Toll-Like Receptors and Emerging Viral Infections

Abstract

Emerging viral infections are considered a global threat, and they have gained more importance after the coronavirus outbreak in 2019, which affected the whole world. The innate immune system recognizes invading pathogens via pattern recognition receptors (PRRs) expressed on different immune cells extracellularly and intracellularly. Out of several PRRs, Toll-like receptors (TLRs) are one of the critical PRRs recognizing diverse pathogen-associated molecular patterns (PAMPs) varying from viruses, bacteria, and fungi. Viral pathogens possess specific molecular signatures such as dsRNA and high CpG content that differentiate them from mammalian cells. TLRs play their role in innate immunity against pathogenic viruses by producing antiviral cytokines and chemokines. Most emerging viral pathogens are RNA viruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These viruses are recognized by TLR 2, TLR 3, TLR 4, TLR7, and TLR8 with the coordination of other PRR members resulting in the activation of costimulatory molecules that initiate immune response. This chapter provides insights into the TLRs’ role in developing and regulating the immune response against emerging viral pathogens. It exploits their roles in innate immunity to develop treatment strategies against deadly emerging viral pathogens.

1. Introduction

It has been established that the recurrence and emergence of viral diseases unquestionably play a significant role in influencing the human world, from Walter Reed’s discovery of the first human virus, known as the yellow fever virus, in 1901 to the coronavirus disease-2019 (COVID-19) pandemic today caused by SARS-CoV-2. They not only have an immense effect on the economy and society in today’s connected world, but they also have the potential to cause high mortality as they spread. Despite significant advancements in medical research, newly emerging and reemerging viral infections continue to have catastrophic effects on human populations all over the world. Unfortunately, there is currently no treatment for most of these viral infections.

A developing infective condition is brought on by novel disease-causing microbes that have either never before arisen in a population and are now affecting it, or that have previously existed but are now quickly spreading to new geographic areas [1]. Either locally or globally, they are to blame for serious public health issues. Rarely can it be determined whether an illness is new to humans or if it has always existed but gone unnoticed by the world of science. However, many newly emerging illnesses are assumed to be the result of a rise in the frequency of close interaction among people and pathological repositories found in nature. This proximity makes it easier for an agent to successfully “jump” from an animal or arthropod to a person and pass the interspecies border [2].

Additionally, the past 50 years have seen exceptional pandemic outbreaks, including HIV in early 1990 [3]. H1N1 “swine flu” in 2009, Zika in 2015, chikungunya in 2014, and as well as Ebola fever outbreaks that resembled pandemics in wide areas of Africa from 2014 to the present [4]. In light of the fact that there are four endemic coronaviruses that afflict people globally, coronaviruses must have originally emerged and circulated extensively during the era when viruses were first acknowledged as causes of human diseases. The SARS-CoV caused a near-pandemic that afflicted 26 nations between 2002 and 2003 before going away in accordance with public health control measures. The animal host was most likely a domestic cat at the time.

The associated Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) first developed in people in 2012 after emerging from dromedary camels [5]. By the end of 2019, COVID-19, which was identified, as just the most recent instance of unforeseen, innovative, and fatal global epidemic illness. SARS CoV-2 has been linked to about 6.96 million fatalities and around 771 million cases as of 04 October 2023 [6]. The root reasons for this novel and risky scenario are varied, and intricate, and deserve careful consideration [7].

The innate immune system serves as the first line of defense against invading pathogens. In viral infection, innate immunity plays a vital role in inducing adaptive immunity. Upon entry immune cells recognized the conserved regions of invading pathogens named PAMPs, that are not present in the host cells. Immune cell receptors known as PRRs engage with pathogen PAMPs to activate a number of intracellular signaling pathways, that trigger the production of pro-inflammatory cytokines and type I interferons (IFNs), that ultimately initiate the antiviral immune response to clear the invading agent [8]. The clearance of viral cells is accomplished within a few weeks in typical infections however, certain viruses learn how to evade or circumvent the host’s immunological defenses, which helps them replicate and infect the host persistently.

2. TLRs in pro-inflammatory immune response

The host’s innate immune system serves as its first line of defense contrary to infectious diseases caused by microbes. The innate immune system is essential and plays an important part in viral infections by locating and eliminating contaminated cells as well as organizing an adaptive immune reaction. The innate immune system comprises different innate immune cells (basophils, eosinophils, mast cells, neutrophils, monocytes, dendritic cells (DCs), natural killer cells) and secreted humoral components such as complement proteins, cytokines, IFNs, and chemokines. These innate immune cells recognize pathogens via different PRRs including nucleotide-binding oligomerization domain (NOD)-like receptor family proteins (NLRs), Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) [9].

PAMPs are persistent architectural motifs that may trigger the body’s natural defenses against infections by identifying characteristics of bacteria or viruses, such as nucleic acids, peptides, and lipoproteins, that differentiate the pathogen from the host. Host PRRs expressed in epithelial cells and first-line immune cells such as DCs, macrophages, and natural killer cells were able to recognize PAMPs during viral infection [10]. Antigen-presenting cells (APCs) including macrophages and DCs as well as certain kinds of T cells express the transmembrane receptors known as TLRs.

The innate immune system is also an initial defense mechanism in emerging viral infections including SARS-CoV. It also prevents the entry, translation, replication, and assembly of the virus. They also assist in the identification and elimination of infected cells along with coordinating and hastening the progression of adaptive immunity. In response to PAMPs, PRRs in the cytosol and on cell surfaces, in endosomes, trigger inflammation and promote apoptosis that stops infections caused by viruses and helps with the removal of viral debris [9, 10, 11].

TLRs were the first to be identified and have been the subject of the most in-depth research, even though vertebrate hosts also express many other classes of PRRs, including the NLRs, RLRs, and C-type lectin receptors [12, 13]. Initially discovered in Drosophila, the TLRs have since been demonstrated to be crucial for the host’s defense against fungus [14]. Researchers identified 10 homologs in humans (TLR1–TLR10) and 12 in mice (TLR1–TLR9 and TLR11–TLR13). These toll receptor homologs were named as TRLs [15, 16]. TLRs are characterized as type I transmembrane proteins consisting of a Toll-interleukin-1 receptor (IL-1R) homology (TIR) domain in the cytoplasmic carboxy-terminal region, which triggers downstream signal transduction. They also possess a transmembrane domain and an amino-terminal ectodomain that is rich in repeats of the amino acid leucine, which makes it easier to recognize PAMPs [17, 18].

The six major families of TLRs found in vertebrates are TLR (1, 3, 4, 5, 7, and 11) [19]. The members of the TLR1 family are TLR (1, 2, 6, and 10). These are found on cell membranes and can identify peptidoglycans and lipoproteins that are found in microbial cell walls and membranes. Furthermore, TLR4 and 5 (located extracellularly on the plasma membrane) recognize LPS and flagellin to initiate TLR-dependent pro-inflammatory immune response [16]. On the other hand, endosomes and lysosomes express TLRs from the TLR (3, 7, and 11) families, which are cytoplasmic TLRs. Transportation of these TLRs from the endoplasmic reticulum to endolysosomal spaces, where they are modified by proteases to turn into functional receptors, is facilitated by UNC93B1, a polytopic membrane protein [20]. Double-stranded RNA (dsRNA) is detected by TLR3. The TLR7 family contains TLR (7, 8, and 9). Single-stranded RNA (ssRNA) is detected by TLR (7 and 8), while TLR9 interacts with unmethylated CpG DNA TLR (7 and TLR8 detect single-stranded RNA (ssRNA), while TLR9 interacts with unmethylated CpG DNA [16]. TLRs in humans with their respective ligands and adaptor molecules are summarized in Table 1. The Toxoplasma gondii parasite’s profilin is sensed by TLR (11 and 12) forming a heterodimer and belongs to the TLR11 family [30], whereas bacterial 23S ribosomal RNA is recognized by TLR13 [31, 32].

Localization	TLRs	Ligands	Adaptor molecules	References
Plasma membrane	TLR1/TLR2	Diacyl/triacyl Iipopeptides	MyD88	[19, 21, 22, 23]
Plasma membrane	TLR2	peptidoglycan, lipoteichoic acid, Lipoproteins, Zymosan	MyD88	[21, 22, 23]
Endosome	TLR3	DsRNA	TRIF	[24]
Plasma membrane	TLR4	LPS, Envelope glycoproteins, endogenous HSP, HMGB1, β-defensin 2	MyD88/TRIF	[25]
Plasma membrane	TLR5	Flagellin	MyD88	[26]
Plasma membrane	TLR6/TLR2	lipoproteins, zymosan, lipoteichoic acid	MyD88	[21, 22, 23]
Endosome	TLR7	SsRNA	MyD88	[16, 27]
Endosome	TLR8	SsRNA	MyD88	[16, 27]
Endosome	TLR9	Unmethylated CpG DNA, MtDNA	MyD88	[16, 28]
Plasma membrane	TLR10	HIV-1 gp41, H1N1/H5N1	MyD88	[29]

Table 1.

Toll-like receptors (TLRs) in human respective natural ligands.

Human TLRs that can recognize viral nucleic acids, such as TLR (3, 7, 8, and 9), are mostly expressed in the endosomal compartment, in contrast to the TLRs that recognize bacteria and are primarily expressed on the cell membrane [33]. Viral dsRNA taken up into sentinel cell endosomes is detected by TLR3, leading to the activation of downstream signaling pathways dependent on the cytoplasmic adaptor protein TRIF. Downstream signaling pathways for TLR (7 and 8), and ssRNAs, rely on MyD88. TLR9, the sole known DNA sensor, specifically detects the unmethylated CpG DNA of DNA viruses [34]. Furthermore, viral structural and nonstructural proteins have been associated with the production of inflammatory cytokines, mediated by TLR (2 and 4) within the TLR family.

TLR signaling is carried out through two mechanisms: the (TRIF)-mediated route, which involves the Toll-interleukin-1 receptor (TIR)-domain-containing adaptor inducing IFN-β, and the (MyD88)-mediated pathway, which relies on the myeloid differentiation factor 88 [35, 36]. The former induces IFNs, which when stimulated, put cells into an antiviral state. The latter MyD88-mediated pathway triggers the activation of the tumor necrosis factors (TNF) such as NF-κB, which subsequently initiates the activation of multiple genes involved in inflammatory reactions. Only the TRIF-mediated pathway is activated by TLR3. IRF-3, a crucial for TF for IFN- β, is activated by TLR3 signaling, inducing the synthesis of IFN. TLR2 exclusively activates the MyD88-mediated pathway, whereas TLR4 activates both the MyD88-mediated pathway and the TRIF-mediated pathway. As a result, TLR4 agonists have the dual effect of activating NF-κB and promoting the production of IFN [37].

The activation of transcription factors (TFs) like NF-κB, AP-1, and interferon regulatory factors (IRFs), occurs as a result of signaling pathways initiated by PRRs. These TFs play an important part in stimulating the expression of different genes that have a role in antiviral defense such as type I and III interferons (IFNs), pro-inflammatory cytokines, and chemokines [8]. These genes of focus can increase the adaptive immune reaction, prevent the spread of pathogens, and help the host cells to adapt to changes in their environment. Macrophages and DCs primarily produce type I IFNs, causing an antiviral defense in the surrounding cells which start expressing the several genes collectively called IFN-stimulated genes (ISGs). Neutrophil, monocyte, and NK cell recruitment may be aided by the chemokines released at the site of infection.

3. Emerging viral pathogens and Toll-like receptors

Members of the Coronaviridae family that are phylogenetically and antigenically unique include SARS-CoV and MERS-CoV [38, 39]. Similar classes of cellular sensors may be able to detect PAMPs, as they are likely to move through similar spaces in the host cells infected by coronaviruses. Virus and other invading pathogen-specific PAMPs are recognized by innate immune sensors, which then cause transcriptional modifications in host cell signaling pathways that make an antiviral state and reduce the effectiveness of viral reproduction.

The emergence of highly virulent 1918 and 2009 H1N1, H5N1, and H7N9 and influenza A viruses (IAV), as well as the SARS, MERS, and most recent SARS-CoV-2 epidemics, demonstrate the potential danger that respiratory virus infections provide to world health [40]. Essential roles in gas exchange are performed by the human lung, which serves as an intricately complex and highly delicate mucosal surface that interacts with numerous environmental bacteria. The lung’s ciliated cells and type II pneumocytes of the airway epithelium are the primary targets of SARS-CoV and IAV infection [40, 41]. To ensure maximum detection of viruses at different stages of the replication cycle, including virus entrance and genome replication, PPRs which contain various classes of cellular sensors, are allocated at cellular membranes and within the cytosol. When exposed to infectious microbes, innate immune signaling cascades are initiated by these cells [42].

3.1 SARS CoV

The SARS outbreak that occurred in 2003 had a disastrous impact globally as it rapidly spread across the continent, causing over 8000 ailments, and resulting in a mortality rate of 10% [43]. It had severe economic consequences for local and regional areas.

SARS CoV genome comprises linear, single-stranded, and positive sense RNA. They are membrane-enveloped viruses [44]. The innate immune system reacts quickly to viral invasion and is crucial in triggering the adaptive immune system. When PAMPs engage with host PRRs in innate immunity, numerous signaling mechanisms are activated in immune cells. This results in the release of pro-inflammatory cytokines and IFNs, which in turn trigger an antiviral response. The primary innate immune cells with antiviral activity are macrophages, DCs, and natural killer cells [8].

Extracellular membrane-bound TLRs recognize extracellular pathogens. TLR3 has been linked to recognizing changes in the pathogenesis of airway disease brought on by infections with respiratory viruses such as IAV, rhinovirus, and respiratory syncytial virus (RSV) as well as recognition of the range of different RNA viruses [45, 46, 47]. Basal levels of TLR3 expression can be found in the lung tissues including human bronchial epithelial cells and alveolar cells, as well as different immune cells (19). TLR3 is attached to the endosomal membrane in cells, where it detects dsRNA patterns produced by invasive pathogens [48].

TLR 3 dimerizes and draws in the TRIF adaptor protein after engaging the dsRNA motif [49, 50]. TFs such as interferon regulatory transcription factor (IRF3) and NF-κB are signaled to be activated as a result of TRIF recruitment to the endosome [51]. TRIF has been defined as an adapter for TLR4 signaling in addition to TLR3-specific signaling pathways [50, 52]. The basal level of TLR4 expression in alveolar cells and bronchial epithelial cells rises in response to inflammatory cell infiltration brought on by insults like viral infections [53, 54]. TLR4 communicates with TRIF or MyD88 via two sorting adaptors: TRAM for TRIF-dependent signaling, and MAL for MyD88-dependent signaling pathways [55]. An increase in the severity of acute respiratory distress syndrome (ARDS) brought on by influenza virus infections and acid damage models has been connected to the TLR4/TRAM/TRIF signaling pathways in earlier investigations [56]. The involvement of TLR4 in the immunopathogenesis of the influenza virus has been a subject of controversy, and the TLR4 antagonist Eritoran has been suggested as a potential immunomodulatory treatment for influenza viral illnesses [57, 58].

The involvement of TLR4 in the highly infectious SARS-CoV remains uncertain. The activation of proinflammatory cytokines (IL-6, TNF, IFN-, and CCL5), type 1 interferons (IFN-α and IFN-β), and interferon-stimulated genes (ISGs) (RSAD2, IFIT1, and CXCL10) is induced by TLR signaling through TRIF [48, 59]. In the setting of ARDS and respiratory viral illnesses, these effector molecules are of definite importance [40, 60]. In the setting of vaccine adjuvants and antiviral medication, TLR antagonists and agonists have been postulated as substances having wide-range efficacy over diverse respiratory illnesses [57, 61, 62, 63]. Lipopolysaccharide (LPS), a TLR4 agonist, and poly(I:C), a TLR3 agonist, have been proven in mouse experiments to give prophylactic protection against SARS-CoV infection, with poly(I:C) displaying better effectiveness than LPS [61]. Additionally, administration of poly(I:C), a TLR3 agonist that initiates signaling regardless of MyD88, demonstrates antiviral effects in mouse models infected with coronavirus species that are extremely contagious, such as group 2c MERS like coronaviruses [64]. Given the wide range of zoonotic precursors that have the potential to spread into human and cattle populations, it becomes crucial to comprehend how TLR signaling pathways and effector networks could affect coronavirus pathophysiology. Previous studies in SARS-CoV infection in the mouse model have suggested a shielding role for the TLR adapter protein MyD88, which facilitates downstream signaling via multiple TLRs [65]. Research has demonstrated that a powerful cell-intrinsic defense network is activated in response to SARS-CoV illness through MyD88-independent signaling via TLR (3 and 4), facilitated by the TRIF adaptor protein.

3.2 MERS-CoV

Severe respiratory disease with a 30% death rate was first linked to the MERS-CoV in 2012. Human individuals who encounter MERS-CoV develop a severe respiratory illness with a high mortality rate [66]. The most fatal human coronavirus infection to date is MERS [67]. Even though it has a lower rate of human-to-human transmission, almost all MERS-CoV infections can cause severe symptoms, making clinical care difficult. Similar to SARS-CoV, MERS-CoV manifests in humans with serious pneumonia with ARDS, lymphopenia, leukopenia, septic shock, and multi-organ failure [67].

Innate immunity is significantly aided by DCs, which can also significantly increase cytokine and chemokine output. These cells can move from lymphoid tissue to peripheral organs, where they can activate the T cell population [68]. As a result of their role as intermediaries between innate and adaptive immunity, DCs are seen as possible targets for pathogen invasion.

Innate immunity relies on PRRs, such as TLRs and retinoic acid-inducible gene-I- (RIG-I-) like receptors, which play a crucial role [69, 70, 71]. One of the two distinct adaptor molecules MyD88 or TRIF—becomes active following TLR virus identification. Various pathways such as MAPK and NF-κB, which are in charge of encouraging the synthesis of proinflammatory cytokines and IFNs, are further activated by these molecules [72, 73, 74].

The interaction between MERS-CoV and the host cell’s DPP4 receptor via the S protein results in the localization of genomic RNA within the cytoplasm. MERS-CoV replication has the potential to partially trigger an immunological response to dsRNA. When dsRNA sensitizes TLR-3 and triggers a series of reactions involving IRFs and NF-κB activation through TRAF3 and TRAF6, respectively, it leads to the generation of type I IFNs and proinflammatory cytokines. The generation of type I IFNs is crucial for enhancing the secretion of antiviral proteins and protecting cells that are not yet infected.

TLR-3 activates IRF7 and IRF3 following attaching to its specific ligand, irrespective of MyD88 [73]. A possible treatment for MERS-CoV disease in a mouse model has recently been revealed using the TLR-3 agonist poly IC [65]. Type 1 IFN-α and IFN- β expression gets increased after poly IC injection [75]. Consequently, the activation of various effectors such as macrophages, CD8 T cells, and NK cells is initiated, leading to their antiviral effects [76, 77]. The host experiences various detrimental effects due to the proinflammatory cytokine response, including the production of cytokines like TNF-α and IL-6, which can lead to pathological tissue damage during any infection [78]. On the other hand, inflammatory cytokine reactions can restrain the spread of viruses. By comprehending the TLR signaling events in a better way and effectively controlling viral infections, the risk of viral dissemination can be minimized in the picture of infection of MERS-CoV.

TLR-3 activation inhibition and evasion of the immune reaction can occur when auxiliary proteins of the MERS-CoV occasionally bind to the viral dsRNA during replication, leading to interference with TLR-3 signaling. Proinflammatory cytokines may be activated by TLR-4 upon recognition of S protein via the MyD88-dependent signaling pathway. Strong synthesis of immunological mediators results from virus-cell interactions. Following MERS-CoV infection, infected cells are stimulated to release substantial quantities of chemokines (MCP-1, CXCL10) and cytokines (IL-10), which in turn attract lymphocytes and leukocytes to the infection site [79].

3.3 SARS-CoV-2

In the detection of PAMPs from SARS-CoV-2, several TLRs have been implicated, as indicated by recent studies. It has been indicated that an antiviral response to SARS-CoV-2 disease may be triggered by TLR (2, 3, 4, 7/8, and 9) [80]. While TLRs are found all over the human respiratory system, their expression varies among different innate immune cells. For instance, NK cells exhibit a higher abundance of TLR3, while macrophages predominantly express TLR4.

3.3.1 TLR2

In murine macrophages lacking TLR2 and in human macrophages administered with a TLR2 inhibitor exhibit reduced activation of pro-inflammatory signaling events and decreased cytokine production upon the stimulus of the SARS-CoV-2 E protein [81, 82]. This indicates that TLR2 plays a role in detecting the E protein of SARS-CoV-2 and initiating inflammatory responses. Moreover, in vivo, experiments demonstrate that the SARS-CoV-2 E protein promotes inflammation through TLR2-dependency, as evidenced by the decreased levels of IL-6 in the serum of TLR2−/− mice following administration of the E protein [81]. TLR2 plays a role in the activation of innate immunity, according to an independent single-cell computational approach that sought to anticipate potential sites for modification to lessen the dysfunctional innate immune reaction brought on by the virus [83]. These results are supported by the observation that TLR2 inhibitor therapy of K18-hACE2 transgenic mice lowers inflammatory cytokine levels in the blood and increases survivability after this viral infection [81]. To find out whether TLR2 directly interacts with the E protein or other ligands of this virus, more research is needed. Apart from TLR2, the extent of research on the involvement of other TLRs in SARS-CoV-2 infection is not as definitive in both in vitro and in vivo studies.

3.3.2 TLR4

In silico studies indicate that TLR (1, 4, and 6) may be potential binding partners for the SARS-CoV-2 S protein, with TLR4 exhibiting the maximum affinity [84]. The activation of TLR4 in reaction to the S protein is evidenced by the observation that the gene expression of Il1b is reduced in Tlr4−/− murine macrophages stimulated with the S protein compared to wild-type cells in vitro [85].

TLR4 exhibits a remarkable ability to identify various molecular patterns from disease-causing agents and initiates downstream signaling events that involve either of these (MyD88-dependent and MyD88-independent) signaling. These pathways activate TFs such as NF-κB, which govern the expression of the proinflammatory cytokine [86]. Recent findings indicate that the spike glycoprotein, a major infection-causing surface protein of SARS-CoV-2, acts as a ligand for human TLR4 [84], as a result of this protein-to-protein interaction, TFs, including NF-κB, IRFs, and activator protein 1 (AP-1), resulting in the expression of proinflammatory cytokines and IFNs [85]. Intense local inflammation caused by the activation of TLR4 in the alveolar macrophages eventually leads to an accumulation of inflammatory substances that disrupt gas exchange in the respiratory system, respiratory gas, induce issues in breathing, and sometimes result in mortality [84, 87]. The presence of inflammatory agents has the ability to stimulate the immune system including the innate and adaptive immune system, amplifying the inflammation and causing a “cytokine storm”, or overt immunopathology. The major organs such as the heart, kidney, pancreas, etc. are directly damaged by cytokine storm [88].

3.3.3 Intracellular Toll-like receptors

Intracellular TLRs (TLR3, TLR7, and TLR8) that detect nucleic acids are found in endosomes to avoid the detection of self-genetic material. Like other coronaviruses, SARS-CoV-2 is also a ssRNA virus and after replication in their host cells generates double-stranded RNA [89, 90]. Intracellular RNA sensors are likely to play a key role in SARS-CoV-2 infection identification. TLR3 recognizes dsRNA via the TRIF signaling pathway in endosomes and stimulates the production of type I IFNs and proinflammatory cytokines. While TLR7 and TLR8 use MyD88 downstream signaling pathways to recognize ssRNA of SARS-CoV-2. Recently three-dimensional lung multicellular spheroids were used to identify the antiviral role of TLR3 and TLR7 following COVID infection [90]. In SARS-CoV-2-infected multicellular spheroids, NF-κB and IRF3 were found to be involved in the TLR3 and TLR7-mediated downstream signaling. Further, the relative expression levels of both TLRs (TLR3 and TLR7) as well as the production of type I IFNs and proinflammatory cytokines were also high.

In vivo, studies have shown that the TLR3 signaling pathway plays a protective role in the context of SARS-CoV infection [42, 91, 92, 93]. TLR3-deficient mice (Tlr3−/−) exhibit higher viral load and reduced function when exposed to mouse-adapted SARS-CoV [42]. Furthermore, the activation of TLR3 with an agonist leads to a reduction in SARS-CoV load in human alveolar epithelial cells [92]. TLR3 has not been particularly connected with SARS-CoV-2 sensing in any investigations to date, though. Although a genomic investigation of individuals with severe COVID-19 discovered a link between innate defects in TLR3 and the severity of infectious illness, subsequent research was unable to confirm this link. As a result, it is unknown what role TLR3 plays in SARS-CoV-2 infection.

Another study revealed that genomic ssRNA fragments of SARS-CoV-2 infection activated the expression of TLR7 and TLR8 along with the MyD88 downstream signaling pathway in humans [94]. Because it was found that uridine and guanosine-rich RNA fragments activated the expression of TLR7 and TLR8, and to test their hypothesis this group searched the ligand of TLR7/8 and chose two UG-rich regions within the genome of SARS-CoV-2 and named as SC2-RNA. After treating human monocytes-derived DCs (MoDCs) with SC2-RNA the authors discovered that SCV2-RNA therapy promoted the release of the CXCL9 (T cell-recruiting chemokine) and pro-inflammatory cytokines expression including IL-6, IL-12, and TNF-α. After stimulation with SCV2-RNA, MoDCs matured and induced IFN-γ production in cocultured CD4 and CD8 T cells, indicating that SCV2-RNA may regulate DC activation. In pDCs, similar results were obtained as the expression of CD86 and production of TNF-α and IFN-α increased. The authors hypothesized that ssRNA-induced activation in MoDCs is mediated by the TLR8/MyD88/NF-Kb pathway and TLR7 in pDCs. These findings suggest that endosomal RNA sensors are responsible for recognizing the ssRNA and dsRNA produced by SARS-CoV-2.

4. Therapeutic interventions based on TLRs

TLR’s vital role in the viral recognition adaptive immunity regulation makes them strong candidates in vaccine formulation as vaccine adjuvants. TLR agonists are generally accepted because they do not cause side effects while offering dose-saving effects by increasing vaccination effectiveness. TLR4 and TLR9 agonists have been most efficient and have been approved for clinical usage while other TLR agonists including TLR3, TLR5, TLR7, and TLR8 require more research. Increased understanding of TLR expression and function, as well as their involvement in viral infection response, may lead to the creation of vaccination platforms including intrinsic TLR PAMPs TLR agonists to elicit efficient and lasting responses from the immune system [95].

TLR agonist adjuvants are more efficient in producing a humoral immune response as compared to first-generation adjuvants like aluminum adjuvants. Recently TLR agonists such as CpG oligonucleotide (ODN) Pam3CSK4, resiquimod (R848), poly(I:C), and monophosphoryl lipid A (MPLA) are being studied as vaccine adjuvants against SARS-CoV-2 infection. Pam3CSK4, a synthetic triacylated lipopeptide TLR1 & TLR2 ligand that can activate the proinflammatory transcription factor NF-B. XS15, a novel water-soluble synthetic Pam3CSK4-derivative, is being used in conjunction with spike protein peptides of SARS-CoV-2 to create a vaccine format that promotes CD4+ T-cell responses against peptides anticipated to bind to human leukocyte antigen – DR isotype (HLA-DR) [96].

LPS, a TLR4 ligand, can regulate the effector T-cell differentiation and influence inflammation. MPLA is a modified version of LPS that stimulates the immune system while avoiding most inflammatory effects. It is found most effective against hepatitis B and human papillomavirus as an adjuvant vaccine and is licensed for use against these viruses. In the previous study fusion of human IgG FC region (S377-588-Fc) and Alum-adjuvanted spike protein of Middle East respiratory syndrome coronavirus produced a significantly higher titer of specific IgG antibodies against this virus as compared to MPLA-adjuvanted protein. Similarly, a higher Th2 (IgG1)-biased response was evoked followed by MPLA-adjuvanted S377-588-Fc protein [97]. A biomaterial-based COVID-19 vaccine also showed a strong adaptive immune response. Biomaterial mesoporous silica rods loaded with SARS-CoV-2 viral protein, a growth factor (granulocyte-macrophage colony-stimulating factor) and MPLA showed an effective immune response by delayed releases after making subcutaneous scaffoldings. These biomaterials recruited the antigen-presenting cells on the local site and produced a strong adaptive immune response [98].

SARS-CoV-2 continues to mutate; however, MPLA-adjuvanted antigens such as S-trimer/MPLA, RBD/MPLA, and S1/MPLA continue to elicit significant cellular and humoral and cellular immune responses against spike variants (alpha, beta, gamma, delta, and omicron) [99].

TLR7 and TLR8 activation could be examined as a new technique for anti-SARS-CoV-2 vaccinations to solve the present problem of viral escape from the immune system. TLR7 activation exerts an antiviral immune response through Th1 while also exerting favorable broncho-vasodilatory effects [100]. Iquimod, a synthetic TLR7 ligand, has been licensed against perianal and genital wart infection induced by human papillomavirus.

A synthetic agonist of TLR7 and TLR8 named resiquimod has been developed that elicits an effective immune response by IL-6, IFN-α, IFN-β, and proliferation of B cell and T cells. Cytokine profiles induced by the mRNA COVID-19 vaccine and resiquimod are the same [101]. The complex of SARS-CoV-2 spike protein S1 subunit, resiquimod, and nanoparticle-based SARS-CoV-2 virus vaccine exert a greater immune response by activation and maturation of antigen-presenting cells, systemic T cells, and specific B cells response as compared to nanoparticles alone [102].

Anti-coronavirus immunity is most likely provided by CD8+ T cells and neutralizing antibodies. TLR3 and TLR9 ligands Poly(I:C) and CpG ODN and Poly(I:C) significantly increase CD8+ T-cell responses more than other adjuvants among TLR agonists. These ligands CpG ODN and poly(I:C) have therefore been used as adjuvants in vaccines against the influenza virus. Inactivated SARS-CoV-2 vaccine combined with CpG ODN has exponential effectiveness by improving the humoral immune response when administered in animals [103, 104]. Boosting with nanoparticles formulated with CpG 1018 and poly(I:C) results in enhanced T cells and B cell activation and IFN- production, as compared to the alum adjuvanted SARS-CoV-2 S1 vaccination [105].

5. Conclusion

TLRs play a vital role in the innate immune system and recognize a wide range of PAMPs. This chapter has described the role of TRLs in the immunity against emerging viral infections including SARC CoV, MERS CoV, and SARC CoV-2. TLRs (2, 3, 4, 7, 8, and 9) are considered very effective against these emerging viral pathogens. TLR 3 is TRIF dependent while TLRs 7 and 8 are MyD88 dependent for the downstream signaling. Viral components including E protein, S protein, and viral genetic material (ssRNA and dsRNA intermediate) act as ligands for these TLRs. Upon viral invasion, TLRs recognize the infectious agent trigger the downstream signaling, and ultimately initiate the immune response. As a result, the production of pro-inflammatory cytokines and type I interferons (IFNs) starts that ultimately initiate the antiviral immune response to clear the invading agent. However overactive immune system activity may lead to detrimental production of pro-inflammatory cytokines and chemokines that can lead to severe illness and widespread inflammation. TLR antagonists are found satisfactory against these severe infections and most of these therapeutics clinical trials are under different phases.