1.3.1 Toll-Like Receptors (TLRs)

The TLR family was the first group of PRRs to be characterized. The Toll gene was first discovered in Drosophila. Due to the similarity of the cell surface receptors to the toll protein they are named as Toll Like Receptors. So far, 222 TLRs have been characterized in invertebrates and 28 TLRs in vertebrates. Among these, 13 TLRs are found in mammals and humans possess only 10 [11]. All innate immune cells, i.e. dendritic cells (DCs), natural killer (NK) cells, macrophages, neutrophils, mast cells, eosinophils and basophils, express TLRs [12]. TLRs are classified based on their location into membrane-bound or intracellular endosome TLRs. TLR1, 2, 4, 5, 6, and 10 are cell membrane TLRs, while TLR3, 7, 8, and 9 are intracellular TLRs [13]. TLRs have three structural domains, that is, the N terminal domain located on the surface of the membrane for recognition of PAMPs and ligand binding. A transmembrane domain and C terminal cytoplasmic intracellular domain for downstream signaling [14]. TLRs activate two pathways depending upon stimulation with a particular microbial ligand. One is the MyD88-dependent pathway leading to the production of inflammatory cytokines. Apart from TLR3, all TLRs utilize this pathway. The second pathway is TRIF-dependent, associated with the stimulation of interferon type-1 and is used by TLR3 and 4 [15]. The signaling cascade depends upon the ligand interacting TLR type, that carries a leucine-rich repeat (LRR) segment. It recognizes PAMP/DAMP and downstream signaling molecules leading to an inflammatory response. This makes TLRs an excellent target for adjuvants to induce immune response effectively.

TLR1/2 forms a heterodimer recognizing various bacterial lipid structures and cell wall components like triacylated lipoproteins, lipoteichoic acid, and β-glucans. Pam3CSK4 (Pam3-Cys-Ser-Lys4) is a synthetic bacterial lipopeptide and is a well-characterized potential activator of TLR1/2. It is proved that Pam3CSK4 enhances the binding of respiratory syncytial virus. to its target cell, eventually initiating a robust immune response [16]. SMP105 is a TLR2 agonist isolated from M. bovis BCG Tokyo. It is proven to enhance the immune response by activating the nuclear factor-kB promoter and eventually enhancing several interferon-γ producing cells and CTLs [17]. CBLB612, ISA201 and OPN305 are synthetic ligands for TLR2. These agonizts are in the phase 2 clinical trial for cancer therapy [18]. Poly (I:C), the TLR3 agonist not only activates the immune system but also offers apoptosis of cancer cells. Studies have shown that the combination of IFN- α along with poly (I:C) is the most effective strategy for apoptosis of cancerous cells [19]. Studies focusing on the use of poly(IC:LC), a synthetic derivative of poly(I:C), showed that poly(IC:LC), along with antigen-pulsed DCs, could boost antitumour immunity, i.e. expand tumor-reactive T cells in patients with pancreatic cancer. Modified version of poly(I:C)- poly(IC₁₂U), induces the production of 2′,5′-OAS, which serves as an antiviral effector [20]. ARNAX, another TLR3 agonist enhances memory CD8 + T cells and rejects tumor re implantation [21]. TLR4 agonist 3-O-desacyl-4′-monophosphoryl lipid A (MPLA) effectively cleared bacterial infection with the help of myeloid cells with effective phagocytic functions [22]. FP molecules, synthetic monosaccharide-based TLR4 agonizts, bind to the TLR4/MD-2 dimer with high affinity, stabilize its active conformation. This binding activate MyD88- and TRIF-dependent signaling pathways and the NLRP3 inflammasome. This exhibits adjuvant activity with antibody response with potency comparable to MPLA, while showing no in vivo toxicity [23]. Research on improved tuberculosis vaccines used GLA-SE adjuvant and TLR4 agonist, which proved to induce robust T-helper cellular immune response [24]. Adjuvant System 04 (AS04) is a well-known TLR4 agonist-based adjuvant licensed for use in human vaccines. It incorporates MPLA and aluminum salt [25]. OK-432 is yet another synthetic TLR4 agonist which is proven to have immunostimulatory effects [26]. A derivative of flagellin, CBLB502 is a TLR5 stimulant being developed for anticancer applications [27]. Mobilan-VM3 is a secreted version of TLR5 ligand is demonstrated to be useful not only as a therapy, but also as a prophylactic anticancer vaccine [27]. 852A, the TLR7 agonist prevents in vitro proliferation of some tumor cells (Hs294T and 769-P) and delays tumor growth in vivo in a DCs and interferon-α-dependent manner [28]. Another TLR7 immune system modifier, gardiquimod, serves as a reverse transcriptase inhibitor, offering a promising path for therapeutic development against HIV-1 [29]. Motolimod, also known as VTX-2337, is a TLR8 activator. It initiates the immune response with the help of effectors immune cells such as NK cells, DCs, and monocytes [30]. Another TLR 8 agonist VTX-1463, an intranasal formulation, is in Phase I clinical evaluation for allergic rhinitis. VTX-763, a member of the VTX family, is in preclinical studies for its potential to decrease the severity of autoimmune inflammation. It is also demonstrated to activate both neonatal and adult leukocytes [31]. R848, one more TLR8 agonist provides antitumor properties in experimental models of breast cancer [32]. It has been demonstrated that CpG oligodeoxynucleotides stimulate TLR9. This causes B cells to secret interleukin-6. Research proved that a low dose of radiation along with CpG-C ODN leads to reduced T regulatory cell counts and induction of tumor-specific CD8+ and CD4+ effector T-cells [33]. In preclinical studies on a DNA-based TLR9 agonist, MGN1703 shown promise in anticancer therapy [34].

1.3.2 Retinoic Acid-Inducible Gene 1 (RIG-I)-Like Receptors (RLRs)

TLRs can recognize viruses present on the cell surface or those which are in the endosomal compartment of the host cell. However, viruses usually replicate inside the cell. Hence effective intracellular receptors are required for an effective antiviral response. RIG-I recognizes viral RNA and is specific to RNA viruses. RIG-1 does not bind to viral dsDNA except for Epstein-Barr virus (EBV). RIG-I was first discovered in the cells of acute promyelocytic leukemia induced by retinoic acid [35]. RIG-like receptors (RLRs) comprises of RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of Genetics and Physiology 2 (LGP2) [36]. The structures of all three RLRs are similar, consisting of a C-terminal domain called the repressor domain (RD) and a DExD/H-box helicase domain. RIG-I and MDA5 have caspase activation and recruitment domains (CARDs), which carry out downstream signaling cascades. LGP2 lacks a CARD domain. The CARDs engage with the CARD of the protein known as the mitochondrial activator of viral signaling (MAVS), a crucial signaling adapter protein for RLRs. The signaling cascade leads to the transcription of various innate immune response genes, including IFNs, direct antiviral genes, and pro inflammatory genes [37]. The expression of RLR is usually low in resting cells, but it increases after exposure to a viral infection or IFN. RLR signaling is enhanced by ssRNA PAMPs as observed in influenza virus, Ebola virus and other RNA viruses by poly-uridine-rich RNA motifs [38]. RIG-I in the cell primarily recognizes influenza, vesicular stomatitis, Sendai, and Japanese encephalitis viruses. MDA5 mainly detects short RNA viruses like poliovirus. MDA5 also produces polycytidylic acid (poly I:C), the dsRNA analog [36]. Separate but overlapping groups of viruses activate RIG-I and MDA5. Although they share similar structure and function, they identify different RNA ligands. RIG-I strongly recognizes ligands such as blunt-ended dsRNAs of at least 10 bp with a 5^| triphosphate group. RIG-I is also activated by long dsRNA molecules that are more than 200 bp long, regardless of the 5^| end, as well as 30-monophosphorylated ssRNAs and poly U/UC-rich stretches of ssRNA. Certain sections of negative sense viral RNAs can activate MDA5, which can detect lengthy dsRNA molecules, preferably with higher-order RNA structures. AU-rich ssRNAs are likewise preferred by both RLRs [38].

1.3.3 Nucleotide-Binding and Oligomerization Domain NLRs

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular PRR in the cytosol. These receptors are essential in recognizing PAMPs associated with bacterial origin [39]. NLRs were first discovered in plants as resistance (R) genes. These R genes play a vital role in plant defense against microbial and parasitic pathogens [40]. To date 23 NLR family members are discovered in human genome [40]. NLRs are predominantly expressed on phagocytes. They are also expressed on macrophages and neutrophils. NLR proteins have central NOD NACHT that is, NLR apoptosis inhibitory protein (NAIP), Class II Transactivator (CIITA), heterokaryon incompatibility protein (HET), telomerase-associated protein 2 (TP-2), N-terminal effector domain, and C-terminal LRRs. Oligomerization and dNTPase activity are the two essential roles of NACHT, while the C-terminal LRR domain is involved in ligand binding and activator sensing. N-terminal domain interacts with other proteins and performs effector functions. Based on the N-terminal domain, the NLRs are classified into four sub-families: the baculoviral inhibitory repeat-like domain (NLRB), the acidic transactivation domain (NLRA), the caspase activation and recruitment domain (CARD; NLRC) and the pyrin domain (NLRP). Most NLRs act as PRRs and detect various ligands from the host cells (ATPs, uric acid etc.), microbial components (peptidoglycan, flagellin), while some instead respond to cytokines such as interferons. The activated NLRs are also broadly divided into four functional categories: inflammasome assembly involved in activation of caspase-1(results in processing and maturation of pro-inflammatory cytokine), signaling transduction, transcription activation and autophagy. NOD1 detects d-glutamyl-meso-diaminopimelic acid (iE-DAP), a peptidoglycan component that is found only in Gram-negative bacteria, and NOD2 recognizes muramyl dipeptide (MDP) from both gram negative and gram-positive bacteria [41]. Stimulation of specific NLRs such as NOD1 and NOD2 triggers the activation of a signaling cascade, which activates NF-κB and MAPKs. These pathways eventually lead to the transcription of various genes involved in both innate and adaptive immune responses [42]. Research demonstrates that, genetic variations within NLR genes have been linked with immune dysregulation, contributing to the development or increased susceptibility to various inflammatory diseases [43]. NOD1 and NOD2 agonizts can synergize with ligands for TLRs. This synergy leads to the enhanced production of pro inflammatory cytokines and antimicrobial molecules. Additionally, NOD1 stimulation has been shown to drive Th2-dependent antigen-specific adaptive immunity. This suggests that NLRs can influence the development of different T helper cell subsets (Th1, Th2, Th17) involved in adaptive immune responses, potentially in cooperation with TLR signaling [44]. NOD1 agonizts FK565, FK156 and NOD2 agonist MDP mimic the peptidoglycan layer of bacteria. These agonizts are proven to be potent adjuvants that can trigger cell-mediated immunity. These agonizts and TLR3, TLR4, and TLR9 immunostimulatory molecules induce DCs to produce IL-12 and IFN-γ and eventually promote Th1-lineage immune responses [45].

1.3.4 C-Type Lectin Receptors (CLRs)

CLRs are a subfamily of PRRs that mainly recognize mannose, fucose and glucan carbohydrate structures. They are expressed in immune cells such as macrophages, DCs, monocytes, and Langerhans cells (LCs) [46]. CLRs have one or more carbohydrate recognition domains (CRD) that can bind to carbohydrates, proteins, lipid molecules and inorganic compounds in a Ca2 + -dependent or Ca2 + -independent manner. CLRs are classified as type I transmembrane proteins with multiple carbohydrate recognition domain (CRDs) and type II transmembrane proteins with a single CRD [47]. Important subclasses of CLR include Langerin, DC-SIGN, Dectin-1, Dectin-2, DCIR, DCAR, and BDCA-2 [48]. These are present on antigen presenting cells, which recognize various PAMPs and play crucial roles in shaping immune responses. These CLRs bind to specific sugars on antigens and trigger the signaling cascade, like uptake, presentation to T cells, cytokine production, and immune modulation. These processes contribute to the intricate interplay between the immune system and infectious agents. CLRs trigger an immune response through two different pathways: (a) immunoreceptor tyrosine-based activation motifs (ITAMs), which induce the activation pathway and (b) myeloid inhibitory C-type lectin (MICL) receptor pathway, which suppresses the inflammatory pathways induced by the activating receptors [48]. CLR can enhance vaccine efficacy by using synthetic agonizts like macrophage-inducible C-type lectin (Mincle) agonist trehalose dibehenate (tDB). Trehalose dibehenate is a synthetic CLR ligand proven to activate new-born human DCs in vitro [49].

1.3.5 Stimulator of IFN Genes (STING) and Cytosolic DNA Sensors

The STING is an intracellular PRR present in the endoplasmic reticulum. It induces the production of cytokines such as type I interferon [50]. The cyclic GMP-AMP synthase (cGAS-STING) signaling pathway initiates with the entrance of pathogenic DNA into the host cytoplasm through infection. GCS catalyzes the synthesis of cGAMP, which binds to STING. STING eventually recruits IκB Kinase (IKK) and TANK-binding Kinase 1 (TBK1). IKK and TBK1 phosphorylates Interferon Regulatory Factor 3 (IRF3) which along with other transcription factors induce the expression of interferons and inflammatory cytokines such as tumor necrosis factor (TNF), IL-1b and IL-6 [51]. STING agonizts are predominantly involved in tumor-targeted therapeutics. DMXAA, a STING agonist, proven to reduce and regress tumor growth [52]. ADU-S100, another agonist induce the local production of antiangiogenic factors [53]. MK-2118, SB11285, GSK3745417, BMS-986301, BI-STING (BI 1387446), E7766, TAK-676, SNX281, SYNB1891, BMS-986301 are the STING agonizts in clinical development against different types of tumor and anticancer therapies [54].

1.3.6 PYHIN Family

The PYHIN family consists of DNA sensors activated by pathogenic infections and cellular stress conditions, such as DNA damage or breaks. These sensors play a crucial role in the innate immune system by detecting cellular damage. In humans, key members of this family include Absent in Melanoma 2 (AIM2), Interferon Gamma Inducible Protein 16 (IFI16), and Myeloid Cell Nuclear Differentiation Antigen (MNDA). AIM2 and IFI16 are well-known for detecting viral or abnormal dsDNA, whereas MNDA regulates immune responses in the myeloid cells [55].

AIM2 is found to be active in assembling inflammasome in response to pathogens such as Francisella tularensis, Listeria monocytogenes, and Staphylococcus aureus. AIM2 is also linked to various conditions, including lupus, psoriasis, cervical cancer and cancers of colon and prostate. It senses dsDNA from viruses such as HPV, HBV and EBV contributing to immune responses. AIM2's molecular mechanism involves binding to dsDNA via its HIN domain, triggering inflammasome assembly with Apoptosis-Associated Speck-like Protein containing a CARD and pro-caspase-1, leading to inflammation. Additionally, AIM2 can promote T regulatory cell stability during inflammation and has been implicated in atherosclerosis progression [55]. IFI16 is a protein involved in the cell cycle regulation and transcriptional control. It contains two hematopoietic interferon-inducible nuclear (HIN) domains (HINa and HINb) interacting with transcription factors Sp1 and p53, and inhibits the oncogenes such as c-MYC and RAS, eventually supressing the tumor growth. IFI16 plays a critical role in innate immunity by sensing dsDNA from viruses like Herpes Simplex Virus Type 1, Kaposi's Sarcoma-Associated Herpes virus, and Human Immunodeficiency Virus (HIV) as well as ssDNA from the damaged cells. Upon detecting viral DNA, IFI16 forms inflammasomes and activates immune pathways, such as the STING/TBK1 pathway, leading to interferon production [56].