September 25, 2023

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Will TLR Agonists become Next Generation Vaccine Adjuvants?

Will TLR Agonists become Next Generation Vaccine Adjuvants?


Will TLR Agonists become Next Generation Vaccine Adjuvants?

The word adjuvant is derived from the Latin words ad and juvare, meaning “to help”.

The role of the adjuvant is to help direct the immune response to the co-administered vaccine antigens needed for protection.

The development of new adjuvants has been stalled due to insufficient understanding of their mechanisms of action.

Recently, the field has been revolutionized by the discovery of Toll-like receptors ( TLRs ), a class of innate immune receptors responsible, directly or indirectly, for detecting pathogen-associated molecular patterns ( PAMPs ) and responding to It reacts.


Naturally occurring and synthetic TLR agonists can exploit these endogenous immune signaling pathways to enhance and modulate vaccine responses, making them excellent vaccine adjuvants.

This has important implications not only for the development of vaccines against infectious diseases, but also for immunotherapies against cancer, allergies, Alzheimer’s disease and other diseases.

Each TLR has its own specific tissue localization and downstream gene signaling pathways, and TLR agonists can be combined with other TLRs or alternative adjuvants to generate combined adjuvants with synergistic or modulatory effects, which provides researchers with precise customization Opportunities for adjuvants with specific immune effects.


TLR receptor family

The TLR receptor family includes six transmembrane TLRs ( TLR-1 , 2 , 4 , 5 , 6 and 10 ) and four TLRs localized to endosomal membranes ( TLC-3 , 7 , 8 and 9 ). Each PAMP is recognized by different TLRs , namely lipopolysaccharide ( TLR4 ), lipopeptide ( TLR2 and TLR6 or TLR1 ), flagellin ( TLR5 ), single-stranded RNA ( TLR7/8 ), double-stranded RNA ( TLR3 ) and CpG motif DNA ( TLR9).


Will TLR Agonists become Next Generation Vaccine Adjuvants?


Upon encountering specific PAMPs, TLRs form homodimers (TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 ) or heterodimers ( TLC1/2 or TLR2/6 ). Ligand-induced dimerization of TLRs enables an adapter protein ( MYD88 ) to bind to the TIR domain.

TLR3 is special, the protein as a general adapter protein of TLRs, using TIR domain-containing inducible interferon-β protein ( TRIF ) as an adapter protein.

The diversity of TLR receptors and adapter proteins means that TLRs can be involved in initiating many different downstream immune pathways.



TLR signaling and its role in immune bias

Activation of TLRs can modulate the type of immune response that vaccines generate. The type of response is determined by the signaling pathways activated by specific TLRs and their adapter proteins.

In general, most TLR pathways lead to Th1 immune responses, except for TLR2 , possibly due to the diversity of TLR2 ligands, TLR2 can induce pro-inflammatory and anti-inflammatory pathways, leading to Th1 , Th0 or Th2 immune responses.


Except for TLR3, most TLRs function through MyD88. Upon TLR activation, MyD88 recruits an oligomeric complex composed of IL1 receptor-associated kinase ( IRAK ) 1, IRAK2, and IRAK 4, and activates MyD88 TNF receptor-associated factor 6 ( TRAF6 ).

Activated TRAF6 subsequently triggers nuclear factor kappa B ( NF-κB ) and mitogen-activated protein kinase ( MAPK ) pathways, thereby inducing pro-inflammatory cytokines such as IL-12 and TNF-α.

TLR7 and TLR9 can activate TRAF3 to phosphorylate interferon regulatory factor 7 ( IRF7 ), thereby producing IFN-α.

Furthermore, TLR3 and TLR4 have been shown to induce IFN-β production by recruiting TRAF3 through their TRIF adapter proteins, thereby activating IRF3.

Type I IFN responses can induce strong Th1 cell/cytotoxic T cell ( CTL ) responses that are important for identifying and clearing infected or cancer cells.

TLR2 receptors mainly induce strong Th2 immune responses characterized by high IL-10 production and low IL-12.


TLRs do not specifically trigger Th1 or Th2 pathways, but can affect multiple pathways. It is the balance of signaling events that determines immune bias.

For example, one study showed that activation of TLR4 and TLR2 agonists can stimulate signaling through the p38 MAPK and ERK1/2-FOS pathways, resulting in the production of IL-12 or IL-10 , respectively ; however, TLR4 is at a higher p38 MAPK signaling is induced at a subthreshold , whereas TLR2 induces ERK1/2 signaling at a higher threshold than TLR4 , resulting in immune polarization.



The role of TLRs in adaptive immunity

TLRs coordinate many functions ranging from immune cell migration to enhancing antibody affinity maturation.

TLRs act as danger signal sensors that increase the trafficking of immune cells to the site of vaccine administration. TLR engagement enhances antigen capture, processing and presentation by MHC class I and class II molecules.

Certain specific types of TLRs can affect antigen cross-presentation to varying degrees, such that TLR3 or TLR4, but not TLR2 or TLR7/8 ligands, reduce antigen uptake and cross-presentation by CD8+ T cells.

TLR activation can also lead to the upregulation of co-stimulatory molecules such as CD80 and CD86 , which are critical for APC priming of naive T cells.

TLRs of DC cells play a key role in self/nonself antigen recognition and whether to generate immune response or immune tolerance.

Different types of APCs express different TLRs, for example, myeloid DCs express TLR2 and TLR4, while plasmacytoid DCs ( pDCs ) express TRL7 and TLR9.

Thus, preferential activation of TLRs to different DC lineages can influence subsequent immune bias.


TLR expression on B cells is more limited in humans compared to mice, humans mainly express TLRs 2, 7, 9, and 10, and TLR2 ligands require crosslinking of the B cell receptor ( BCR) and TLR7 For additional sensitization, TLR7 requires type 1 interferon ( IFN ) for priming. These TLR agonists induce proliferation, activation and differentiation of B cells in vitro.

Initially, TLRs were thought to promote only the extrafollicular response of B cells, which is characterized by the rapid production of low-affinity antibodies; however, recent studies have shown that TLRs can also enhance germinal center responses, resulting in the production of high-affinity antibodies.

TLRs cooperate with BCRs to induce class switch recombination and maturation of antibody responses TLR4 also enhanced the migration of B cells to draining lymph nodes and accelerated antibody class switching in the process. Inappropriate B cell TLR signaling has been implicated in the development of autoreactive B cells and autoimmune disease; therefore, it is important to avoid excessive TLR stimulation in vaccine formulations.


TLRs have also been shown to enhance various T cell subsets.

Several studies have shown that TLRs can act as co-stimulatory molecules for CD8+ T cells, promoting increased cell proliferation/survival, effector function, cytokine production, and memory formation.

The role of TLRs in Treg cells is still controversial. Several studies have shown that TLR2 agonists induce a temporary loss of Treg suppressive activity; whereas others have shown that Treg cells retain their suppressive function despite TLR2 agonists increasing antigen-specific proliferation.




The latest research progress of TLR adjuvants

TLR agonists used in vaccine formulations come in a variety of forms, ranging from lipopeptides to single-stranded DNA and RNA . The main categories of TLR adjuvants are described below .



TLR2 is expressed on the surface of a variety of cells, including monocytes, macrophages, endothelial cells, epithelial cells, natural killer cells, dendritic cells, myeloid suppressor cells, platelets, and mast cells. The repertoire of PAMPs recognized by TLR2 is the broadest because of its ability to form heterodimers with TLR1 and TLR6 (and TLR10 in humans).


Will TLR Agonists become Next Generation Vaccine Adjuvants?


The range of TLR2 adjuvants is broad and includes synthetic lipopeptides ( PAM3CSK4 and PAM2CSK4 ), arabinomannolipids, lipoteichoic acid, GPI membrane anchors, zymosan, and peptidoglycan. The major trends in TLR2 research in recent years include the discovery of small synthetic TLR2 agonists, the improvement of the properties of traditional TLR2 agonists, and the bioconjugation of TLR2 to vaccine antigens.

For example, XS15 ( PAM3CS-GDPKHPKSF ) is a novel PAM3CS-based TLR-1/2 agonist in which the tetralysine ( K4 ) of PAM3CSK4 is replaced by nonapeptide ( GDPKHPKSF ) to change the water solubility of the conjugate , not only to promote uptake, but also easy to purify.

Limitations of using TLR2 agonists as adjuvants include the size, complexity, and hydrophobicity of most ligands.



TLR3 is an intracellular recognition system that responds to viral nucleic acids ( dsRNA, ssRNA, and ssDNA ) as well as endogenous double-stranded RNA. Many TLR3 agonists have been developed, such as RGC100 and ARNAX, a synthetic DNA-RNA hybrid compound. In the past two years, however, researchers have returned to using traditional dsRNA mimic ligands, such as poly IC, with a focus on improving their delivery modes and new disease applications.





TLR4, the most studied member of the TLR family, recognizes lipopolysaccharide ( LPS ).

TLR4 is located on the plasma membrane and is mainly expressed on myeloid cells, but not on pDCs and naive B cells. TLR4 recognizes LPS through its co-receptors myeloid differentiation factor-2 ( MD-2 ) and CD14.

Recent work on TLR4 agonists has focused on the development and evaluation of modified products such as monophospholipid A ( MPLA ) and glucopyranose lipid A ( GLA ), which are structurally related to LPS but are not hyperpyrogenic The originality and maintain the strong immunoenhancing properties, thus increasing the feasibility of its clinical application.

Will TLR Agonists become Next Generation Vaccine Adjuvants?




TLR5 recognizes flagellin and is expressed on epithelial cells and immune cells such as macrophages and immature DCs , generating immune responses through MyD88-dependent signaling pathways. Recent work on TLR5 agonists has largely focused on improving tolerance.

Unfortunately, flagellin induces unwanted hyper-reactogenicity, as a protein it can induce antibodies against self, interfering with its function as an adjuvant.

This issue was addressed by removing the B-cell epitope region from flagellin, and the deimmunized flagellin retained its TLR5 adjuvant activity.



TLR7 and TLR8 are located on the endosomal membrane of immune cells. TLR7 is mainly expressed in pDC and B cells, while TLR8 is expressed in myeloid dendritic cells and monocytes, and to a lesser extent in pDC.

TLR7/8 recognizes single-stranded ribonucleic acid ( ssRNA ) and signals through a MyD88-dependent pathway. Among natural ligands, adenosine- and uridine-rich oligonucleotides ( ORNs ) are able to activate TLR8 without any effect on TLR7, whereas guanosine-rich ORNs activate TLR7 and TLR8-dependent signaling.

TLR7 exists as a monomer and dimerizes in the presence of ligand, whereas TLR8 exists as a naturally occurring weak dimer that undergoes a conformational change upon ligand binding.

More than 15 novel heterocyclic molecules, such as imidazoquinoline, pterin, pyrimidine, pyridine pyrimidine, pyrropyrimidine, and benzimidazole, have been discovered and identified as TLR7/8 agonists.


Will TLR Agonists become Next Generation Vaccine Adjuvants?



A common disadvantage of TLR7/8 agonists is reactogenicity, and in recent years, a large number of studies have attempted to overcome these side effects.

Encapsulation of TLR7/8 agonists in nanoparticles of cationic DOEPC-based liposomal formulations, or covalent attachment of these small molecules to hyperbranched polymers, could avoid their deleterious systemic responses while maintaining their potential for humoral immunity. 



TLR9 is intracellularly localized in endosomal membranes and recognizes single-stranded unmethylated CpG oligonucleotides of bacterial and viral DNA.

TLR9 is expressed on immune cells such as dendritic cells, macrophages, natural killer cells and other APCs.

Synthetic CpG sequences can be used as TLR9 agonists to enhance immune responses to vaccines, and each unique combination of sequence variants has been shown to have distinct structural and biological properties.


Recent developments in TLR9 agonists over the past two years have largely focused on the efficient delivery and uptake of CpGs to cells.

For example, a study introduced a novel method for conjugating CpG-ODN to novel cationic liposomes, a complex capable of inducing robust immune responses at low antigen and adjuvant doses.


Combined TLR Adjuvant

Combining agonists of different TLRs in a single vaccine produces a synergistic effect that can drive a robust vaccine immune response.

For example, a liposomal adjuvant containing 1V270 ( a TLR7 agonist ) and 2B182C ( a TLR4 agonist ) induced balanced anti-HA and anti-NA IgG1 and IgG2a responses against influenza without hyperresponsives common to Th1 proinflammatory responses sex. Similarly, a study evaluated the co-encapsulation of ovalbumin ( OVA ) with ten unique combinations of two to three TLR ligands, including Pam3CSK4 (a TLR2 agonist ), MPLA ( a TLR4 agonist ), imiquimolar TLR7 /8 agonist ) and CpG ( TLR9 ​​agonist ), while the triple combination promotes antigen-specific antibody titers with an overall balanced Th1/Th2 response. Thus, combinations of TLR adjuvants can provide a broad range of tailored immune responses.


Combinations of TLR agonists with other PAMPs such as NOD2 and macrophage-inducible Ca²⁺-dependent lectin receptor ( Mincle ) ligands are also promising.

Covalently linking CL239 ( TLR7 agonist ) and muramyl dipeptide ( NOD2 agonist ) and combining the dual agonist as nanoparticles with NP-p24 HIV vaccine synergistically enhanced protection in mice.




TLR agonists exploit the endogenous innate immune pathway to enhance the adaptive immune response to vaccines.

For the past two years, the field of adjuvants has been dominated by research into COVID-19 vaccines.

The dynamics of the pandemic have rapidly advanced the field of adjuvants, with multiple new adjuvants now incorporated into licensed new COVID-19 vaccines.

Beyond COVID-19, the need for more potent adjuvants continues to grow, especially in the area of ​​cancer and hard-to-prevent infectious diseases such as malaria, tuberculosis and HIV.

Unraveling the mechanism of action behind TLR -combined adjuvants, coupled with more safety and efficacy research data, will help drive the development of next-generation adjuvant platforms.







1.Toll-like receptor (TLR) agonists as a driving force behind next-generation vaccine adjuvants and cancer therapeutics. Curr Opin Chem Biol.2022 Oct;70:102172

Will TLR Agonists become Next Generation Vaccine Adjuvants?

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