From defense to offense: Modulating toll-like receptors to combat arbovirus infections

ABSTRACT

Arboviruses are a significant threat to global public health, with outbreaks occurring worldwide. Toll-like receptors (TLRs) play a crucial role in the innate immune response against these viruses by recognizing pathogen-associated molecular patterns and initiating an inflammatory response. Significantly, TLRs commonly implicated in the immune response against viral infections include TLR2, TLR4, TLR6, TLR3, TLR7, and TLR8; limiting or allowing them to replicate and spread within the host. Modulating TLRs has emerged as a promising approach to combat arbovirus infections. This review summarizes recent advances in TLR modulation as a therapeutic target in arbovirus infections. Studies have shown that the activation of TLRs can enhance the immune response against arbovirus infections, leading to increased viral clearance and protection against disease. Conversely, inhibition of TLRs can reduce the excessive inflammation and tissue damage associated with arbovirus infection. Modulating TLRs represents a potential therapeutic strategy to combat arbovirus infections.

KEYWORDS:

• Infectious disease
• arboviruses
• toll-like receptors
• modulation
• agonist
• antagonist
• adjuvant

Introduction

Toll-like receptors

Toll-like receptors (TLRs) were first discovered in 1997 and the discovery was a significant breakthrough in immunology, as it provided insights into how the immune system recognizes and responds to pathogens.¹ Hoffmann and his colleagues were studying the innate immune system of fruit flies (Drosophila melanogaster) when they found that a gene called Toll, which had previously been known for its role in embryonic development in fruit flies, also played a role in the immune response.¹ Observation of the mutations in the Toll gene resulted in increased susceptibility to fungal infections in fruit flies and this led to the investigation of Toll function in the innate immune response of other organisms, including humans.² Although toll-like receptors are highly expressed on innate immune cells such as macrophages and neutrophils, they are also present in certain nonimmune cells including epithelial cells.³ These receptors recognize pathogen-associated molecular patterns (PAMPs), such as viral structural proteins, lipopolysaccharides, and flagellin, which are present on the surface of many types of bacteria and viruses.⁴ The activation of TLRs triggered an immune response that included the production of cytokines, chemokines, and other inflammatory mediators.⁵ The discovery of TLRs opened new avenues for understanding the mechanisms underlying innate immunity and paved the way for the development of new strategies for combating infectious diseases.

In humans, 10 known TLRs have been identified so far, and classified into two groups namely transmembrane and intracellular TLRs. The former comprises TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 whilst the latter consists of TLR3, TLR7, TLR8 and TLR9.⁶ Each receptor has a distinct role in the recognition and response to different types of pathogens.⁷ TLRs function by recognizing specific PAMPs that are present on the surface of various pathogens, such as bacteria, viruses, and fungi.⁸

TLRs on the cell surface predominantly recognize membrane components of the said microbes. To illustrate, TLR2 forms dimers with TLR1 or TLR6 to recognize a diverse array of PAMPs including peptidoglycans, lipoproteins, mannan, and fungal zymosan.⁹ On the other hand, intracellular TLRs in the endosomes recognize nucleic acid from viruses and bacteria, in addition to self-nucleic acids from damaged cells known as damage-associated molecular patterns (DAMPs).¹⁰ Table 1 provides an overview of the natural ligands associated with the respective TLRs.

Table 1. The natural ligands associated with each TLRs.

TLRs	Natural ligands and references
TLR1/2 heterodimer	Bacterial triacyl lipopeptides^Citation11
TLR2/6 heterodimer	Bacterial diacylated lipopeptides,^Citation11 fungal zymosan,^Citation12 bacterial modulin,^Citation13 bacterial lipoteichoic acid (LTA)^Citation14
TLR3	Viral double-stranded RNA (dsRNA),^Citation15 polyinosinic-polycytidylic acid,^Citation15 mRNA^Citation16
TLR4	Bacterial LPS,^Citation14 fungal mannan^Citation14
TLR5	Bacterial flagellin^Citation14
TLR7	Viral single-stranded RNA (ssRNA),^Citation14 small interfering RNA (siRNA)^Citation17
TLR8	Viral single-stranded RNA (ssRNA),^Citation14 small interfering RNA (siRNA)^Citation17
TLR9	Bacterial and viral DNA,^Citation14 HMGB1,^Citation18 CpG motifs^Citation14
TLR10	Unknown natural ligands

When a TLR recognizes a PAMP, it triggers a signaling cascade within the immune cell that leads to the activation of transcription factors and the production of cytokines and chemokines, which help to coordinate an immune response against the invading pathogen.¹⁹ TLRs are made up of several different structural domains, including a transmembrane domain, an extracellular domain, and an intracellular signaling domain.²⁰ The extracellular domain of the TLR is responsible for recognizing and binding to PAMPs.²¹ This domain is composed of leucine-rich repeats (LRRs), which form a horseshoe-shaped structure that is thought to interact with the PAMPs.²²

The intracellular signaling domain of the TLR is responsible for initiating an immune response once the TLR has bound to a PAMP.¹⁹ This domain is composed of a Toll/interleukin-1 receptor (TIR) domain, which interacts with downstream signaling molecules to activate immune cells and trigger the release of cytokines.²³ In addition to the LRRs and TIR domain, some TLRs also contain additional domains that can help to enhance their activity. For example, TLR4 has a co-receptor called MD-2 that can help to bind to lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria.²⁴ As previously mentioned, TLRs also play a role in the recognition of endogenous molecules that are released by damaged or stressed cells, known as DAMPs.²¹ This recognition can lead to inflammation and tissue repair processes.²⁵

TLRs work by using adaptor molecules to initiate downstream signaling cascades. The signaling cascades initiated by TLRs and their adaptor molecules are complex and involve multiple steps. There are several different adaptor molecules that can be used by TLRs, but the two main ones are MyD88 and TRIF.²⁶ When a TLR recognizes a PAMP on the surface of a pathogen, it undergoes a conformational change that allows it to recruit an adaptor molecule to its intracellular signaling domain.^27,28 The adaptor molecule then interacts with downstream signaling molecules, leading to the activation of transcription factors and the expression of genes involved in the immune response.^27,28 The adaptor molecule MyD88 is used by most TLRs, apart from TLR3, which uses the adaptor molecule TRIF.²⁵ Once recruited to the TLR, MyD88 interacts with the interleukin-1 receptor-associated kinase (IRAK) family of proteins, leading to their activation and subsequent phosphorylation of the transcription factor NF-κB. This results in the expression of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α).^27–30

In contrast, TRIF recruits another downstream signaling molecule called TRAF3, which leads to the activation of interferon regulatory factor 3 (IRF3) and the expression of type I interferons (IFN-α and IFN-β).³¹ These interferons play a crucial role in the antiviral immune response by inducing the expression of genes involved in viral clearance and the activation of immune cells.³² In addition to MyD88 and TRIF, there are several other adaptor molecules that can be used by TLRs, such as TRAM and TIRAP.³³ These adaptor molecules can also interact with downstream signaling molecules to activate various immune responses.³³ Overall, TLRs are important components of the innate immune system, helping to identify and respond to a wide range of potential pathogens and maintain tissue homeostasis.

Arboviruses

Viruses, including arboviruses, are small obligate intracellular parasites, relying on host cells for multiplication and subsequent spread of infection within the host. The seven stages of viral replication are attachment, penetration, uncoating, replication, assembly, maturation, and release. This interaction with host cells inevitably triggers the activation of TLRs via the various viral PAMPs at different stages.³⁴ Arboviruses are RNA viruses that are transmitted to humans and other animals by arthropods, such as mosquitoes, ticks, and sandflies.³⁵ The term “arbovirus” is derived from “arthropod-borne virus.” Arboviruses can cause a range of illnesses in humans, from mild febrile illnesses to severe neurological diseases.³⁶ Examples of arboviruses that affect humans include the dengue virus (DENV), Yellow Fever virus (YFV), Zika virus (ZIKV), West Nile virus (WNV), and Chikungunya virus (CHIKV).³⁷ The geographic distribution of arboviruses is determined by the distribution of the arthropod vectors that transmit them. DENV from the family Flaviviridae is a positive single-strand RNA. It is transmitted by Aedes mosquitoes and is endemic in tropical and subtropical regions around the world, particularly in Southeast Asia, the Western Pacific, and the Americas.³⁸ DENV can cause a range of clinical symptoms, from mild febrile illness to severe dengue hemorrhagic fever and dengue shock syndrome, which can be fatal.³⁸ Dengue virus infections are caused by any of the four serotypes (DENV 1–4) of the virus. However, when someone gets infected by a certain serotype, they will only gain lifelong immunity to that specific serotype and not to others.³⁹

ZIKV is another single-stranded positive-sense RNA virus that belongs to the family Flaviviridae.⁴⁰ This virus has evolved into two genotypes, namely the African and Asian genotypes.⁴¹ It is transmitted by Aedes mosquitoes and has been responsible for recent outbreaks in the Americas, Africa, and Asia.³⁴ ZIKV infection is usually mild and self-limiting, but it can cause neurological complications, such as Guillain-Barré syndrome and congenital Zika syndrome, which can result in severe birth defects in newborns.⁴² CHIKV is an enveloped, positive single-stranded RNA virus from the family Togaviridae.⁴³ Three major genotypes have been identified which are West African, East/Central/South African (ECSA), and Asian genotypes.⁴⁴ It is transmitted by Aedes mosquitoes and is endemic in Africa, Asia, and the Indian subcontinent.⁴⁵ In recent years, CHIKV has caused large outbreaks in the Caribbean and South America.⁴⁵ CHIKV infection can cause severe joint pain, fever, rash, and in some cases, long-term joint pain, and disability.⁴⁵ WNV from the family Flaviviridae is an enveloped, positive single-strand RNA, and is transmitted by Culex mosquitoes and is found in Africa, Europe, the Middle East, and North America.^46,47 WNV infection is usually asymptomatic or mild, but in some cases, it can cause severe neurological diseases, such as encephalitis and meningitis.⁴⁷ Belonging to the family Flaviviridae, Yellow fever virus (YFV) shares the characteristic of being an enveloped, single-stranded positive RNA virus.⁴⁸ Thus far, 7 genotypes have been described including five African lineages and two South American.⁴⁹ It is transmitted by Aedes and Haemagogus mosquitoes and is endemic in tropical regions of Africa and South America.⁵⁰ YFV infection can cause a range of clinical symptoms, from mild febrile illness to severe hemorrhagic fever and liver failure, which can be fatal.⁵⁰

The geographical distribution and impact of arboviruses vary depending on the specific virus and its transmission cycle, the distribution of the arthropod vector, and the susceptibility of the human population.⁵¹ There are several factors that have contributed to the spread of arboviruses by their vectors, such as globalization and increased travel, urbanization and population growth, climate change, and changes in land use.⁵¹ Various measures have been taken to control the spread of arboviruses and their vectors including vector control, vaccine and therapeutic development, surveillance, and early warning systems, public education, and behavior change as well as research and innovation.⁵² Despite these measures, the continued emergence and spread of arboviruses highlight the need for sustained investment in research, surveillance, and public health interventions to address this ongoing global health threat.

Methods

Literature was searched in Web of Science, PubMed, and Google Scholar, using the combinations (benefitting from Boolean operators: AND, OR) of the following search terms: TLR, arbovirus, DENV, ZIKV, WNV, CHIKV, YFV, innate immunity, antiviral, agonist, and antagonist. The references were checked from all sources to find eligible articles. The reference lists of all articles published in peer-reviewed journals selected were reviewed 31,108 not published in English were excluded, all the duplicates were excluded, and a total of 72,347 references were identified. Among these eligible references, only 22 summarized or investigated the pathway system of toll-like receptors, 17 discussed the general information about arboviruses, 19 offered detailed research addressing the interplay between innate immunity on DENV, 16 on ZIKV, 9 on WNV, 14 on CHIKV, and 8 on YFV. Also, 13 publications discussed innate immunity in general, 7 on its perspective to develop antivirals, 13 studies on existing agonists, and 13 on existing antagonists. (Figure 1). A total of 123 high-quality research results published in authoritative journals were summarized and preferred cited in the review. In addition to searching sources in Web of Science, PubMed, and Google Scholar, we actively tracked the latest updates on outbreaks and case progress by regularly visiting the websites of reputable organizations such as the World Health Organization (WHO), the United States Centers for Disease Control and Prevention (CDC), and the Ministry of Health, Malaysia (MOH).

Figure 1. Flowchart of database search, screening, and selection process. This figure depicts the systematic approach used to identify, screen, and select relevant studies for inclusion in the review manuscript.

The relationship between TLR and arbovirus infection

TLRs elicit immune responses during viral infections by activating their respective signaling pathways which promotes the production and release of inflammatory cytokines and chemokines.⁵³ Despite their important role in producing the protective effect of immune defenses, exaggerated activation may lead to undesirable and harmful effects such as in West Nile Virus, where TLR3-dependent inflammation lead to lethal encephalitis.⁵⁴

Previous research has established that during dengue virus infection, the signaling pathways of TLR2 and TLR6 were up-regulated via the presence of DENV non-structural protein 1 (NS1), which is essential for virus replication. This in turn increases the IL-6 and TNF-α production by peripheral blood mononuclear cells (PBMC) which is attributed to increased vascular permeability and leakage.⁵⁵ On the contrary, another study suggested that instead of the aforementioned TLR2 and TLR6, TLR4 is the receptor that triggers the inflammatory cytokine release in the presence of NS1.⁵⁶ Additionally, studies have shown that TLR3,^57–60 TLR7,^{57,58,61–63} and TLR8^57,64–66 are involved in the recognition of DENV RNA and the activation of downstream signaling pathways, leading to the production of antiviral cytokines and type I interferons (IFN). IL-8 and distinct IFNα/β release are triggered after viral recognition in human monocytic cells by TLR3 following endosomal acidification.⁵⁷ Strong signaling through TLR3 and RIG-1, but not Mda5, was experienced by skin fibroblasts infected with DENV-2, leading to over-expression of IFNβ, TNFα, defensin 5 (HB5), and β-defensin 2 (HβD2).⁵⁸ Moreover, DENV-infected fibroblasts displayed enhanced nuclear translocation of IFN regulatory factor 3 (IRF3) but not interferon regulatory factor 7 (IRF7) compared to mock-infected fibroblasts.⁵⁸ The TLR-induced response was specifically characterized by increases in myeloid DC subset activation together with increased serum levels of CXCL-10 and IL-1Ra.⁵⁹ An experimental indication that poly (I:C) may be a promising immunomodulatory drug against DENV infection and might be useful for clinical prophylaxis is that TLR3 activation is efficient in preventing DENV2 replication via IFN-β.⁶⁰ It is likely that certain genotypes of TLR7 and 8 polymorphisms contribute to elevated dengue/chikungunya virus loads in patients who are also infected with those diseases.⁶¹ Uncleaved prM-containing immature particles may help to control the course of dengue virus infection by functioning as a carrier to the endolysosome-localized TLR7 sensor.⁶² As a first line of innate defense against DENV infection in vivo, TLR7‘s RNA helicase-mediated sensing works in a tissue-dependent manner.⁶³

However, some studies have suggested that DENV can also evade or modulate the TLR pathway to promote viral replication and evasion of the host immune response. For example, DENV can induce the expression of suppressor of cytokine signaling (SOCS) proteins, which can inhibit the TLR signaling pathway and the production of antiviral cytokines.⁶⁷ One of the mechanisms used by DENV involves the expression of viral proteins that can interfere with the TLR signaling pathway. For example, DENV non-structural protein 4B (NS4B) has been shown to inhibit the TLR3-mediated activation of interferon regulatory factor 3 (IRF3) and the production of type I interferons.⁶⁸ DENV NS1 can also inhibit the TLR3-mediated induction of interferon-stimulated genes (ISGs) and the production of type I interferons.⁶⁹ In addition, the DENV envelope (E) protein has been shown to inhibit TLR4-mediated activation of NF-κB and the production of pro-inflammatory cytokines, such as IL-6 and TNF-α.⁷⁰ These viral proteins may interfere with the TLR pathway to allow for increased viral replication and evasion of the host immune response.

In the case of ZIKV infection, TLR3 and TLR7 have been found to recognize the viral RNA, initiating a signaling cascade that leads to the production of pro-inflammatory cytokines and interferons.^71,72 TLR3 recognizes double-stranded RNA, which is a viral replication intermediate, while TLR7 recognizes single-stranded RNA, such as that produced during viral transcription. Recent studies suggest that TLRs may also play a role in the pathogenesis of ZIKV infection, with aberrant activation of TLR signaling pathways contributing to disease severity.⁷³ The mechanisms by which ZIKV proteins interact with TLR pathways are complex and not fully understood. The NS5 protein of ZIKV has been shown to inhibit TLR3-mediated signaling, which is involved in the innate immune response to viruses.⁷⁴ This inhibition is thought to help the virus evade detection by the host immune system. The NS3 protein of ZIKV has also been shown to interact with TLR3, TLR7, and TLR8.⁷⁵ This interaction leads to the activation of the NF-κB and IRF3 pathways, which are involved in the production of pro-inflammatory cytokines and type I interferons. These cytokines and interferons are important for the clearance of viral infections. On the other hand, the ZIKV envelope protein (E protein) has been shown to activate the TLR2 pathway, leading to the induction of pro-inflammatory cytokines.⁷⁶ This activation is thought to contribute to the immune response to ZIKV infection and can help limit viral replication.⁷⁶

WNV is known to activate TLR pathways in infected cells. The viral envelope protein (E protein) of WNV has been shown to activate the TLR3 pathway, leading to the production of interferons and other pro-inflammatory cytokines.⁷⁷ These cytokines play a role in the host’s immune response to viral infection, limiting viral replication. The WNV NS5 protein has been shown to interact with the TLR3 pathway, inhibiting the production of type I interferons.⁷⁸ This is thought to help the virus evade detection by the host immune system, and contribute to the pathogenesis of WNV infection. The WNV NS4B protein has also been shown to interact with the TLR3 pathway, inhibiting the activation of IRF3, a transcription factor involved in the production of type I interferons.⁷⁹ This interaction is thought to contribute to the ability of WNV to evade the host immune response and establish infection.

Research has shown that the CHIKV E2 protein interacts with TLR2, leading to the production of pro-inflammatory cytokines such as IL-6 and TNF-α.⁸⁰ In addition, CHIKV has been shown to activate the TLR4 pathway, leading to the production of pro-inflammatory cytokines and type I interferons.⁸¹ CHIKV may “hijack” the TLR pathways in infected cells to evade the host immune response.⁸² For example, the CHIKV nsP2 protein has been shown to interact with the TLR3 pathway, inhibiting the production of type I interferons.⁸³ This is thought to help the virus evade detection by the host immune system and contribute to the pathogenesis of CHIKV infection. Other CHIKV proteins, including nsP3 and E2, have been shown to interact with the TLR7/8 pathway, which plays a role in the recognition of single-stranded RNA viruses.⁸⁴ This interaction may contribute to the immune response to CHIKV infection.

YFV envelope protein E is the main protein responsible for the activation of TLRs. The current Yellow Fever vaccine utilizes a weakened form of the yellow fever virus, which can trigger various TLRs and generate a well-balanced Th1/Th2 response in dendritic cells of mice. Notably, when TLR2 is absent in knockout mice, there is a significant enhancement in Th1 responses, coupled with reduced production of IL-6 and IL-12.⁸⁵ Additionally, the YFV E protein has been shown to activate the TLR3 pathway, leading to the production of interferon-beta (IFN-β). It appears that the YFV E protein can activate TLRs directly without the need for accessory molecules.⁸⁶ The activation of TLRs by the YFV E protein seems to occur through the recognition of PAMPs present in the virus, such as viral RNA or glycoproteins. This leads to the activation of downstream signaling pathways, including the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways.⁸⁶ Overall, it appears that the YFV E protein can hijack or navigate TLR pathways to its liking, leading to the production of pro-inflammatory cytokines and IFN-β.⁸⁷ This can help the virus to evade host immune responses and establish infection in the host.

Understanding the mechanisms by which DENV, ZIKV, WNV, CHIKV, and YFV can evade or modulate the TLR pathway (as represented in Figure 2) may help in the development of new strategies to prevent or treat DENV infection.

Figure 2. Evasion and modulation of TLR pathway by the arboviruses. This figure illustrates the intricate mechanisms by which these arboviruses evade or modulate the TLR pathway during infection. Also included are the natural ligands for respective TLRs.

TLR agonists as adjuvants for arbovirus vaccines

Many vaccines contain a component called adjuvants that increases the efficacy of the vaccine by stimulating the immune response more effectively. These include mineral salts, emulsions, and immune potentiators such as TLR agonists.⁸⁸ TLR agonists trigger the TLRs to activate the innate immune response and promote immunity against infectious diseases, cancer, and other conditions.⁸⁹ TLR agonists have been investigated as potential vaccine adjuvants, as they can enhance the immune response to the vaccine antigen and improve vaccine efficacy.⁹⁰ In addition, TLR agonists have been studied as potential therapies for cancer and other diseases.⁹¹ For example, TLR7 and TLR9 agonists have shown promise in the treatment of some cancers (e.g. melanoma and lymphomas), as they can activate immune cells to recognize and attack tumor cells.^92,93 This extends to previous studies having explored the usage of TLR9 agonist as monotherapy for skin cancers and hematologic malignancies, as well as in combination with other therapies such as radiotherapy and certain chemotherapies.⁷⁵ Imiquimod (IMQ), a TLR7 agonist demonstrated this by increasing the expression of autophagy-related genes and autophagosomes, leading to cell death in melanoma cells.⁷⁴ The innate immune response toward cancer and viruses exhibits parallels in their mechanisms, focusing on the recognition and removal of abnormal or potentially harmful cells- specifically, malignant cells and viral pathogens. Consistent with the immune response toward cancer, TLR-assisted cell death of the virus-infected cells, may restrict further virus multiplication in the host.⁹⁴ TLR agonists have also been investigated for their potential use in treating autoimmune and inflammatory disorders.⁹⁵

There are TLR agonists that have been approved to treat medical conditions. Imiquimod is a TLR7 agonist that is approved for the topical treatment of certain skin conditions, including genital warts and actinic keratoses.^95,96 Resiquimod is a TLR7 and TLR8 agonist that is approved for the topical treatment of external genital and perianal warts caused by human papillomavirus (HPV).⁹⁷ Bacillus Calmette-Guérin (BCG) is a vaccine that contains a TLR2 and TLR4 agonist and is used to prevent tuberculosis.⁹⁸ MEDI9197 is a TLR7 agonist that is being studied as a potential treatment for advanced solid tumors.⁹⁹ VTX-2337 is a TLR8 agonist that is being studied as a potential treatment for various types of cancer.¹⁰⁰

Vaccines are an effective way to prevent arbovirus infections, and several arbovirus vaccines are currently available. However, some of these vaccines have limitations, such as a lack of efficacy against all strains of the virus, the need for multiple doses, and the risk of adverse effects.¹⁰¹ One strategy to overcome these limitations is to use adjuvants. TLR agonists are one type of adjuvant that has been investigated for use in arbovirus vaccines.^102–104 TLR agonists can enhance the immune response to arbovirus vaccines by activating innate immune cells, such as dendritic cells, to produce cytokines and chemokines that promote the activation of antigen-specific T cells and B cells.¹⁰⁵ TLR agonists can also enhance the production of antibodies against the vaccine antigen, which is an important component of the immune response to arboviruses.¹⁰⁵

Several TLR agonists have been studied as adjuvants for arbovirus vaccines. For example, TLR9, TLR2/1, and TLR7/8 agonists have been shown to enhance the immune response to the live-attenuated YFV vaccine 17D (YF-17D). A phase 3 observer-blind, placebo-controlled randomized study involving 900 participants took place at 11 sites across the US from February 2018 to May 2019. The administration of YF-17D vaccine and TAK-003, whether sequentially or concomitantly, proved to be immunogenic and well-tolerated.⁸⁵ In addition, a combination of TLR4 agonists and saponin adjuvants have been shown to enhance the immune response to the WNV vaccine in Syrian hamsters.¹⁰⁶ The use of TLR agonists as adjuvants for arbovirus vaccines has several potential benefits. First, TLR agonists can enhance the efficacy of existing vaccines, which could lead to better protection against arbovirus infections. Second, TLR agonists can reduce the number of vaccine doses needed, which could improve vaccine coverage and compliance. Third, TLR agonists could be used to develop new vaccines against arboviruses that are currently difficult to vaccinate against.

However, there are also some potential challenges and risks associated with the use of TLR agonists as adjuvants for arbovirus vaccines. For example, some TLR agonists can cause inflammation and adverse reactions, and the optimal dose and timing of TLR agonist administration may need to be carefully considered to avoid these effects.¹⁰⁷ The use of TLR agonists as adjuvants for arbovirus vaccines is an active area of research that holds promise for improving the prevention and control of arbovirus infections. Further studies are needed to fully understand the potential benefits and risks of TLR agonists as adjuvants for arbovirus vaccines and to develop safe and effective TLR agonist-based vaccine formulations.

TLR antagonists as therapeutic agents for arbovirus infection

TLR antagonists are substances that inhibit the activity of TLRs to modulate the immune response to infectious agents by blocking the activation of TLRs.¹⁰⁸ By inhibiting the activity of TLRs, TLR antagonists can reduce inflammation and tissue damage that can occur as a result of an excessive or dysregulated immune response to infection. TLR antagonists can also potentially reduce the severity of infections caused by certain pathogens, such as bacteria and viruses.

There are several different types of TLR antagonists that have been developed and studied for various applications. Small molecule inhibitors are synthetic compounds that bind to TLRs and block their activity. Small molecule inhibitors have been developed for various TLRs, including TLR4 and TLR7, and they have been studied for their potential to treat infectious diseases, inflammatory disorders, and cancer.¹⁰⁹ Antibodies that target TLR2 and TLR4 have been developed and studied for their potential to treat sepsis, a life-threatening condition that can occur as a result of an overwhelming immune response to infection.¹¹⁰ In particular, eritoran tetrasodium (E5564) is suggested to completely block cellular activation by LPS via TLR4 signaling, hence reducing the following production of pro-inflammatory cytokines and chemokines.¹¹⁰ Some natural compounds, such as curcumin and epigallocatechin gallate (EGCG), have been shown to have TLR antagonistic activity.^111,112 These compounds have been studied for their potential to treat inflammatory disorders and cancer by way of cell death induction including apoptosis and autophagy.^111,112 This can be briefly illustrated by EGCG inhibiting NF-κB activity through the TLR4 signaling pathway and activating caspases, resulting in the apoptosis of epidermoid carcinoma cells.¹¹¹

Currently, there are no TLR antagonists that have been approved by regulatory agencies for the treatment of medical conditions. However, several TLR antagonists have been developed and studied in preclinical and clinical trials for various applications. A TLR4 antagonist called eritoran tetrasodium has been studied in clinical trials for the treatment of sepsis.¹¹³ Although early clinical trials showed promising results, larger phase III clinical trials failed to demonstrate a significant improvement in patient outcomes.¹¹³ Another TLR4 antagonist called TAK-242 has been studied in preclinical and clinical trials for the treatment of various inflammatory conditions, such as rheumatoid arthritis¹¹⁴ and Crohn’s disease.¹¹⁵

TLR7 antagonists have also been developed and studied for the treatment of viral infections, such as hepatitis B and C. One TLR7 antagonist called GS-9620 has shown promising results in preclinical and clinical trials for the treatment of chronic hepatitis B infection.¹¹⁶ While TLR antagonists have shown promise in preclinical and early clinical trials for the treatment of various medical conditions, more research is needed to fully understand their efficacy, safety, and potential side effects. Further clinical trials are needed to determine whether TLR antagonists can effectively treat various diseases. A TLR7 antagonist, IRS661, was found to reduce IFN-I and other cytokines in the lung by acting through pDCs and monocytes, which resulted in a reduction of inflammation and its intensity.¹¹⁷ Moreover, inhibiting TLR7 demonstrated a reduction in mortality and inflammation in the mouse model’s infected lung, specifically by diminishing neutrophil recruitment and monocyte-produced chemo-attractants, even in the absence of IFN-I signaling.¹¹⁷

A TLR4 antagonist, eritoran (also known as E5564), was found to block pulmonary pathology, clinical symptoms, cytokine and oxidized phospholipid expression, influenza-induced mortality in mice, and lower viral titers.¹¹⁸ It was also discovered that eritoran-mediated protection required CD14 and TLR2, as CD14 directly bound eritoran and prevented ligand binding to MD2.¹¹⁸ Eritoran in addition to another TLR4 antagonist, Rhodobacter sphaeroides lipopolysaccharide, attached to a hydrophobic region in MD-2 and dose-dependently inhibited RSV F-protein-mediated TLR4 activity in HEK293T-TLR4-CD14-MD-2 transfectants through TNF-mediated signaling.¹¹⁹

Convinced by the inhibitory effects of TLR antagonists against other viruses, they have the potential to revolutionize the treatment of arbovirus infections. Further research is needed to utilize TLR antagonists for the treatment of arbovirus infections in humans and to fully understand the efficacy, safety, and potential side effects of TLR antagonists as therapeutic agents for arbovirus infections. Clinical trials are needed to determine whether TLR antagonists can be effective treatments for arbovirus infections in humans.

Challenges and future direction for TLR-based strategies against arbovirus infection

The use of TLR-based strategies against arboviral and other viral infections has shown promising results in preclinical and clinical studies (Table 2), but several challenges need to be addressed before their clinical application. One of the main challenges is the complexity of arboviral infections. Arboviruses have complex life cycles, involving both the insect and mammalian hosts, and they have evolved to evade the host’s immune response.¹²⁰ Different arboviral species have different mechanisms of infection and immune evasion, making it difficult to develop a universal TLR-based strategy. Therefore, it is essential to develop a detailed understanding of the immune response to specific arboviruses and identify the PAMPs recognized by TLRs to design effective TLR-based strategies.

Table 2. TLR-based strategies against arboviral and other viral infections in preclinical and clinical studies.

No.	Viral infection	TLR-based strategy	Stage	Finding(s)	References
1	Human papillomavirus	TLR7 and TLR8 agonist: Resiquimod	FDA-approved	Resiquimod is an FDA-approved topical treatment for viral and tumor lesions. Topical resiquimod stimulated TLR7/8-expressing skin APCs, upregulated surface activation markers, overexpressed pro-inflammatory cytokines/chemokines (TNF-α, IFN-α), induced tumor-directed antibodies, Th1 CD4+ T-cells and Th1 CD8+ CTLs to cure viral skin lesions/tumors.	79
2	Yellow fever virus	TLR 9 agonists CpG A (ODN 2336), and CpG B (ODN 2006) DNA; TLR2/1 agonist Pam-3-cys, and TLR7/8 agonist (3 M–003) as a vaccine adjuvant	FDA-approved	An effective vaccine against YFV (even in modern days), administered as a single subcutaneous or intramuscular injection since its establishment in the year 2000. YF-17D stimulated multiple DCs through TLRs 2,7,8 and 9 with MyD88 and TIRAP, inducing proinflammatory cytokines and antigen-specific CTLs with mixed Th1/Th2 profile. MyD88 activated while TLR2 inhibited Th1 responses.	88
3	Influenza virus	TLR4 (2B182C) and TLR7 (1V270) agonists as a vaccine adjuvant	preclinical	Intramuscular IIAV-1V270-2B182C immunization exhibited minimal reactogenicity, blocked homologous virus-induced mice lethality, decreased lung viral titers and cytokine expression, enhanced nodal B cell clonal expansion with broadly cross-reactive humoral responses against numerous HA epitopes, HA and NA subtypes.	103
4	West Nile virus	TLR4 agonist as a vaccine adjuvant	preclinical	Single WN-80E-SLA-SE vaccination facilitated 100-fold antigen dose-sparing, decreasing serum virus to minimal concentrations with higher neutralizing IgG2c titer and Th1 CD4+ T-cell response alongside long-term protection through increased BM-derived APCs.	68
5	Hepatitis B virus	TLR7 antagonist: GS-9620	preclinical and clinical	Short-term oral treatment enhanced infected hepatocyte apoptosis continuously suppressed viral DNA and secreted viral antigen levels, elevated ISG, NK cell, and T-lymphocyte subset expression, and produced IFN-α and other cytokines/chemokines.	98
6	Influenza virus	TLR4 antagonist: eritoran (also known as E5564)	preclinical	Even when administered 6 days post-infection, Eritoran-CD14-TLR2 interaction prevented mice mortality and severe clinical manifestations, reduced viral titers, preserved lung architecture and function, and suppressed IFN-β/-γ expression and phospholipid oxidation.	100
7	Respiratory syncytial virus	TLR4 antagonists: Eritoran in addition to Rhodobacter sphaeroides lipopolysaccharide	preclinical	The binding of antagonists to MD-2 hydrophobic pocket prevented direct interaction between MD-2 and N-terminal domain of RSV F protein, dose-dependently inhibiting F protein-MD-2-mediated TLR4 activity and TLR4-dependent NF-κB activation.	101

Another challenge is the potential side effects of TLR agonists. TLR agonists are potent immune stimulators and can induce systemic inflammation and cytokine storms, which can cause tissue damage and lead to severe side effects.¹²¹ Therefore, it is important to identify safe and effective TLR agonists and determine the appropriate dose and route of administration. The use of TLR-based strategies in combination with other treatments, such as antiviral drugs or monoclonal antibodies, may enhance their efficacy. However, the optimal combination and sequence of treatments need to be determined, and potential drug interactions and side effects must be considered.

The development of TLR-based strategies also requires appropriate animal models to evaluate their efficacy and safety. Animal models that mimic human arboviral infections are essential to understand the immune response and testing TLR-based strategies. However, the availability of appropriate animal models for arboviral infections is limited.¹²² Future directions for the development of TLR-based strategies against arboviral infections include the identification of novel TLR agonists and the development of TLR agonist-based vaccines. The use of adjuvants, such as TLR agonists, in vaccines, can enhance the immune response and increase the efficacy of vaccines. Therefore, TLR agonist-based vaccines may provide a promising approach to the prevention of arboviral infections.

Conclusions

In summary, TLR-based strategies hold promise as a potential approach to combat arboviral infections. However, several challenges need to be addressed, including the complexity of arboviral infections, potential side effects of TLR agonists, optimal combination and sequence of treatments, appropriate animal models, and identification of novel TLR agonists. Future research should focus on addressing these challenges to develop safe and effective TLR-based strategies against arboviral infections.

Acknowledgments

We would like to express our deepest gratitude to the staff of the Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya, and Tropical Infectious Diseases Research and Education Centre (TIDREC), Universiti Malaya for providing the necessary facilities and resources in bringing this review to fruition. We acknowledge the funding from the Ministry of Higher Education, Malaysia for niche area research under the Higher Institution Centre of Excellence (HICoE) program (MO002-2019 & TIDREC-2023).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the Ministry of Higher Education, Malaysia for niche area research under the Higher Institution Centre of Excellence (HICoE) program [MO002-2019, TIDREC-2023].