Recognition of microbial pathogens and vaccine antigens is an essential element for initiating adaptive immune responses as well as determining the quality of resulting host immune responses. Dendritic cells (DCs) and macrophages play a critical role in inducing protective adaptive immune responses of both T and B cells. We found that presenting HA antigen on VLPs was significantly much more effective in inducing protective T cell and B cell responses than the soluble HA protein antigen [110
]. Effective induction of protective immunity by antigens in a VLP form is probably because particulate antigens are more likely to activate innate immune cells such as DCs.
Antigen-presenting cells (APCs) such as DCs and macrophages prominently express toll-like receptors (TLRs) which recognize pathogen, vaccine antigen, or adjuvant components. APCs play critical roles in activating innate immunity by recognizing and capturing pathogens or vaccine antigens at the site of infection or vaccination via receptors such as TLRs. Antigen-loaded APCs initiate the events of inducing the activation of adaptive T and B cells [111
]. In cases where vaccine antigens (such as subunit vaccines) are not effective in stimulating APCs, adjuvants should be included in the vaccine formulation to activate APCs. Some compounds enhancing antigen depot or activating innate immunity by interacting with pathogen recognizing receptors (PRRs) such as TLRs have shown some promise. For example, alum, MF59, or AS04 (TLR agonist) adjuvants are often added to the subunit vaccine formulations. But live attenuated (cold-adapted) influenza vaccines are likely to activate TLR3 and 7 during viral replication intra-cellularly leading to the up-regulation of inflammatory cytokines [112
], and thus adjuvants are not needed. Also, the live attenuated yellow fever vaccine, one of the most effective vaccines, was demonstrated to activate multiple DC subsets via TLRs 2, 7, 8, and 9 [113
]. Activated DCs up-regulate costimulatory molecules through receptor-mediated recognition of a pathogen or vaccine antigen and can elicit the differentiation of naïve T cells into different effector T cells leading to the generation of cytotoxic CD8 T cells and activated B cells [111
B cells are one of the important adaptive immune cells. Previous studies suggest that follicular B cells express TLR 1, 2, 4, 7, and 9. These B cells also showed proliferative responses and isotype switching upon the in vitro
stimulation with TLR2 (Pam3Cys), TLR3 [poly(I:C)], TLR4 (lipopolysaccharide [LPS]), TLR7, and TLR9 agonists [114
]. There have been controversies concerning whether TLR signaling is essential for B cell responses after vaccination. The addition of TLR9 ligand CpGs to B cells in vitro
cultures induced the production of IgG2a, IgG2b and IgG3 antibodies [115
]. An innate adaptor signaling molecule, myeloid differentiation factor-88 (MyD88), is known to be a key adaptor componentfor activation from the most TLRs except the TLR3 [116
]. MyD88-deficient mice have been shown to have a defect in inducing T helper type 1 immune responses to ovalbumin plus complete Freund adjuvant [117
]. Importantly, we found that MyD88-deficient mice showed a significant defect in generating T helper type 1 isotype switched antibodies, and interferon (IFN-γ) secreting T cell responses as well as for eliciting long-lived antibody secreting plasma cells and protective immunity after vaccination with influenza VLP vaccines [118
]. Better understanding of stimulating innate immune components by vaccine antigens will provide informative insight into developing effective and safe vaccines.
Initially, TLRs were recognized as a family of PRR host cell proteins that recognize a wide range of pathogen-associated molecular patterns (PAMPs) in the microbial pathogens or vaccine antigens [116
]. To date, 10 and 13 functional TLRs have been identified in humans and mice, respectively. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 are expressed on the host cell surface. TLR3, TLR7, TLR8, TLR9, and orphan receptor TLR13 are expressed within intracellular compartments such as endosome and recognize nucleic acids [116
]. TLR1, TLR2, and TLR6 recognize pathogen-derived molecules such as lipoproteins. TLR3 binds to double-stranded (ds) RNAs that are present in many RNA viruses including influenza virus. TLR4 recognizes bacterial LPS, fusion protein of respiratory syncytial virus, fibronectin, and heat-shock proteins. TLR5 is known to recognize bacterial flagellin molecules. TLR7 and TLR8 are activated by single-stranded (ss) RNA molecules that are present in RNA viruses such as influenza virus whereas TLR9 recognizes bacterial unmethylated DNA molecules. Once TLRs recognize various components of vaccine antigens or microorganisms including viruses, inflammatory cytokines are produced, which initiate the activation of signaling cascade leading to the induction of adaptive immune responses to pathogens or vaccine antigens.
Another important family of PRRs includes the C-type lectin receptors (CLRs), cytosolic proteins such as NOD-like receptors (NLRs) and cytoplasmic RNA helicase retinoic acid inducible gene I (RIG-I) or melanoma differentiation associated gene 5 (named RIG-I like receptors, RLRs) [121
]. The representative CLRs are DC-specific ICAM-3 grabbing non-integrin and DC-associated C-type lectin-1 (dectin-1), both of which play key roles in inducing immune responses in response to fungal, bacterial or virus pathogen molecules. NLRs are expressed intracellulary and respond to various PAMPs to trigger inflammatory responses [121
TLRs are expressed on various immune cells including macrophages, DCs, B cells, specific T cells and even expressed on non-immune cells such as fibroblasts and epithelial cells [114
]. All TLRs except TLR3 use the downstream adaptor molecule MyD88, whereas TLR3 and TLR4 in part use Toll receptor-associated activator of interferon (TRIF). After TLRs-ligand interaction, TLR activation results in triggering of downstream signaling cascades through the engagement of signaling intermediates, MyD88, Toll-interleukin (IL)-1 receptor-associated-protein (also known as MAL), TRIF, Toll receptor-associated molecule, IL-1 receptor associated kinase, and tumor necrosis factor receptor-associated factor 6. TLR ligation can result in activation of transcription factors (IRF3, IRF7, activator protein-1, nuclear factor-κB) and consequently produce the proinflammatory cytokines and chemokines. Furthermore some TLRs (TLR3, 4, 7, 8, 9) are capable of inducing type 1 interferons (IFN-α/β) to elicit antiviral responses [116