccine is one of the most important tools to control the global spread of dengue disease. The development of dengue vaccine, however, is complicated by the interference among four dengue serotypes and an incomplete understanding of host immune responses. This review provides an overview of current work on host immunity against dengue viral infection. The knowledge will help to modify the existing vaccine design strategies and the development of novel dengue vaccine modalities in the future.

Overview

Dengue virus (DENV) is one of the most important flaviviruses transmitted by Aedes aegypti and Aedes albopictus mosquitoes in tropical and subtropical regions. Approximately one third of the world population are at risk of dengue virus infection. Dengue disease is endemic in over 100 countries, mainly in South-east Asia, Western Pacific, South America, and Africa [1-4]. It causes 50-100 million infections, and 500,000 hospitalizations each year [5,6]. Its rapid global expansion may have been accelerated by climate change, increased urbanization, globalization, and international travels [3,4].

DENVs are comprised of four serotypes, DENV-1, DENV-2, DENV-3, and DENV-4, based on their differences in neutralization and complement fixation tests. These viruses co-circulate in endemic regions. DENV has an approximately 10 kb single positive stranded RNA genome, from which one polyprotein is translated and processed into 10 viral proteins: Core (C), pr-membrane (prM), envelope (E), nonstructural protein 1 (NS1), NS2a, NS2b, NS3, NS4a, NS4b, and NS5 [7]. While the vast majority of dengue infections are asymptomatic, some show febrile illness named dengue fever (DF), among which a small proportion can also develop into life-threatening severe disease as manifested by vascular leakage, thrombocytopenia and shock, collectively called dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [8]. The risk of severe dengue disease increases in secondary infections [9,10].

Immune responses elicited by dengue virus infection

Once dengue viruses invade human bodies, innate immune responses, B cell responses, and T cell responses are triggered. Much effort had been placed on describing antibody responses to dengue virus in animal model, or in human subjects either during or afterrecovery from dengue viral infection. In contrast, innate and cellular immunity have not been as intensively studied.

During viral replication, dengue ssRNA is sensed by Tolllike receptor 7 (TLR7), while dengue viral dsRNA is recognized by TLR3, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation associated gene 5 (MDA5) [14-18]. The activation of such pathogen recognition receptors (PRRs) triggers downstream signaling pathways and stimulates the production of interferon α/β. Once produced, soluble interferons bind to IFN-α receptor (IFNAR), stimulates interferon-stimulated gene (ISG) transcription through Janus kinase/signal transducer and activators of transcription (JAK/STAT) signaling. ISG proteins such as 2′, 5′-oligoadenylate synthetase (2′, 5′ OAS), and interferon-inducible trans-membrane protein (IFITMs) are capable of inhibiting dengue virus infection [19,20]. in vitro studies indicate that dengue virus can inhibit type I IFN production by cleaving stimulator of interferon genes (STING) [21], which is an adaptor molecule of several PRRs in the cytoplasmic nucleic acid sensing pathway, activating downstream transcription pathway to induce type I IFN production [22,23]. The expression of dengue viral proteins NS2A, NS4A, NS4B or NS5, impairs JAK/STAT signaling by reducing the amount of phosphorylated STAT1 [24], or inducing degradation of STAT2 [25,26]. Dengue virus may also circumvent type I interferon response through an idiosyncratic Fc gamma receptor (FcγR) signaling pathway. When immune complexes ligate FcγR, two suppressive pathways are activated to interrupt IFN production: (1) deoxyadenosine kinase (DAK) and the autophagyrelated genes 5 and 12 (Atg5-Atg12) complex inhibit MDA5 and RIG-I pathways, respectively. And, (2) activation of sterile alpha and armadillo-motif- containing protein (SARM) and TNF receptor associated factor (TRAF) family member associated NF-кB activator (TANK) blocks the expression of TLR-3, -4,-7 [27,28].

Nevertheless, dengue virus fails to subvert host defensive mechanisms in immune competent mice, where total viral clearance is observed. Although using high titer viral infection and artificial infection route such as intracerebral injection help dengue virus to infect wild-type mice, symptoms of human dengue disease cannot be fully replicated [29-31]. Similarly, DHF/DSS are difficult to be reproduced in non-human primates or humanized mice, despite detectable viral replication in peripheral tissue and some signs of DF [32,33]. Other alternative models are to use mouse adapted dengue viral strains or immune deficient mice such as AG129, and STAT1-/- mice that are devoid of type I/II IFN receptors (AG129) or STAT1 (STAT1-/-) function. Infection with mouse adapted DENV-2 strains in AG129 or STAT1-/- mice replicate antibody dependent enhancement and to some extent the disease profiles observed in human DF and DHF/DSS [34-36]. Such in vivo evidence also suggests that IFN pathway is an essential component of host immunity to control dengue infection.

NK cell

Natural killer (NK) cells are part of innate immunity. It can recognize ‘self’ or ‘non-self’ patterns through the major histocompatibility complex I molecules expressed on cell surface. They lyse target cells by secreting cytotoxic granules that contain perforin and granzyme. Alternatively, they can stimulate the apoptotic receptor on target cell surface. However, these processes are normally inhibited, when the self MHC I molecules on target cell are detected by an inhibitory receptor on NK cells [37]. Additionally, NK cells mediate antibody dependent cell mediated cytotoxicity (ADCC) through FcγRIII on their surface [38]. Early activation of NK cells is important in the defense against primary dengue infection [30]. Conversely, dengue infection has been reported to up-regulate the cell surface expression of MHC I molecules and thereby inhibits NK mediated cell lysis [39]. Moreover, studies in a murine model of dengue viral infection showed that infiltrating NK cells are associated with the liver cell death [40]. Thus, the definitive role of NK cells in dengue viral infection requires further investigation

Complement activation has three cascades, classical, alternative, and lectin pathways. All the processes trigger subsequent cleavage of pro-proteins and generate a series of active factors to form a membrane attack complex (MAC), phagocytes are then recruited to lyse target cells [45]. Only the classical pathway requires specific antibodies to initiate the cascade. During dengue infection, soluble NS1 protein circulating in blood plays a vital role to modulate the complement pathways. DENV NS1 was found to interact with clusterin, a regulator that inhibits MAC formation [46]. NS1 antibody was necessary in the activation of complement pathway [42], possibly by blocking the interaction of soluble NS1 with other regulators or factors in the pathways besides the classical one. In fact, studies have demonstrated that NS1 inhibits classical and lectin pathway by binding to C4 directly [47]. Some complement splitting products, such as C3a and C5a, have activities of anaphylatoxin to increase vascular permeability [48-50]. Whether the modulation of complement pathways by NS1 and the possible aggregation of splitting products are responsible for the vascular leakage developed in DHF cases is yet to be clarified by direct evidence.

In summary, innate immunity plays different roles during dengue virus infection. It can be protective if it is stimulated at the early stage of infection, but pathogenic if it is activated too late.

The distribution of epitopes targeted by neutralizing, crossreactive or serotype specific antibodies is different in humans and mice. Early studies of dengue specific antibodies in mice showed that most of the strongly neutralizing monoclonal antibodies (MAb) are serotype specific and EDIII specific [51,52]. The main target regions include the lateral ridge and A strand that are partially overlapping within domain III [51-53]. Interestingly, studies on human MAb (hMAb) isolated from patient sera during dengue infection revealed only a few serotype specific neutralizing antibody, and the conserved EDII fusion loop is the major target of the serum antibodies [54]. EDIII antibody is not abundant in human sera, but it has the highest neutralizing potency among all other antibodies in sera [55]. These data have drawn question on whether anti-EDIII antibody is essential for the neutralizing ability of human sera. A study uses recombinant EDIII protein to adsorb EDIII antibodies has demonstrated that the depletion of EDIII hardly affected the neutralizing ability of human sera, indicating other vital neutralizing epitopes exist outside the domain III region [56,57]. In fact, several newly identified epitopes of human neutralizing antibodies are mapped to the EDII fusion loop, and antibody binding to which blocks virus fusion post entry [54,58]. Recent studies also showed that some flavivirus neutralizing antibodies can only be induced in humans with whole virion but not the ectodomain of E protein [59,60]. Two neutralizing antibodies had been found to recognize the complex structure of adjacent E dimers, including residues in the hinge region of EDI and EDII, which is only presented on an intact dengue virion surface, thus may have been missed in previous studies using purified protein to determine the antibody affinity or using linear epitopes [60].

Antibody response in primary and secondary infection

In a primary infection, dengue specific IgG antibody appears one week after the onset of fever, reaches the peak level within weeks, and then declines to a lower level that persists for decades. In contrast, in a secondary heterologous infection, IgG antibodies are detectable earlier (< Day 3 of fever) and at higher levels [71,72]. Although higher levels of antibodies are elicited earlier in a secondary infection, the majority of which are cross-reactive against the dengue serotype causing primary infection [73]. Such cross-reactive antibodies produced in a secondary infection may react with conserved epitopes, but generally bind to heterotypic virus inefficiently. In addition, the presence of such antibodies may also sequestrate new antigens that are needed for stimulation of naïve B cells. Consequently, B cell responses specific to the virus serotype in secondary infection are weaken and delayed. This phenomenon is called “original antigenic sin” [74-76]. Indeed, the highest neutralizing antibody titers in acute and convalescent sera from children experiencing secondary dengue infection are to the primary infection virus serotype [76]. A recent study of anti-EDIII antibodies in a cohort of patients during primary or secondary dengue infections also revealed stronger binding and enhancement activities against the primary infection DENV serotypes during secondary infections [77], thus providing support to the theory of “original antigenic sin”.

As most cross-reactive antibodies generated in primary infection do not effectively neutralize heterotypic dengue viruses in secondary infection, such “original antigenic sin” could partially contribute to increased risk of severe dengue disease in a secondary infection. Nonetheless, a recent study has reported that flavivirus group crossreactive antibodies isolated from patients with primary dengue infection are of low avidity and weak neutralization activity, whereas group cross-reactive antibodies from secondary infection have higher neutralization potencies compared to cross-reactive antibodies produced during primary infection [78]. Further clinical studies are needed to clearly define the similarity and difference between antibody responses during primary and secondary infections.

Cell immunity

In the course of infection, dengue viruses elicit high levels of T cells that are cross-reactive among four dengue serotypes. Whether these T cells cause pathogenesis or confer protection remains controversial.

CD8+ T cell response

In contrast to CD4+ T cells, a majority of CD8+ T cells target non-structural proteins, while a smaller proportion of them targets structural proteins [100]. CD8+ T cells, recognizing structural proteins, are generally serotype specific, while those that recognize non-structural proteins (predominantly NS3) are highly crossreactive [102]. Many studies have speculated that broadly crossreactive T cell responses contribute to the pathogenesis of dengue disease by triggering inflammatory cytokine storm or lysing virus infected cells directly. Indeed, a recent cohort study showed that a lower magnitude of HLA restricted CD8+ T-cell response is associated with an increased disease susceptibility [97], suggesting a protective role of CD8+ T cell response against dengue viral infection [97,103]. Furthermore, immunization with immunodominant CD8+ T cell epitopes enhanced viral clearance in experimental dengue viral infection in mice [104]. However, in another murine study, the infiltration of CD8+ T cells was associated with liver damage during dengue viral infection [40].

Similar to what has been observed for B cells, T cell response induced by dengue infection is also thought to be affected by the “original antigenic sin” [76,89]. Some data have showed that T cell responses induced during secondary infection are dominated by an expansion of T cells specific to the primary infection serotype [89,90,105], this may lead to diminished response to the virus causing the secondary infection and resulting in a less effective viremia control. However, in a recent study that screened human dengue specific T cell epitopes using overlapping peptides covering the full length of DENV genome, no differences were found between CD8+ T cell responses targeting serotype specific and conserved epitopes in terms of magnitude, phenotype, multifunctionality, or avidity, thus questioning whether an original antigenic sin exists in the context of dengue-specific T cell response [97,103].

In sum, for aiding rationale dengue vaccine design, both CD4+ and CD8+ T cell immunity need further in-depth analysis. Additionally, other factors such as host (human or mouse), primary/secondary infection, host HLA type, and sampling time must be taken into consideration

Current dengue vaccine development

To contain the spread of dengue virus around the world, researchers have tried to develop an effective dengue vaccine for years. From inactivated vaccines, live attenuated vaccines, recombinant vaccines to subunit vaccines and DNA vaccines, various candidates have been examined, and several general concepts of dengue vaccine design have been developed. First, it must be tetravalent to cover all four dengue serotypes. Second, it should elicit potent and balanced neutralizing antibody responses to all four dengue serotypes. And third, it must not induce enhancing antibodies. However, many unanticipated problems have been revealed in the process of dengue vaccine development. How to overcome these problems have become current goals of vaccine research.

The CYD23 recombinant vaccine had been made by swapping dengue virus prME genes with that of Yellow Fever (YF) viral genes on YF17D backbone. This vaccine has finished Phase IIb clinical trial and produced partial protection against 3 serotypes of dengue viruses [109]. The major problem of CYD23 is the lack of protection against DENV-2 in the endemic region where this serotype has the highest prevalence. One explanation offered by the trial investigators is the interference among four dengue viral serotypes in vivo when the four vaccine viruses were inoculated within one admixture. Another possibility is that antibodies elicited by the DENV-2 viral strain used in the vaccine could not neutralize the contemporaneous DENV-2 virl strain in Thailand [109,110]. Additionally, compare to live attenuated viruses or chimeric DENVs, CYD23 only incorporated dengue viral structural antigens, most of which stimulate B cell and CD4+ T cell responses. As the protective role of dengue specific CD8+ T cells has been implicated in recent studies [97], CD8+ T cell epitopes may have to be added to a dengue vaccine.

Structural proteins including prM from all four dengue serotypes were included in all aforementioned live attenuated or recombinant vaccines, because they are the main antigens recognized by neutralizing antibodies. However, more recently, evidences support prM protein elicits cross-reactive antibodies and facilitates the infection of immature virus were reported [61,106-108]. A new E85 vaccine that has removed prM and retained ectodomain of E protein and expressed in alphavirus vector, has been shown to induce robust protective immunity against dengue virus infection in a macaque model of dengue viral infection [111]. Direct comparison between vaccines with or without prM with respect to immunogenicity and protective efficacy will help to determine whether prM is a necessary component in a dengue vaccine

Subunit vaccine and DNA vaccine

Subunit protein vaccines and DNA vaccines have been explored more recently. Different from attenuated vaccines, subunit vaccines and DNA vaccines avoid the potential risk of virulence caused by reversion virus, have fewer adverse effects, and can be produced outside a biosafety level III laboratory.

Obstacles in Dengue Vaccine Development

Intra-serotype variation

Current tetravalent dengue vaccines have been designed based on the assumption that neutralizing antibodies induced by one strain of virus can protect people from infection by any viral strains within the same serotype. Although from cohort studies, it is observed thatinfection by one serotype can protect patient from re-infection by the same serotype of virus for decades or a lifetime [121], different neutralization activity was observed for different viral genotypes. It is common to find that one monoclonal antibody cannot neutralize a large panel of viruses from the same serotype [53,121], and the result of neutralization test also depends on the panel of viruses selected. To obtain a tetravalent vaccine with broad coverage, representative strain for each of the four components needs be selected at an early stage of vaccine development.

Future Perspective of Rationally Designed Dengue Vaccines

T cell vaccine

Before recent studies indicating a protective role of CD8+ T cells against dengue viral infection, T cell immunity had been to a large extent neglected as it was thought to be associated with viral pathogenesis. In view of the failure of chimeric vaccine CYD23, which was designed principally to stimulate neutralization antibodies and did not include nonstructural proteins that can stimulate T cells, a new focus on T cell immunity in dengue vaccine development may be needed. Inclusion of antigens capable of eliciting T cell response in a vaccine (besides live attenuated dengue viruses), or selection of optimal T cell epitopes for a T cell vaccine, may help to create a better dengue vaccine.

The roles of antigen presenting cells (APC) in dengue vaccine design

When dengue virus is transmitted to humans through mosquito bite, the first infected cells are thought to be Langerhans cells, which are specialized skin dendritic cells (DCs) [123]. In experimental models, dengue viruses enter immature DCs via dendritic cell specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). Upon maturation, DC-SIGN is down regulated and Fcγ receptors are upregulated. Through Fcγ receptor, mature DCs facilitate ADE in a secondary infection [124]. Dengue infection of monocyte derived DC (mDC) and plasmacytoid DC (pDC) lead to a diminished upregulation of MHC and co-stimulatory molecules [125], and render them incapable of priming CD4+ Th1 cells [126]. In an attempt to initiate stronger cellular and humoral protective immunity against dengue virus, some have started to test a new vaccine strategy by delivering dengue antigens to dendritic cells directly [127]. Whether such a manipulation can improve the balance and potency of dengue specific immune responses is yet to be tested experimentally.

Summary

After a half century’s basic research and clinical trials, the mechanisms underlying dengue protective immunity have been gradually revealed, these knowledge provide foundation for an improvement in the design of future dengue vaccines. Besides traditional human pathogen based vaccine and antiviral drugs, expanded knowledge of flavivirus infection in mosquitoes [128-132], modulation of dengue infection in mosquitoes through infection by Wolbachia bacterium or genetic modification [133-135], will also provide additional novel strategies to control dengue virus transmission. The more we know, the further we can move forward in the direction of making an ideal dengue vaccine. Based on current knowledge of DENV specific immunity, either induced by natural viral infection or elicited by candidate vaccines, new dengue vaccine design and modification of current vaccine candidates can be made oeventually obtain an efficacious human dengue vaccine.

Acknowledgements

This study is supported in part by a research grant “NSFC 81071359 and NSFC 81128008” from National Science Foundation of China (No.31300757).

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