Host Immune Response to Dengue Virus Infection: Friend or Foe?

Abstract

DENV belongs to the Flaviviridae family and possesses a single-stranded RNA genome of positive polarity. DENV infection manifests in mild subclinical forms or severe forms that may be dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Despite a lot of effort worldwide, the exact mechanism underlying the pathogenesis of severe DENV infection remains elusive. It is believed that both host and viral factors contribute to the outcome of dengue disease. The host factors are age at the time of infection, sex, nutrition, and immune status, including the presence of pre-existing antibodies or reactive T cells. Viral factors include the serotype, genotype, and mutation(s) due to error-prone RNA-dependent polymerase leading to the development of quasispecies. Accumulating bodies of literature have depicted that DENV has many ways to invade and escape the immune system of the host. These invading strategies are directed to overcome innate and adaptive immune responses. Like other viruses, once the infection is established, the host also mounts a series of antiviral responses to combat and eliminate the virus replication. Nevertheless, DENV has evolved a variety of mechanisms to evade the immune system. In this review, we have emphasized the strategies that DENV employs to hijack the host innate (interferon, IFN; toll-like receptors, TLR; major histocompatibility complex, MHC; autophagy; complement; apoptosis; RNAi) and adaptive (antibody-dependent enhancement, ADE; T cell immunity) immune responses, which contribute to the severity of DENV disease.

1. Introduction

The dengue virus (DENV) infection is a major public health threat in tropical and subtropical regions of the world [1,2]. Most of the global populations inhabiting dengue-endemic areas are exposed to DENV infection each year. For instance, DENV is endemic in more than 100 countries, and reported estimates predict about 390 million infections per year [2]. The WHO has reported that there are 50–100 million dengue infections per year worldwide, with the greatest impact on children [3]. Based on the amino acid similarity, four different serotypes of DENV (Figure 1) (DENV1, DENV2, DENV3, and DENV4) have been reported [4]. Furthermore, based on the phylogenetic analysis, each of these serotypes is further classified into different genotypes. Each DENV serotype consists of four to six genotypes (with varying terminology between the authors) that differ from one another by ≤10% at the amino acid level across the envelope protein [5,6,7]. DENV-1, DENV-2, DENV-3, and DENV-4 can be divided into five (I, II, III, IV, and V), six (Asian I, Asian II, Cosmopolitan, American, American/Asian, and Sylvatic), four (I, II, III, and V), and four (I, II, III, Sylvatic) genotypes, respectively [6,8,9,10]. These genotypes originate from different locations and at different times due to mutations and have substantially contributed to the pathogenicity of the DENV infection [11].

DENV is a member of the Flaviviridae family. Flaviviruses (FV) are transmitted by arthropod vectors. The other medically important members of this family include Zika virus (ZV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLEV), and tick-borne encephalitis (TBE) viruses [14,15,16,17]. Aedes mosquitoes act as biological vectors and aid in DENV transmission (Figure 2) [18]. The alarming rate of the DENV burden calls for proper diagnosis and treatment of infection. Clinical presentation of DENV infection involves a broad spectrum of symptoms and is divided into three phases: the initial (febrile) phase, the critical phase, and the recovery phase [19] (Figure 3). Increasing research studies have implicated the involvement of nearly all organs as targets of DENV [20,21,22,23,24,25,26,27,28].

Despite enormous efforts by virologists worldwide, the pathogenesis of DENV is still elusive [35,36,37]. The outcome of DENV infection is the result of the interaction between the virus and host factors. Viral factors augmenting the manifestations include the serotype and mutations in the structural and non-structural proteins; the host factors include the age and immune profile of the host [35,38]. Reports have demonstrated the importance of the immune system in the pathogenesis and outcome of DENV infection [35,38]. In this review, we emphasized the various ways in which the host mounts an immune response following DENV infection, and the various strategies employed by DENV to evade the host and their deleterious consequences.

2. Dengue Virus (DENV)

The dengue virus has a small spherical (50 nm) structure composed of a lipid envelope and a nucleocapsid core that encapsulates the single-stranded positive RNA genome of approximately 10.7 kb [39] (Figure 1). Moreover, the genome consists of a single long open reading frame, flanked by type 1 capped 5′- and 3′ tail-less-untranslated regions (UTRs) that encode a single polyprotein. This polyprotein is cleaved co- and post-translationally to yield mature structural and non-structural proteins [40] (Figure 4). The structural proteins include the envelope (E), membrane (M), and capsid (C) proteins, and the non-structural proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [41,42]. All of these proteins have varied functions in viral replication and virulence, as illustrated in

Table 1. Dengue virus proteins in replication and virulence.

	Proteins	Functions	References
Structural	E	DENV receptor binding and fusion site	[43]
	consists of three domains (I, II, III)	host range, tropism, virulence
	prM	forms protruding trimers with E giving a spiky appearance	[44]
	part of immature virions	form a cap that prevents the premature fusion of E before virus release
	M	arrangement/maturation	[43]
	sits below the E protein		[44]
	C	Nucleocapsid formation	[45]
	Homo-dimeric protein	encapsidation of virus
Non-Structural	NS1	viral replication	[46]
	dimer in the initial stage and hexamer in the latter stage	interacts with NS4A, and NS4B to form a virus replication complex (RC)	[46,47]
	occurs in many different forms (membrane-bound, soluble form, NS1′)	interacts with E and prM to facilitate the production of infectious virions	[48]
			[49]
			[50]
	NS2A	RNA synthesis	[51]
	transmembrane protein	RNA packaging/virion assembly	[49]
			[50]
	NS2B	interacts with NS3 to make it functional	[52]
	membrane-associated protein	a cofactor in the structural activation of the DENV serine protease of NS3	[53]
			[53,54]
	NS2B3 protease complex	cleaves hSTING	[55]
	NS3	polyprotein and host protein cleavage at specific site	[52]
	bifunctional with protease activity at the N terminus while helicase activity at the C terminus	unwinding the RNA duplex during replication	[56]
	NS4A	membrane alterations necessary for viral replication	[53]
	integral membrane protein		[54]
	NS4B	interacts with NS3 helicase domain	[56]
	integral membrane protein		[53]
			[54]
	NS5	5′ capping of new viral genomes	[57]
	bifunctional with the N terminus as methyl transferase and C terminus as RNA-dependent RNA polymerase		[54]

.

The viral glycoprotein is composed of the envelope (E) and the pre-membrane (prM) proteins. The envelope (E) protein is organized into three domains (EDI, EDII, and EDIII) that are attributed to different roles [58]. Likewise, EDI facilitates the conformational change that is required for virus penetration. The fusion loop (FL) is in the EDII domain, which is essential for the fusion of the virus with the host cell membrane. The last domain, EDIII, interacts with the host-receptor molecule [59,60,61,62,63]. The prM protein consists of the ‘pr’ peptide followed by the surface structural M protein, which has a very crucial role in virion maturation and assembly. The function of the prM protein is to hide the fusion loop (FL) of the EDII to prevent premature fusion. In the last step of the maturation, at the trans-Golgi site, prM is cleaved by the host-encoded furin. This cleavage leaves behind a peptide (pr) that still covers the FL. Once ejected from the infected cells, the pr peptide is detached from the virion, which is now a mature virion [64].

The seven non-structural proteins of DENV are designated as NS1, NS2A, NS2A, NS3, NS4A, NS4B, and NS5. It is established that most of these proteins have functions in viral replication and host immune evasion. Likewise, during the viral replication cycle, a replication complex is formed by the association of different NSPs with host co-factors on interconnected lipid vesicles derived from the ER [65,66,67,68]. Among the seven NS proteins, it has been reported that NS1 has a major role in viral replication. Moreover, the importance of the latter has been elucidated by deleting NSI from the genome, resulting in the suppression of the replication cycle [46]. NS1 occurs in two different forms (membrane-bound and soluble) at varied locations in the infected cells that govern the vitality of NS1 among NS proteins [69,70]. An extended form of NS1, designated as NS1’, has been reported from the extracellular space during the replication of DENV and other FVs like WNV and JEV [71,72]. Laboratory cultures of infected mammalian and insect cells also demonstrate the occurrence of a secreted form of NS1′ [71,73]. Moreover, NS2 is further subdivided into NS2A and NS2B. NS2A participates in viral physiology by ameliorating viral RNA synthesis and facilitating the virion assembly [51]. NS2B interacts with NS3 to function as viral proteases [52].

NS3 proteins have a multifunctional role, as NS3 demonstrates chymotrypsin-like serine protease activity, RNA helicase activity, and RTPase/NTPase activity [53,54]. The N terminus of NS3 has protease activity, while the C terminus is harnessed with helicase characteristics, both of which are crucial in viral replication [52,56]. Another protein bestowed with enzymatic activity is NS5, which is the largest and most highly conserved NS protein among the flaviviruses [74,75]. NS5 has a significant role as a bifunctional enzyme that works as RNA-dependent RNA polymerase (RdRp) and methyltransferase located at the N terminus which facilitates the 5′-RNA capping of the naive viral genomes [14,57].

3. Pathogenesis

The pathogenesis of DENV is the outcome of both the host and the virus factors [76] (Figure 5). It has been reported that cell and tissue tropism have a pivotal role in facilitating DENV infection [77]. Elevated viremia is the hallmark of severe dengue disease and indicates the involvement of different organs, such as the liver and brain [78]. Furthermore, the immune system, liver, and endothelial cell lining of the blood vessels are the major key players underlying the outcome of DENV pathology [79]. DENV is inoculated into the host’s skin by a bite from an infected mosquito, which initially replicates in the skin cells, such as keratinocytes and Langerhans cells [80,81]. One in vitro study directed at delineating the cell population infected by DENV revealed the productive infection of CD14(+) and CD1c (+) DCs, Langerhans cells, and dermal macrophages [82]. After infection, these cells make their way toward the lymph nodes, where they infect the monocytes and macrophages [79]. Moreover, through amplification and dissemination into the lymphatic vessels, many different cells are infected which include several cells of the mononuclear lineage, including blood-derived monocytes [83], myeloid DC [84,85,86,87], and splenic and liver macrophages [20,88,89,90,91]. Furthermore, studies incorporating murine models enumerated that blood-resident mononuclear cells and cells dwelling in the spleen, lymph nodes, and bone marrow stromal cells are also targets of DENV [92,93]. This infection triggers some host factors that work in agreement to combat the infection. The host-derived defensive apparatus is a form of proinflammatory, anti-inflammatory cytokines, and chemokines [83,94].

Studies involving human patients, non-human primates, and mice have been directed to delineate the organ tropism of DENV. Some scientific studies tried to unravel the fate of DENV in various organs in humans and revealed that following severe DENV infection, the infectious virions were isolated from cells derived from skin [21], liver [20,22,23,24,89,90], lymph node [22], kidney [23,25,90], bone marrow [22,25,26,90], lung [22], thymus [27], and brain [28]. Furthermore, non-human primates demonstrated the presence of DENV infectious particles in the skin and gastrointestinal tract, spleen, thymus, and several peripheral lymph nodes [102]. The wild-type mouse is not susceptible to DENV infection. Therefore, interferon-deficient mice are the preferred choice for the DENV-related studies [22,92,103,104,105]. Mouse models depicted the presence of DENV antigen in the skin, liver, spleen, lymph nodes, kidney, bone marrow, lung, thymus, brain, stomach, and intestine as reported by various research groups [22,84,92,103,104,105,106].

Studies indicate that the dengue virus (DENV) also contributes to liver pathology. In DENV infection, hepatocytes are primarily infected, leading to cell apoptosis and necrosis, which disrupt liver function [90]. The damage to the liver depicted in the DENV infection may be either due to viral factors or due to the host’s immune response to the virus [107]. Liver damage is one of the cardinal symptoms of severe dengue and has been observed in human cases and mouse studies. Evidence supporting liver involvement is hepatitis, accompanied by elevated levels of liver enzymes following exposure to DENV infection. For example, a study conducted on mice demonstrated that liver transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) were elevated in the injured liver caused by DENV. In mice infected with DENV, the majority of the DENV virion population dominates the endothelial cells of the liver [108]. Moreover, reports indicate that DENV infection can lead to significant damage to liver cells. This damage is characterized by several key findings: moderate midzonal hepatocyte necrosis, which refers to localized cell death in the middle area of the liver lobules; microvesicular steatosis, where small fat droplets accumulate within the liver cells, potentially disrupting their normal function; and the presence of Councilman bodies, which are indicative of apoptotic hepatocytes. These findings underscore the impact of the dengue virus on liver health and the importance of monitoring liver function in affected patients [25,92,109,110,111]. Furthermore, necropsies of the fatal dengue disease cases have demonstrated signs of liver damage, such as mononuclear cell infiltration and mitochondrial swelling, the presence of DENV antigen in hepatocytes and in surrounding necrotic foci [89,112] along with deformed liver architecture and red blood cell extravasation conjugated with damaged reticular formation [113].

Vascular leakage is an important sign of severe dengue and is due to the impairment of the vasculature lining the blood vessels. DENV induces a handful of changes in the endothelial cell (EC) permeability, which covers the vasculature system, culminating in edema or hemorrhagic disease [114]. A vast number of host cells, including peripheral leukocytes, dendritic cells, liver cells, and endothelial cells, are infected by the dengue virus (DENV) in patients, murine models, and in vitro studies. Among these, peripheral leukocytes and dendritic cells are critical in mediating immune responses. Infected leukocytes release proinflammatory cytokines, which can lead to systemic inflammation and increased vascular permeability, contributing to severe dengue manifestations [22,90,115,116,117,118,119,120]. There is no specific study that reported DENV infects endothelial cells (ECs) but some of the studies have deciphered the role of ECs in dengue disease pathogenesis, where DENV antigen was not detected in ECs but was detected in cells surrounding the microvasculature [25,121,122]. Even necropsy samples revealed the presence of DENV-infected ECs [22,90]. Moreover, ECs dwelling in the lungs and the spleen have been shown to express viral proteins [22,90,123,124]. The exact mechanism of how ECs contribute to DENV pathogenesis is unclear. However, some studies suggest a transient disruption in the function of the endothelial glycocalyx layer due to the binding of DENV-NS1 protein to heparan sulfate, a key component of glycocalyx [125,126].

Very sparse information is available on the interaction of DENV in endothelial cells, their kinetics, timing, and replication of dengue viruses within a patient’s endothelial cells [114]. It is unclear what receptors facilitate the entry of the virus into ECs. The possibility of attachment of DENV antigen-complex immune mediators is also ruled out as ECs lack Fc receptors. One possibility is that it is speculated that the virus ingresses these cells via pinocytosis [90]. Whether ECs facilitate the fate of DENV in the initial stages or at the late stages of infection is still unclear. Nonetheless, DENV-infected ECs can alter endothelial cell barrier functions, resulting in an enhanced immune response and augmenting the further dissemination of the infection [127].

4. Invasion and Evasion in DENV Infection

DENV infection of the host in the initial stages is accompanied by the amalgamation of the specific viral proteins with those of their counter receptors on the host cell surfaces. To ameliorate the interaction of the DENV, the host receptors like the mannose receptor, DC-SIGN (Dendritic cell and heparan sulfate), and viral E protein play a profound role [128,129,130]. Once the infection is initiated, an array of host-derived reactions against the virus takes place on every level of the immune response (Figure 6). DENV infection pathology is dependent on both host and viral factors, with host factors including age, sex, immune profile, and underlying conditions [131]. On the other hand, DENV has evolved with varied mechanisms to counteract the host immune response. In this section, we have discussed the host immune response against DENV infection and the mechanism that DENV uses to dodge the host response and facilitate its spread, leading to severe forms of DENV infection (

Table 2. Dengue virus proteins in immune invasion.

	Functions	References
Proteins
NS1	vascular hyperpermeability	[125,132,133]
Interaction with TLR4	suppression of the complement pathways	[134]
NS2A	IFN antagonist	[53]
		[54]
NS2B	inhibits type I IFN production by targeting cyclic GMP-AMP synthase (cGAS) for lysosomal degradation and prevents mitochondrial DNA sensing	[135,136]
NS3	NS3 interacts with chaperone protein 14-3-3e to inhibit the translocation of RIG-I to the adaptor protein MAVS.	[136]
NS4A	NS4A inhibits the interaction of RIG-I with the adaptor protein MAVS by binding to the N-terminal CARD-like domain and C-terminal transmembrane domain of MAVS, resulting in the suppression of IRF3 activation and IFN production	[137]
NS4B	Blocks IFN-induced signal transduction	[53]
integral membrane protein		[54]
NS5	Blocks production of ISGs by interrupting the transcription complex PAF1C	[138]

).

5. Innate Immune Responses

5.1. IFNs and Activation of ISGs

The innate immune response serves as the first line of defense against pathogens. During DENV infection, the virus enters tissue-resident macrophages, monocytes, and Langerhans cells after an infected mosquito feeds on the host’s skin [139]. These cells possess pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) that include double-stranded or single-stranded RNA and diphosphate structures generated during virus replication [140,141,142,143]. Once the virus binds to these host recognition molecules, a cascade of immune mediators is activated, initiating the host’s antiviral response. Among these mediators, type I IFN is secreted by all the nucleated cells and is the signature of early DENV infection [139,144]. Major sources of type I IFN are DCs and plasmacytoid dendritic cells (pDCs) [145]. On the other hand, macrophages, monocytes, and DCs produce type III IFNs, but epithelial cells are the main source [146]. Alongside the type III IFN system, type I IFN triggers signaling pathways that lead to the secretion of numerous proinflammatory cytokines, creating an inhospitable environment for viral replication [139,144,147].

The activity of the IFN system during viral infection resembles a chain reaction, generating a variety of molecules. Similarly, it has been documented that the signaling pathways induced by the IFN system enhance the activation of transcription factors—IRF3 (interferon regulatory factor), IRF7, and NF-κB [148]. The activity of type I IFN induces interferon-stimulated genes (ISGs) in the nucleus of infected cells [147], with approximately 100 genes activated by IFN-α, leading to the establishment of an antiviral state [149]. The cascade of reactions occurring following the attachment of the type I IFN with that of the IFNAR includes the activation of the Janus kinase (JAK)/signal transducer and activator of the transcription (STAT) pathway. Furthermore, phosphorylation and dimerization of STAT1 and STAT2 is mediated by the activation of JAK1 and tyrosine kinase 2 [150]. It is demonstrated that phosphorylated and dimerized STAT1 and STAT2 conjugate with IRF9, and then this conjugate makes its way to the nucleus where ISGs are secreted [151]. Apart from the activation of ISGs facilitated by the IFNs, other mediators that are ameliorated during the DENV infection phase are some cytokines and chemokines [148].

DENV has evolved ways to suppress the IFN response. This notion is supported by the fact that once the viral infection is established, IFN alpha/beta has a very minimal effect on DENV replication [119,152]. Likewise, the DENV multiplies in humans, reaching high titers in the presence of IFN alpha [153,154,155]. As a result, the addition of exogenous IFN after DENV infection does not provide protection. In vitro experiments by Diamond and Roberts showed that pretreatment with IFN-alpha/beta or IFN-gamma can protect human cells from DENV infection [119]. The non-structural proteins of DENV—NS2A, NS4A, and NS4B—interfere with the phosphorylation and nuclear translocation of STAT1, thereby disrupting JAK/STAT signaling [156]. Additionally, N-terminal components of NS5 act as degraders of STAT2 [157,158].

STING is a transmembrane protein found in the endoplasmic reticulum (ER) that functions as a stimulator for IFN [144,159,160,161] and has a role in DENV infection. It is reported that the polyubiquitination of the DENV NS3 protein enhances the production of the NS2B3 protease complex that degrades the STING [162]. DENV can degrade human STING (hSTING), while murine STING is resistant to DENV degradation [55], which explains the pathogenesis of DENV specifically in humans and not in mice. Moreover, in the study conducted by Aguirre and co-workers, they explained the mechanism of inhibition of type I IFN production by DENV in primary human and mouse cells [55]. Furthermore, they identified the human STING as a target of the DENV NS2B3 protease complex, which is important for the cleavage and degradation of STING and impairs the production of type I IFN in DENV-infected cells [163]. Following the sequence alignment between human and mouse STING, the authors showed a difference in the amino acid sequence in this region (94-HCMA-99), which explains the inability to cleave the mouse STING by the DENV NS2B3 [55]. Furthermore, a study conducted by Stabel and colleagues demonstrated that NS2B3 proteases of human (DENV1-4) and sylvatic dengue viruses cleave human STING but not STING derived from chimpanzees, macaques, or marmosets [164].

It was initially established that STING directly interacts with DNA but not with ds RNA (poly I: C), establishing that STING acts as an adaptor that recognizes the RNA viruses through interaction with RIG-1 [165,166]. Moreover, interaction activates the MAVS (mitochondrial antiviral signaling) proteins on mitochondria with the aid of caspase activation recruitment domains (CARD). This triggers the production of TANK-binding kinase 1 (TBK1), IκB kinase ε (IKKε), phosphorylating IFN regulatory factors (IRF3), and IRF7, which enter the nucleus to induce the production of type I IFNs such as IFN-β [167]. It is a well-established fact that STING also attaches cGAMP, which is synthesized by Cyclic GMP-AMP synthase (cGAS). Interestingly, cGAS is a cytosolic DNA sensor that is stimulated by the presence of DNA in the cell’s cytoplasm [168,169]. Although DENV is an RNA virus, it has been reported that DENV has antagonist activity against the cyclic GMP-AMP (cGAMP) synthase (cGAS)/stimulator of interferon genes (STING) pathway [170]. For example, a study demonstrated cGAS as one of the most potent restriction factors for all positive-sense RNA viruses [171,172]. A handful of studies about the interaction of STING with RNA viruses have been proposed, including RIG-1-dependent and independent pathways like cGAS [171,172,173].

5.2. Toll-Like Receptors

DENV is deposited into the host’s skin by the mosquito bite and the initial cells that interact with DENV are tissue-resident macrophages and Langerhans cells [139,144]. These cells carry pattern recognition receptors (PRRs) that recognize the pathogen-associated molecular patterns (PAMPs) on the surface of the DENV [174]. The fact that PRRs recognize the PAMPs is the nexus between the innate immune signal and the initiation of the adaptive immune response [175].

Therefore, antiviral defense is significantly influenced by innate immune responses, which are mediated by PRR. Some PRRs that are crucial in DENV infection are Toll-like receptors (TLRs), cytoplasmic retinoic acid-inducible gene I (RIG-I), NOD-like receptor protein 3 (NLRP-3)-specific inflammasome, and melanoma differentiation-associated protein 5 (MDA-5), [176] and act as important innate immunity regulators. Previous research has indicated that TLR2, TLR6, and TLR4 are linked to detrimental consequences for the host and may be targeted for treatment during DENV infection. Numerous studies have also shown that TLR3 is essential for DENV clearance and TLR7 is necessary for type I IFN generation. For instance, a recent study pointed out that TLR3 Leu412Phe polymorphism has a higher inclination for CNS involvement leading to dengue-induced encephalitis [177]. These seemed to have a role in the development of effective and safeguarding innate immune responses against viruses. Studies indicate that a variety of TLR agonists aid in anti-viral responses against viruses. For example, agonists for TLR3 and TLR9 were shown to induce potent anti-viral responses to HSV-2 [178]. Moreover, a derivative of the analog of the synthetic TLR3 agonist, poly ICLC, confers protective immunity to a range of viruses, including influenza, RSV, and SARS [179]. TLR9 agonists and analogs of the synthetic TLR3 agonist poly (I: C) have been reported to be useful in the treatment of HIV [178,180]. However, in the case of DENV, TLR-mediated signaling during infection is still not fully understood. Therefore, developing therapeutic and preventive methods requires a thorough understanding of the interactions between TLR and DENV [181,182].

6. Major Histocompatibility Complex (MHC) and Natural Killer (NK) Cell Activity

NK cells are a part of the innate immune defense mechanism against viruses, including the Flaviviruses. It has been reported by a group of researchers that intact prM triggers the upregulation of MHC class I in DENV infection [183]. However, the mechanism by which the structural protein regulates MHC-1 is unclear. The association of MHC-I with DENV is also highlighted in incidences of DENV-infected cells expressing only non-structural proteins, which depicted augmented MHC-1 expression [184]. Overexpression of MHC-1 is associated with reduced natural killer (NK) cell lysis, which is explained on the basis of the observation that upregulation of MHC-1 is related to heightened binding of specific NK-inhibitory receptors [19].

7. Mast Cells

In DENV infection, mast cells are a crucial part of the innate immune system, which is also infected, particularly in the presence of pre-existing antibodies [185]. It is demonstrated that mast cells have both positive and negative impacts on the host. These cells contribute to immune surveillance and recruitment by engaging more immune cells and triggering an anti-viral response [186]. Mast cells in the DENV infection are one of the key players in severe DENV manifestations. Heightened levels of mast cell-associated protein chymase have been reported in human DHF cases. This kind of observation is correlated with mouse experiments where liver and spleen-expressing markers of mast cells, MCPTI, were infected after DENV infection [186]. The latter observation in mice was further supported by studies on immunocompromised mice that depicted signs of vascular leakage and mice lacking mast cells, which were lacking vascular leakage. Accumulating evidence pointed out the debatable role of mast cells in DENV. It is still elusive whether these mast cells have a protective or deleterious effect on the DENV infection pathology [187].

8. Complement System

Another efficient component of the innate defense mechanism is the complement system, which acts as an antagonist to the DENV infection. The mannose-binding lectin (MBL) pathway is initiated by MBL which interacts with the carbohydrate structures on the surface of the microbes [188]. In the case of DENV infection, the MBL-mediated neutralization occurs in the presence of complement activation. Additionally, the presence of high-mannose (N-linked glycan) at Asn-67 in DENV, facilitates its binding to DC-SIGN, the receptor for DENV [189]. Studies have shown a cascade of reactions involving complement components and MBL. For example, MBL-associated serine protease-2 cleaves C4 and C2 complement components. Furthermore, another enzyme C3 convertase is generated by the deposition of C4b and C2a components on the virion surface. Following a couple of reactions, in the complement system, C5 convertase is generated leading to the development of C5b-9, which is a part of the membrane attack complex (MAC) [190,191]. MAC has a substantial role in viral infection as it aids in engaging the phagocytes to facilitate the rupture of the infected cells [191].

Studies have demonstrated a link between complement activation and NS1 in severe DENV [133,192]. However, the exact mechanism is still elusive [193,194]. DENV requires the aid of NS1 antibodies for proper utilization and generation of complement and C5b-9, respectively [133]. DENV derived from NS1 diminishes the effect of classical and lectin pathways by directly forming a complex with C4. The combination of NSI and C4 has many negative effects on the complement system: (1) deposition of C4 is declined, (2) diminished C3 convertase (C4b29) activity [195], and (3) NS1-C4 complex interacts with CISC, which facilitates the cleavage of C4 to C4b in solution [196]. Furthermore, NSI is shown to interact with clusterin which is an activated component for MAC formation [197].

DENV NS1 protein is highly immunogenic and occurs in two forms: soluble (sNS1) and membrane (mNS1) [198,199,200,201,202]. Avirutnan and colleagues reported that soluble NS1 (NS1s) and membrane-associated NS1 (NS1m) activate human complement, and plasma levels of sNS1 and terminal SC5b-9 complement complex correlate with disease severity (vascular leakage) [133]. Furthermore, Kurosu and coworkers have demonstrated that sNS1 interacts with human clusterin (Clu), a complement regulatory protein. Clu inhibits the formation of the terminal complement complex (TCC). The authors proposed two possibilities: the NS1-Clu complex is eliminated by NS1 antibodies, and NS1 may inhibit the interaction of Clu and C7 complement components. The interaction of Clu and C7 inhibits the formation of TCC [197]. Therefore, in the second situation, if there is competition between NS1 and C7, there is partial inhibition of Clu leading to vascular leakage since the low concentration of TCC can increase the leakage [203].

9. RNA Interference (RNAi)

RNA interference (RNAi) is a phenomenon of gene regulation accomplished by either miRNA or siRNA [204]. miRNA is a small single-stranded non-coding RNA molecule with approximately 22 nucleotides that functions in post-transcriptional regulation of gene expression [205]. RNA interference (RNAi) plays a role in both invertebrate and invertebrate immunological responses [206]. When dsRNA is employed as the starting material, the Dicer can create siRNA; miRNA is created by both Dicer and Drosha. The interference of viral replication by these small non-coding RNAs is due to their association with RNA-induced silencing complex (RISC) in the cytoplasm to target mRNA for inhibition or degradation by Argonaute (Argo) proteins [204]. DENV RNA is recognized by the latter complex, which governs silencing and degradation, thereby inhibiting DENV infection. The substantial role of RNAi in suppressing the DENV infection has been documented by in vitro experiments. For instance, the knockdown of essential components of RNAi (Dicer, Drosha, Argo1, Argo2) resulted in increased DENV viral titer in the human cell line Huh7 [207].

Furthermore, more examples of the role of miRNA in DENV infection have been documented in cell culture experiments where upregulation of miRNA-30e* was demonstrated in HeLa and U937 cells upon DENV infection, which restored type I IFN production and suppressed DENV replication through targeting Ikba and subsequent activation of NF-kB signaling [170]. Moreover, another study showed that upregulation of miRNA-155 blocked DENV replication by specifically targeting BACH1, which induced the HO-1-mediated suppression of NS2B/NS3 and enhanced antiviral IFN responses [208]. Another report pointed out that miRNA-34 ameliorated type I IFN production and ISG expression by inhibiting the Wnt signaling, resulting in the inhibition of viral replication [209]. Another study reported that miRNA-424 downregulated the expression of the SIAH E3 ubiquitin protein ligase 1 (SIAH1), which abrogated the DENV infection [210]. Other miRNAs like Let-7c also inhibit the DENV-2 and DENV-4 in Huh-7 cell infection by modifying the host fa heme oxygenase-1 (HO-1) and BTB domain and CNC homolog 1 (BACH1). The heme-oxygenase-1 (HO-1) enzyme interferes with the viral NS2B/NS3 protease activity, resulting in the stimulation of antiviral IFN responses [211].

10. Autophagy

The physiology of the cells is bestowed with unique physiological phenomena that are essential to sustain proper functioning [212,213]. The process involves small bi-membrane structured autophagosomes, which engulf the cellular material that harbors foreign material, including pathogens [18]. Once the cell components are engulfed, these structures fuse with lysosomes, where further degradation is accomplished [18]. In the case of DENV infection, autophagy has both anti-viral and proviral activity depending upon the specific cell involved [214]. In liver cells, autophagy has a pro-viral effect, where DENV blocks the autophagosome from fusing with the lysosome and instead uses the vacuoles for its infection process involving replication [215], assembly, and maturation [216], and evading neutralizing antibodies during transmission [217]. In endothelial cells, autophagy employs specialized receptors FAM134B to inhibit DENV infection [218].

11. Apoptosis

Another unique cellular phenomenon is apoptosis, or programmed cell death, which occurs in the situation of tumors or pathogen-infected cells and involves (1) DNA fragmentation, (2) cytoskeleton degradation, (3) formation of apoptotic bodies, and (4) phagocytosis [219]. Furthermore, apoptosis has extrinsic and intrinsic pathways that are interconnected and lead to cell death [219]. In the case of DENV infection, viral protein C (capsid) activates apoptosis by interacting with death-associated protein 6 and triggers Fas-mediated apoptosis in liver cells [220]. Moreover, non-structural DENV proteins like NS2b-NS3 protease precursor and NS3 protease induce apoptosis [221], most likely through the caspase-8 pathway [222] or NF-κB [223]. In the case of DENV-2 CNS infection, it has been observed that this serotype induces neuronal apoptosis by the following mechanisms: (1) phospholipase A2 (PLA2) activation; (2) superoxide anion generation; (3) cytochrome c release; (4) caspase 3 activation; and (5) NF-kappa B activation [224]. In some instances, apoptosis has been linked to severe forms of dengue disease. For instance, apoptotic cells have been reported from the liver, cerebral, and endothelial cells from autopsies of patients with dengue hemorrhagic fever (DHF)/dengue shock syndrome [225]. A possible mechanism of immune evasion in the case of DENV-induced apoptosis is that DENV activates P13K signaling at an early stage of infection, which leads to the blockage of caspase-dependent apoptotic cell death. The latter activity acts as a trigger for initiating a survival signal for the infected cells which sustains and supports longer viral replication [19]. Therefore, from this evidence, it may be concluded that DENV inhibits apoptosis for their survival.

12. Adaptive Immune Response

Following the innate immune response, the adaptive immune response, and the second line of defense is activated. DENV glycoproteins are recognized by the host immune response and trigger the immunological memory which lays the foundation of the adaptive immune response [182]. The adaptive immune response is composed of two components: humoral immune response (antibody-mediated) and cellular immune response (cell-mediated). Both of these arms of the adaptive immune response have a substantial role in eliminating the DENV infection from the host.

12.1. Humoral Immune Response

The humoral immune response is impacted by the original antigenic sin (OAS). According to the concept of the original antigenic sin (OAS), when a slightly different antigen encounters the host, due to prior exposure to a similar kind of antigen before, the body manifests defensive mechanisms including innate and adaptive immune responses, which are initially beneficial to the host. However, the challenge arises when this so-called slightly different antigen is far more varied from the previous one. In that case, an inadequate immune response is generated, which is unable to clear the pathogen from the host [226]. In other words, the capacity of the immune system to recognize a pathogen is impaired, which leads to the evasion of the host immune system [226]. Many viruses, including DENV, have demonstrated that the perilous nature of the disease is bestowed by the phenomenon of the OAS [227,228,229,230,231,232]. An example where OAS impacted the outcome of DENV infection was identified in DENV-infected Thai children, who mounted a poor immune response and subsequent severe DENV infection following secondary infection with a distinct DENV serotype [233]. It is obvious that severe DENV manifestations are the product of OAS, which is fueled by an imbalance of the host’s immune response towards the DENV infection.

Antibody-dependent enhancement (ADE) type of immune response is mediated by the antibodies. ADE is only evident in the case of secondary infection with a heterotypic DENV strain. Supporting the latter concept, it has been observed that neutralizing antibodies from a previous infection are protective in secondary infection due to a homotypic DENV strain [234]. In the case of DENV, the neutralizing antibodies are mainly directed to the epitopes on domain III of the E, prM/M, and NS1 proteins [235]. Neutralizing antibodies are protective and induce long-term immunity in the case of primary DENV infection with a homotypic serotype. However, in secondary DENV infection with a heterotypic strain, these neutralizing antibodies offer term short-term immunity and partial protection. In certain situations, these antibodies mediate the phenomenon of antibody-dependent enhancement (ADE), which results in severe consequences of DENV infection [19,236,237]. Neutralizing antibodies from a previous DENV infection combines with the epitopes and form a complex that facilitates the entry of the virus into the immune cells bearing Fc receptors [236,237]. Scientific studies supporting ADE have been documented from animal and human cases. For instance, studies conducted on passively immunized non-human primates and mice demonstrated higher viremia and transition of non-lethal DENV infection to lethal one, respectively [238,239]. Moreover, DENV clinical cases also reported a similar finding [234,240,241]. This evidence demonstrates that ADE transforms mild self-limited DENV infection into a severe form.

Employing ADE in secondary infection, DENV infection is enhanced and leads to severe outcomes like vascular leakage and cytokine storms [242]. Adding to this, ADE also evades the host immune response in different ways, leading to malfunction of the immune machinery. First, the introduction of the DENV antigen–antibody into the monocytic cells triggers the release of negative signaling molecules, leading to the inhibition of the IFN system. Some of these signaling regulators are selective androgen receptor modulator (SARM), dihydroxyacetone kinase (DAK), TANK, and autophagy-related 5-autophagy-related 12 (Atg5–Atg12) [19,243]. The modes of action of these released components are different; for instance, SARM and TANK function to disrupt the toll-like receptor (TLR) signaling cascade, whereas Atg5-Atg12 and DAK function to inhibit the RIG-I/MDA-5 signaling cascade, but overall, they aid in suppressing the host antiviral response [19,243]. Furthermore, ADE disrupts the secretion of IL-12 and IFN-gamma, which act as inflammatory mediators. Moreover, interleukins like IL-10, which are involved in degranulation and cytokine production along with inhibition of T-cell activation, also play a major role in augmenting ADE [19,243]. Also, IL-10 activates the expression of the suppressor of cytokine signaling 3 (SOCS-3) gene, leading to the suppression of the JAK-STAT pathway [19]. Furthermore, certain cytokines like B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) contribute to increased production of DENV-specific antibodies, leading to ADE [244] by provoking the transformation of the resting B cells into antibody-secreting plasma cells.

One aspect of the DENV replication inside the host that contributes to the ADE is the generation of immature particles, which are facilitated into the cells by prM and FLE antibodies [245,246]. Complete cleavage of prM protein is vital for DENV infectivity. FL epitope (FLE) is a highly conserved moiety among the Flaviviruses, and anti-FLE antibodies account for around 20–30% of the DENV antibodies [247,248,249]. In certain instances, the prM is not fully disrupted, which leads to the generation of immature DENV particles [250,251]. These immature particles are inefficient at penetrating the cell without the aid of antibodies. Therefore, in primary infection, these are not contributors to the disease severity. However, in secondary infection, pre-existing prM and FL epitope antibodies combine with the immature DENV particles to form a complex, which is then transported to the host monocytes and macrophages, thereby augmenting the infectivity and disease severity [252,253]. Studies indicated that among cross-reactive DENV antibodies, against prM and Fl epitopes are poor neutralizers and potent triggers of ADE [116,237,245,254].

Another aspect contributing to the ADE is the antigenic variation. Due to the error-prone nature of RNA polymerases, DENV lacks proofreading activity during genome replication, resulting in rapid adaptation and evolution of these viruses [255,256]. These evolved viruses harbor specific and significant mutations that help them escape neutralization by neutralizing antibodies [255,256]. One of the notable examples is the occurrence of mutation in the domain III of the E protein of the members of the FV, including WNV, DENV, YFV, and TBEV [257,258,259,260]. The previous example is explained because FV has low-fidelity RNA polymerase that generates quasispecies containing distinct antigenic epitopes that contribute to the immune evasion of the host. In the case of DENV serotype 1, the presence of these escape mutants has been reported from clinical samples of infected DENV patients [261].

12.2. Cell-Mediated Immunity (CMI)

Another arm of the adaptive immune response involving various lineages of T-T-cells is cell-mediated immunity (CMI). Specific HLA haplotypes have a specific role in DENV T-cell immunity. For example, HLA-A11/NS3133 induced a tetramer-positive population that appeared to associate with disease outcomes in one population of patients [97], but not others [96,98], and cytokine responses, but not degranulation, to this epitope were associated with disease outcomes. Moreover, the frequency of HLA-B7-restricted tetramer-positive T cells appeared to correlate with disease severity only in HLA-A11-negative subjects and not in HLA-A11-positive individuals [96]. Occurrence of genetic variants of the human major histocompatibility complex class I–related sequence B and phospholipase C epsilon 1 gene are risk factors for severe DENV infection [99,100,101].

As mentioned in the previous section, OAS also impacts the CMI. It has been demonstrated that memory T cells and effector CTL generated after primary infection can lyse infected cells through the secretion of cytokines and lytic enzymes. If a second infection by a somewhat different virus occurs, these pre-existing memory CTL sometimes lead to the immune response over the new virus. This situation is well documented in HIV, lymphocytic choriomeningitis virus (LCMV), and DENV infection, where heightened immune response results in CTL anergy, uncontrolled virus replication, and cytokine storms, respectively [35,254,262,263].

A lot of effort over the past years has been directed to delineate the role of T cells in DENV pathology [264,265,266,267,268]. Most of the epitopes for the T cells are concentrated in the non-structural proteins of DENV [269]. Studies involving human cases from natural DENV infection and live attenuated monovalent vaccine receivers unrevealed NS3 as the immunodominant non-structural protein with multiple epitopes [187,264,265,266,268,270,271,272,273]. A study conducted in Thailand on DENV-infected children revealed the T cell response to multiple DENV proteins, with NS3 as the dominant one [153,265,266,267,268,271]. Another finding involving peripheral blood mononuclear cells (PBMC) from DENV-2-infected adult patients with secondary infection demonstrated that CD8+ cell responses were directed toward the NS3 and NS5 proteins [274]. The same study pointed out that epitopes from structural proteins envelope (E), capsid (C), and non-structural protein NS1 are potent triggers of CD4+ T cells [274]. Furthermore, elucidation of the ex vivo levels of IFN gamma-derived Sri Lankan population from the hyperendemic areas revealed that about two-thirds of the total T-cell responses were directed to NS3, NS4B, and NS5 [262].

T-cell responses following multivalent vaccine trials have demonstrated a lot of insight into DENV pathology. The National Institute of Allergy and Infectious Diseases (NIAID)developed live attenuated vaccines targeting all four DENV serotypes (DENV-1, 2, 3, and 4). In clinical studies, a low dose of 10 plaque forming units (PFUs) of DENV-1 showed a strong immune response, with CD4+ T cells producing key cytokines (IFN-γ, TNF-α, and IL-2) at 21 days post-vaccination, but minimal cross-reactivity to other serotypes [275]. Another multivalent vaccine under investigation was developed by Sanofi Pasteur, combining four chimeric live yellow fever viruses (YFV) with the prM and E proteins of each DENV serotype (DENV-1 to DENV-4). This tetravalent chimeric dengue vaccine, CYD-TDV, successfully induced CD4+ and CD8+ T cell responses specific to both DENV and YFV17D204. Notably, CD8+ T cell responses specific to YFV 17D NS3 and T helper responses specific to each DENV serotype were also detected in the PBMCs of vaccinated individuals [276]. Recently, the tetravalent dengue vaccine TAK-003 has shown sustained efficacy and an acceptable safety profile throughout a 4.5-year phase 3 trial. All four dengue virus serotypes were successfully protected against the vaccination, which markedly decreased the incidence of severe dengue. Safety issues persisted in line with previous findings, demonstrating the efficacy of TAK-003 as a long-term, dependable dengue preventative treatment in endemic areas [277].

In an experimental study with a mouse model, NS3 was the prime target of DENV-specific H-2k cytotoxic lymphocytes (CTL). From these examples, it can be interpreted that NS3 is the potent trigger for the T cells in DENV cases [278]. The exact reason for the involvement of immunodominant NS3 and NS5 in T cell activation in the DENV infection is still elusive. However, one of the explanations by the researchers is the large size of the NS3 and NS5 proteins that offer more varied types of epitopes as compared to the other DENV-derived proteins. Moreover, a reason for the cross-reactivity of the T-cells to NS3 and NS5 proteins may be accounted for by the highly conserved nature of these proteins among the Flaviviruses [279].

13. Conclusions

Interactions between viral and host factors are complex, often leading to severe outcomes from dengue virus (DENV) infections in both humans and animals. Despite recent advancements in our understanding of DENV, areas remain to be explored regarding its evasion strategies and the immune responses they elicit. Each immune response ranging from antibody production to T cell activation may contribute differently to controlling the virus and influencing the severity of the infection. However, the relationship between the immune system and DENV infection still requires extensive research. DENV has evolved various strategies to evade the host immune response, facilitating its replication and spread. Future research should prioritize delineating the mechanisms employed by each of the four DENV serotypes to evade host defenses.

Additionally, investigating the antagonistic mechanisms of other emerging viruses can provide valuable insights into viral pathogenesis and host immune evasion. Understanding how DENV, particularly through its non-structural proteins, inhibits interferon signaling will be vital in identifying new therapeutic targets. Addressing these knowledge gaps is essential for developing effective strategies to combat DENV and safeguard against future viral threats.