Veterinary Immunology: Development of Vaccines and Diagnostic TechniquesView this Special Issue
Review Article | Open Access
Afonso P. Basto, Alexandre Leitão, "Targeting TLR2 for Vaccine Development", Journal of Immunology Research, vol. 2014, Article ID 619410, 22 pages, 2014. https://doi.org/10.1155/2014/619410
Targeting TLR2 for Vaccine Development
Novel and more effective immunization strategies against many animal diseases may profit from the current knowledge on the modulation of specific immunity through stimulation of innate immune receptors. Toll-like receptor (TLR)2-targeting formulations, such as synthetic lipopeptides and antigens expressed in fusion with lipoproteins, have been shown to have built-in adjuvant properties and to be effective at inducing cellular and humoral immune mechanisms in different animal species. However, contradictory data has arisen concerning the profile of the immune response elicited. The benefits of targeting TLR2 for vaccine development are thus still debatable and more studies are needed to rationally explore its characteristics. Here, we resume the main features of TLR2 and TLR2-induced immune responses, focusing on what has been reported for veterinary animals.
The innate immune system senses microorganisms through germ-line encoded receptors, the pattern recognition receptors (PRRs), which include the membrane associated toll-like receptors (TLRs) . Based on the knowledge that stimulation of PRRs by pathogen-associated molecular patterns (PAMPs) has a determinant role in shaping the profile of the subsequent adaptive immune response [2, 3], the conjugation of antigens with PRR ligands has been extensively explored in the last decades for the development of improved vaccines [4–7]. For that purpose, much attention has been paid to PRR ligands inducing strong polarized Th1 and cytotoxic T lymphocyte (CTL) responses, for example, ligands for TLR3, TLR7/8, or TLR9, since these are immune mechanisms poorly induced by vaccination with nonlive, inactivated or subunitary, vaccines. Activation through TLR2 is not recognized as a strong polarizing stimulus, resulting in Th responses with variable characteristics. However, TLR2 offers unique properties to be explored in vaccine development. The possibility to covalently attach TLR2 ligands to antigens, the enhancement of direct- and cross-presentation of antigens coupled to TLR2-targeting lipid moieties, the capacity to induce balanced Th responses and even regulatory mechanisms, and the mucosal imprinting properties of TLR2 stimulation are characteristics that have potential to help solving actual vaccine challenges. Here, we will review the present knowledge on the modulation of the immune response by immunogenic formulations targeting TLR2 and discuss its potential for the development of immunization strategies in the veterinary field.
2.1. The Receptor
TLRs are transmembrane type I glycoproteins with a structure composed by three domains. The N-terminal extracellular domain, which is involved in the recognition of their ligands, consists of leucine-rich repeats (LRR) with the conserved motif “LxxLxLxxN” with around 20 to 30 amino acids. This domain is followed by a transmembrane region then extended intracellularly by a cytoplasmic toll/IL-1 receptor (TIR) domain, needed for signal transduction [1, 8, 9].
Phylogenetically, TLR2 belongs to a TLR family that includes TLR1, TLR6, TLR10, TLR14, and possibly the avian TLR15 . TLR2 is located at the surface of the cell and, upon binding of its ligands, dimerises with TLRs of the same family (see below). In consequence, the juxtaposition of the cytoplasmic TIR domains recruits the signaling adaptors MyD88 and TIRAP, initiating a signaling pathway that leads to activation of NF-κB transcription factor and MAPK which activate the AP-1 transcription factor [8, 11, 12].
Like other TLRs, TLR2 evolved under strong selection pressure, being preserved in all the vertebrate species tested so far [10, 13]. Sequence information on TLR2 is available for the generality of domestic animals, including cattle, sheep, goat, buffalo, horse, pig, chicken, dog, and cat [14–18].
Species-specific variations in TLR2 have been reported, mainly at the extracellular domains, possibly reflecting adaptation to different microbial environments . Peculiarities in chicken TLRs include the existence of two types of TLR2 (TLR2a and TLR2b) and of TLR1 (TLR1La and TLR1Lb), originated by gene duplication, and the absence of TLR6 . TLR15, apparently unique to avian species, is phylogenetically related to the TLR2 family .
2.2. Cells and Tissue Expression
TLR2 expression has been reported in antigen presenting cells (APCs), namely, macrophages, monocytes, and dendritic cells (DCs), including CD8α+, CD8α−, and plasmacytoid DCs in the mouse and interstitial and Langerhans DCs but not plasmacytoid DCs in humans . TLR2 immunomodulatory influence can also be exerted directly on B cells, CD4+ and CD8+ T cells, Treg cells, γδ T cells, natural killer (NK) cells, neutrophils, basophils, and some epithelial cells [21, 22].
Tissue and cell distribution of TLR2 expression in domestic animals follows in general terms what has been described for mice and humans (for comprehensive reviews, see [14, 15]). Most of the information available, summarized in Table 1, has been obtained by reverse transcription (RT)-PCR and data on distribution and levels of the protein itself is sparse due to the lack of characterized specific antibodies for domestic animals. However, in the last few years, efforts have been made to fill this gap and anti-TLR2 antibodies have been used to assess TLR2 expression in different species, namely, bovine and ovine , porcine [17, 24, 25], chicken , and dogs .
|RT-PCR: reverse transcription-PCR; FC: flow cytometry; IHC: immunohistochemistry; WB: Western blot.|
In the bovine, differences in TLR2 expression on monocytes, macrophages, and DCs were found by RT-PCR. Monocytes and monocyte-derived macrophages have shown a higher signal, alveolar macrophages and bone marrow-derived DCs an intermediate signal and monocyte-derived DCs, as well as CD172a+ and CD172a− DC subsets of afferent lymph, have shown weaker signals . These differences were later confirmed by flow cytometry using anti-TLR2 antibodies . Expression of TLR2 on ovine and bovine peripheral blood mononuclear cells (PBMCs) was detected only in CD14+ monocytes . No differences were observed comparing different sheep breeds . Das et al.  analysed TLR2 sequences from nilgai, buffalo, sheep, and goat and, interestingly, found that nilgai immune cells and tissues express more TLR2 transcripts than buffalo.
Studying the expression of TLR2 in gut-associated lymphoid tissues from adult swine, Tohno et al.  showed that TLR2 mRNA was preferentially expressed in the mesenteric lymph nodes and Peyer’s patches in levels higher than that of spleen, and Western blotting confirmed the high TLR2 expression in these structures. Beside immune cells, like T and B cells, TLR2 expression was also detected in membranous cells (M cells). Its detection in the apical membrane of the pocket-like M cells suggests a possible role in ligand-specific transcytosis and transport in these cells.
After raising a panel of monoclonal antibodies for porcine TLR2, Alvarez et al.  could demonstrate TLR2 expression in monocytes, macrophages, and granulocytes but not on peripheral blood lymphocytes. TLR2 expression was also detected in nonimmune cells lining body entry sites like tracheobronchial and intestinal epithelial cells, bile ducts in the liver, renal tubules, and basal layer of the epidermis . Expression of TLR2 and TLR6 was demonstrated in porcine alveolar macrophages by Western blot and using antibodies against these receptors, their relevance in the sensing of Mycoplasma hyopneumoniae was shown . Also in the horse, TLR2 expression was detected by RT-PCR in alveolar macrophages , as well as in respiratory epithelia  and PBMCs .
Expression of chicken TLR2a and TLR2b was detected in high levels by Western blot in heart, liver, gizzard, and muscle  and was also identified by RT-PCR in heterophils, monocytes, macrophages, and B and T cells [34, 35].
Ishii et al.  studied the mRNA expression of canine TLR2 in different dog tissues and found it in blood mononuclear cells, lymph node, lung, liver, spleen, bladder, pancreas, small intestine, large intestine, and skin. Bazzocchi et al.  found that TLR2 mRNA is constitutively expressed in canine blood neutrophils and, by flow cytometry, it was detected on the blood neutrophils, monocytes, and, at lower levels, lymphocytes.
For the cat, TLR2 expression was reported in lymphoid tissues (spleen and thymus), in lymphocytes (CD4+ and CD8+ T cells and, in higher levels, CD21+ B cells) , in bone marrow-derived DCs , and in the oral mucosa .
2.3. The Natural TLR2 Ligands
TLR2 is usually described as the TLR recognizing the largest range of ligands. These include components from bacterial cell walls such as lipoproteins, peptidoglycan (PGN), lipoteichoic acid (LTA), lipopolysaccharides (LPS) from some bacterial species (e.g., Porphyromonas gingivalis), porins from Neisseria, lipoarabinomannan from mycobacteria, and zymosan and phospholipomannan from yeast cell walls, among others [6, 8, 39]. The recognition of such a variety of ligands is attributed to the formation of heterodimeric structures with other membrane molecules, like TLR1, TLR6, CD36, CD180/RP105, or dectin-1 . This is however a controversial issue, since some authors argue that bacterial lipoproteins are the only ligands recognized by TLR2 at physiological concentrations . In addition, TLR2 stimulation by most of other ligands was attributed to contamination with lipoproteins [40–42].
Lipoproteins are membrane structural components of bacteria with diverse molecular structure but with a common lipidic modification at an N-terminal cysteine [43, 44]. In the diacylated specimens, that is, lipidated with two fatty acid residues, the modification consists in a di-O-acylated-S-(2,3-dihydroxypropyl) cysteine. The triacylated specimens have a third fatty acid, bound through an amide link to the same N-terminal cysteine. Examples of diacylated forms are the M161Ag lipoprotein of Mycoplasma fermentans from which the macrophage-activating lipopeptide (MALP)-2 is derived [45, 46], the LP44 lipoprotein from Mycoplasma salivarium from which the fibroblast-stimulating lipopeptide (FSL)-1 is derived , and the synthetic lipopeptide di-palmitoyl-S-glyceryl cysteine (Pam2C) SK4. The Braun’s lipoprotein from Escherichia coli is the prototype of the outer membrane triacylated lipoproteins from Gram-negative bacteria and some synthetic lipopeptides used as TLR2 stimulators, for example, tri-palmitoyl-S-glyceryl cysteine (Pam3C) SK4, have a lipid modification analogue to this lipoprotein [48–50]. Other examples of triacylated lipoproteins are OspA from Borrelia burgdorferi  and the 19 kDa lipoprotein from Mycobacterium tuberculosis [52, 53]. The capacity to stimulate through TLR2 of both diacylated and triacylated lipoproteins is conferred by the lipidic N-terminal moiety [54, 55]. The initial studies pointed that diacylated lipoproteins are signalized through TLR2/6 heterodimers, while the triacylated molecules do so through TLR2/1 heterodimers [52, 56–58]. However, later studies suggested that lipopeptide activation through TLR2 may occur independently of TLR1 and TLR6 .
In 2007, Jin and collaborators determined by crystallography the structure of the complex TLR1-TLR2-lipopeptide, allowing a structural comprehension of the heterodimerisation induced by the ligand . The binding of the triacylated lipopeptide (Pam3CSK4) induces the formation of an “m” shaped heterodimer of the TLR1 and TLR2 ectodomains. This dimerisation occurs by the insertion of the two ester-bound fatty acids in a pocket in TLR2 and the insertion of the amide-linked fatty acid in a hydrophobic channel in TLR1 . The role played by the three lipid chains thus explains the incapacity of the diacylated peptide Pam2CSK4 to dimerise TLR2 and TLR1. On the other hand, Kang et al.  showed that TLR2/6 heterodimer has a reduced affinity for triacylated lipopeptides because TLR6 lacks a proper binding site for the amide-bound lipid chain. They also showed that, in the TLR2-TLR6-diacylated lipopeptide complex, the increased hydrophobic area found in the interface of the two dimerised receptors appears to compensate the absence of interaction between a lipid chain and TLR6. For TLR2 ligands without patent hydrophobic regions for TLR2 binding, such as PGN and zymosan, a structural support for the receptor dimerisation is lacking . TLR10 is nonfunctional in the mouse, but in the human, it was shown to form dimers with TLR2, recognizing triacylated lipopeptides and other microbial components . However, this receptor fails to activate typical TLR-induced signaling and its role remains elusive.
In general terms, TLR2 ligands are the same across different vertebrate species; however, some species specificities have been reported. For example, by cotransfection of bovine TLR1 and TLR2 in HEK293 cells, Farhat et al.  showed that the ester-bound acid chains of triacylated lipopeptides need to have at least 12 carbon atoms to activate the bovine heterodimer, contrasting with the murine heterodimer that could already be activated by lipopeptides with fatty acids of only 6 carbon atoms. Willcocks et al.  showed that, for some TLR2 ligands, bovine primary macrophages or cells transfected with bovine TLRs respond at lower levels than humans.
Irvine et al.  cloned equine TLR2, TLR1, and TLR6 and addressed their responses to classical TLR2 ligands. Functionality of TLR2/1 and TLR2/6 heterodimers was demonstrated, with LTA inducing responses similar to those observed with human heterodimers. Pam2CSK4 activation of TLR2/6 was identical for receptors of both species, while, in opposition to what is observed in human, Pam3CSK4 was less potent than Pam2CSK4 in activating the equine TLR2/1 heterodimer.
Different studies addressed the ligand recognition by both types of chicken TLR2 and TLR1 [26, 66, 67]. Together, these studies show that the avian TLR2a and TLR2b form heterodimers with the avian TLR1La and TLR1Lb, allowing the recognition of the same range of ligands that bind the mammalian TLR2 heterodimers, including Pam3CSK4, MALP-2, FSL-1, and PGN.
3. Immunogenic Formulations Targeting TLR2
As occurred with other TLR ligands, the use of lipoproteins and lipopeptides of bacterial origin as adjuvant molecules is well prior to the knowledge of their receptors and mode of action. Soon after the pioneer studies describing and characterizing a lipoprotein present in the cell wall of E. coli, published by the groups of Braun and Inouye around 1970 [50, 68–75], it was demonstrated that this lipoprotein, then called Braun’s lipoprotein, presented mitogenic properties in mouse B cells . The study of the fragments obtained by hydrolysis of the native lipoprotein enabled Bessler et al.  to attribute its mitogenic capacity to the N-terminal triacylated moiety and this was confirmed soon later through the chemical synthesis of lipopeptides with structure analog to the lipid moiety of that region . Following these works, the adjuvant properties of the lipid moiety were tested in vivo. The mice inoculation with synthetic lipopeptides covalently bound to a nonimmunogenic peptide of the epidermal growth factor receptor led to the induction of specific antibodies two weeks later after one single administration  and the inoculation of guinea pigs with synthetic peptides from the foot-and-mouth disease virus (FMDV) conjugated with the Pam3CSS lipopeptide resulted in the induction of neutralizing antibodies and protection against viral infection . At the end of the 1980s, it was reported that the lipopeptides stimulated in vitro not only lymphocytes but also human monocytes and mouse macrophages  and Deres et al.  demonstrated the possibility of inducing in vivo CTL responses, restricted to MHC class I, by the inoculation of mice with synthetic lipopeptides conjugated with epitopes from the influenza virus nucleoprotein. Based on these works, as well as in earlier demonstrations of the immunomodulatory effect of protein lipidation [82–85], many studies using synthetic lipopeptides in different disease and immunization models were carried out.
However, it was only at the end of the 1990s that, shortly after the publication describing the cloning and characterization of the human receptor homolog to the Drosophila toll , different studies reported that TLR2 is a receptor for bacterial lipoproteins [48, 54, 87, 88]. This definitely contributed to understand the adjuvant properties of lipopeptides and lipoproteins and drove a renewed interest in their use as adjuvants. Meanwhile, many other molecules have been claimed to bind TLR2, including nonlipidated molecules, and, although their interaction with the receptor is not entirely elucidated, some have also been used experimentally as adjuvants (e.g., [89, 90]). Among the strategies proposed to explore TLR2 stimulation for the modulation of immune responses, the different systems allowing covalently link lipid moieties to proteic antigens present the most promising applications in vaccinology.
3.1. Recombinant Bacterial Lipoproteins Expressed in Fusion with Heterologous Antigens
The first plasmidic vectors for the expression of proteins in fusion with bacterial lipoproteins in E. coli aiming at vaccine development were reported in the 1990s. Some of these works had the single objective of exhibiting antigens at the surface of the host bacteria [91, 92], but others were developed also with the purpose of making use of the adjuvant properties of the lipid moiety for the induction of immune responses against heterologous antigens [93, 94]. These vectors consist in partial or complete sequences derived from bacterial lipoprotein genes followed downstream by coding sequences for the heterologous antigens. Among the lipoproteins used as partners in these chimeric structures or used as sources of the lipidation signals are the colicin E2 lysis lipoprotein from an E. coli colicinogenic plasmid , OprI lipoprotein from Pseudomonas aeruginosa , Braun’s lipoprotein from E. coli [95, 96], 26 kDa lipoprotein (Rv1411) from Mycobacterium tuberculosis , Wza lipoprotein from Vibrio anguillarum , Ag473 lipoprotein from Neisseria meningitidis , and OMP19 lipoprotein from Brucella abortus . Apart from the Rv1411 lipoprotein from Mycobacterium tuberculosis, all the mentioned lipoproteins are originally from Gram-negative bacteria where they are found anchored to the outer membrane. These molecules are first expressed in the cytoplasm as prolipoproteins with an N-terminal signal peptide and are then translocated by the Sec translocon across the inner membrane to the periplasmic side where the processing takes place . The initial lipidation step consists in the binding of a diacylglycerol group through a thioether linkage to the cysteine residue located at the N-terminus of the mature sequence, followed by the cleavage of the signal peptide. The third acyl chain is then attached to the amine group of the same cysteine residue through an amide linkage. Mature lipoproteins are finally transported and anchored on the outer membrane by the Lol System [101, 102]. Due to this maturation process, triacylated forms of the recombinant lipoproteins are only present in the outer membrane of the expression hosts and purification strategies were developed to purify these fully mature forms from the outer membrane . In the cases in which lipoproteins are purified from whole bacterial cell lysates, immature forms, including diacylated lipoproteins, are also present in the final formulations [104, 105].
In some of these cloning and expression systems, multiple cloning sites were included downstream of the lipoprotein gene offering a flexible platform for the cloning of heterologous antigens and were even proposed for shotgun cloning viral genomes and screening for T cell antigens . In some cases, C-terminal hexahistidine tails were also added to enable the purification of the fusion proteins by metal affinity chromatography. When the lipid moieties were characterized, palmitic acid was the predominant fatty acid found, although other fatty acids, including unsaturated, were also present [103, 107].
It is worth mentioning here that there are also examples of vaccine formulations using lipoproteins as homologue antigens, extracted from their native hosts or produced in other expression hosts (e.g., [108, 109]). The classical example is OspA from Borrelia burgdorferi for vaccination against Lyme disease .
3.2. Synthetic Lipopeptides
The chemical synthesis of peptides linked to lipid moieties is another widely used strategy to produce self-adjuvant formulations. Epitopes extended by Pam3C or Pam2C mimic tri- and diacylated bacterial lipid moieties, but many different variations to this structure have also been developed. These include single-chain palmitoyl-peptides and the more complex lipid core peptide (LCP) and multiple antigen lipophilic adjuvant carrier (MALAC) systems. The covalent attachment of TLR2 agonists to intact proteins has also been reported . Their description and the relations between their structural characteristics and activity have been extensively discussed in detailed reviews (see, e.g., [112–114]) and thus we will not focus on that. However, it is important to stress that differences in the lipid moiety structure, such as the length of the fatty acids and chirality of the glyceryl modification, affect the TLR2-activating properties and this may reflect on the immune response elicited.
Another important point to consider is that some of these synthetic ligands have peptidic and lipid structures very different from the typical bacterial TLR2 ligands and, in certain cases, the dependency on TLR2 activation for their immunomodulatory properties remains to be elucidated. However, for monoacetylated lipopeptides and some other lipoamino acid based lipopeptides the activation through TLR2 is documented [115–117].
4. Immunomodulation by Formulations Targeting TLR2
A rational use of adjuvants in the development of better subunit vaccines relies on the understanding of how stimuli exerted at vaccination are translated in specific immune mechanisms, including their magnitude, profile, persistence, and localization. The innate activation through PRRs plays a central role in this shaping of the adaptive immunity and here we resume what has been reported, mainly based on mouse and human studies, concerning TLR2 and TLR2-targeting immunogenic formulations (Table 2).
|rLipo-D1E3 and rlipo-E7m: Domain 1 of the Ag473 lipoprotein from Neisseria meningitidis fused to a sequence of dengue virus envelope protein (E3) or inactive E7 oncoprotein of human papillomavirus (E7m); OprI or OprI-COOHgp63 BLPs: outer membrane lipoprotein I from Pseudomonas aeruginosa, alone or fused with a truncated 32-kDa version, the major cell surface glycoprotein gp63 of Leishmania major; [Th]: CD4+ T cell epitope; [Tc]: CD8+ T cell epitope; [B]: B cell epitope; K: Lysine with a palmitic (Pam), diacylated (P2CSS), or cholesterol (Chol) moiety attached to -amino group; 19-kDa, Tp47, and OspA LP: synthetic tripalmitoyl lipopeptides based on the sequences of 19-kDa lipoprotein from Mycobacterium tuberculosis, 47-kDa lipoprotein from Treponema pallidum, and OspA lipoprotein from Borrelia burgdorferi; 19-kDa and 38-kD BLP: 19-kDa and 38-kD lipoproteins from Mycobacterium tuberculosis; LP40: synthetic lipopeptide CGP 40774; Hda: α-aminohexadecanoic acid; BPPcysMPEG: pegylated synthetic derivative of MALP-2; MAP: multiple antigen peptide; LT-IIa-B5: pentameric B subunit of type IIa enterotoxin of Escherichia coli.|
↗: enhancement; ↘: decrease; BM-DCs: bone marrow-derived DCs; spl. DCs: splenic DCs; Mo-DCs: monocyte-derived dendritic cells; Mo: monocytes; Mo-LC: monocyte-derived Langerhan-like cells; D1 cells: a line of immature DCs derived from spleen cells; PBMC-DCs: PBMC-derived DCs; CB-PBMCs: cord blood-derived PBMCs; THP-1 cells: human monocytic cell line; i.n.: intranasal.
4.1. Modulation of APC Migration and Antigen Internalization
TLR activation has been implicated in the several steps that culminate in the development of specific immune responses, including APC migration. Recently, addressing different adjuvants on influenza subunit vaccines, a role for TLR2 activation in leukocyte migration to inflammation foci was suggested . Pam3CSK4 was shown to be more efficient than CpG and resiquimod, respectively, TLR9 and TLR7/8 ligands, at inducing an early recruitment of CD11b+ blood cells, mainly neutrophils, at the injection site and this observation correlated with the higher capacity of Pam3CSK4 to enhance antibody responses against influenza antigens . TLR ligands were shown to transiently reduce the motility of DCs at the inflammatory sites, allowing for an extended contact between DCs and the antigen at the site of inflammation [119, 120]. However, the DC activation through TLRs downregulates receptors for inflammatory chemokines and upregulates CCR7 promoting their subsequent migration through lymphatic vessels and localization in the T cell areas of the regional lymph nodes [4, 121–123], and this was reported for TLR2 agonists as well [124, 125].
Internalization of pathogens is also regulated by TLR activation at the inflammatory sites. An initial transitory increasing in the internalization of antigens in response to TLR ligands occurs and is followed by the characteristic reduction in the endocytic capacity of mature DCs, which is congruent with a phenotype specialized in processing and presentation of antigens [126–129].
The above-mentioned transitory reduction in DC motility and the enhancement of antigen internalization was observed upon stimulation of different TLRs including TLR2 [120, 129]. Also demonstrating a TLR2 role in antigen internalization, Schjetne et al.  showed that targeting TLR2 with an anti-TLR2 monoclonal antibody leads to the internalization of the ligand into endosomes and its presentation under MHC class II.
4.2. Antigen Processing and Presentation in the Context of MHC Class II
Although still controversial [131–133], growing evidences support a determinant role of TLR activation in the control of phagosome maturation, with consequent impact on the regulation of antigenic presentation by APCs. Studies developed by the groups of Blander and of Medzhitov sustain the existence of two modes of phagosome maturation, one constitutive and the other inducible and controlled by TLR signalling [134–136]. According to these studies, the phagocytosis of bacteria leads to a phagolysosomal fusion rate superior to that observed in phagocytosis of apoptotic cells. This difference is only observed in the presence of TLR signalling and involves activation of MAPK p38 through MyD88 activation. Notably, this control of maturation occurs autonomously in the phagosome; that is, the inducible mode is only observed at the phagosomes containing TLR ligands. In addition, TLR activation has been shown to control autonomously the MHC class II loading in the phagosome  and to stabilize MHC class II at the cell surface . This autonomous control of phagosome maturation and MHC class II loading may contribute to explain the enhancement of specific immune responses when the antigen is covalently linked to the TLR ligands or is incorporated in the same physical particle [138–141]. The TLR2-targeting formulations in which the antigen is chemically linked to lipid moieties or expressed as fusion with a bacterial lipoprotein ensure TLR activation inside the same phagosomes that contain the antigen and thus are particular interesting tools to modulate antigen processing and presentation. In fact, clearly increased antibody and cellular responses were observed in vivo when the antigens were covalently linked to lipopeptides or lipoproteins in comparison to admixed formulations [140, 142, 143].
For the development of an effector response by CD4+ T cells, the recognition of a peptide in the context of MHC class II must be accompanied by the engagement of costimulatory molecules expressed at the DC surface, like CD80, CD86, and CD40. In the absence of costimulation, T cells are instructed towards a regulatory or anergic phenotype, resulting in tolerance to the presented antigen . The upregulation of these molecules and of MHC class II at the surface of DCs is generally induced through TLR activation . TLR2 is not an exception. In several studies, the activation of APCs with synthetic lipopeptides and recombinant lipoproteins resulted in cell maturation, with upregulation of MHC and costimulatory molecules (e.g., [99, 142, 145–152]). Moreover, the presentation of MHC class II epitopes to specific T cells by DCs has been demonstrated to be enhanced in the presence of TLR2 agonists [129, 149].
4.3. CD4+ T Cell Polarization
The fate of CD4+ T cells is a key issue for the type of immunity elicited upon immunization and its adequacy to the challenge is of capital importance for the success of a vaccine. In this respect, the consequence of TLR activation is not the same for the different TLR ligands and this is probably the most controversial point regarding TLR2 activation and the use of TLR2-targeting formulations in vaccination.
Some authors associate the activation through TLR2 with the induction of Th2 responses [151–155]. According to the model proposed by Dillon et al. , the activation via this receptor induces a high level of ERK1/2 signalling resulting in the stabilization of the transcription factor c-Fos suppressing IL-12(p70) production and promoting IL-10 secretion, thus favouring Th2 type responses. Moreover, Gautier et al.  attribute the secretion of IL-12(p70) by DCs, essential for Th1 polarization, to an autocrine-paracrine loop of type I interferon (IFN) initiated in response to TLR activation. Stimulation via TLR2 induces the activation of a MyD88-dependent signalling pathway, with activation of NF-κB and the MAPK pathway, resulting, among other effects, in the production of proinflammatory cytokines but not of type I IFN. This would justify the incapacity to polarize responses towards Th1 type via TLR2.
However, other authors pointed TLR2 stimulation or the use of adjuvants composed of TLR2 ligands as an efficient strategy to induce Th1 responses through APC activation [87, 117, 143, 157–163]. Moreover, according to Imanishi et al. , the stimulation of mouse Th1 cells by TLR2 ligands directly induces IFN-γ production, as well as cellular survival and proliferation in the absence of TCR stimulation. The same is not observed for ligands of other TLR receptors, suggesting an important role of TLR2 activation in the promotion and maintenance of Th1 responses.
In other experimental conditions, the induction of Th17 polarization by IL-1β, TGF-β, and IL-23 produced by human Langerhans cells stimulated via TLR2 was reported . Synthetic lipopeptides or bacterial lipoproteins have also been shown to induce Th1/Treg differentiation and inhibition of Th2 responses, suggesting a potential application in the treatment of asthmatic diseases [147, 166, 167]. In fact, a regulatory role has been attributed to TLR2 in several studies and its targeting for the induction of tolerogenic responses has also been proposed [168–172, 191]. However, experimental evidences support that the direct activation of Tregs by the TLR2/1 ligand Pam3CSK4, together with stimulation through TCR in the presence of IL-2, induces proliferation and temporary loss of suppressive properties, which is restored after removing the stimulus [173, 174]. Furthermore, the same TLR2 ligand was reported to exert antitumor effects, either through the reduction of the suppressive function of Tregs  or by the enhancement of the resistance of T effector cells to Treg suppression .
The observations supporting divergent TLR2 polarizing properties were obtained from distinct models, using various TLR2 ligands and looking at different levels of the immune response, from the molecular signaling level up to in vivo context. As underlined by Mele and Madrenas  and supported by other studies , it is clear that TLR2 plays both proinflammatory and regulatory roles and that through TLR2 activation, alone or in combination with other stimuli, both effector and regulatory immune mechanisms can be elicited in vivo depending on not yet completely clarified factors. To focus on understanding these discrepancies should be a priority for rationally exploring TLR2 stimulation in vaccine development.
4.4. Cross-Presentation and CD8+ T Cell Cytotoxicity
TLR activation has been also implicated in the induction of cross-presentation of antigens by DCs as consequence of enhanced antigen internalization and delivery to the cytosol as well as increase in TAP and proteasome activity [128, 129]. A MyD88-dependent cross-presentation mechanism that requires the dislocation of TAP to the early endosomes was also reported , suggesting a spatial separation between endogenous MHC class I-restricted antigen presentation and cross-presentation of exogenous antigens, the latter being biased toward antigens containing PAMPs. Cross-presentation and in vivo induction of CTL by TLR activation are usually attributed to TLR ligands high inducers of type I IFN [126, 194, 195], which is not the case of TLR2 ligands. However, different studies demonstrate the induction of these mechanisms by antigens conjugated with TLR2 ligands [140, 146, 157, 177, 178]. In fact, this has been from the beginning one of the most appealing characteristics of these immunogenic formulations [81, 196]. Initially, it was suggested that this capacity could be due to the access of lipidated peptides to the cytoplasm of APC, facilitated by the interaction of the membrane lipids with those of the lipoprotein, with consequent entering in the MHC class I processing pathway . Another explanation resided in the physical properties conferred to the formulations by the lipids, for example, the possible formation of micellar structures and the consequent processing by APCs identical to that observed with particulate antigens . Although these mechanisms are fundamentally of physical nature and TLR-independent, the activation of these receptors also plays an important role in the induction of CTL responses. In fact, Khan et al.  showed that and glycerol configurations of Pam3CSK4 are equally internalized by DCs but diverge at their capacity to induce cytokines and maturation markers on these cells, as well as on the induction of CTL responses in vivo. This suggests a determinant role of TLR2 activation on promoting the CTL immune mechanisms. Additionally, it was demonstrated that DC stimulation with the TLR2 ligand MALP-2 induces the expression of proteins of the immunoproteasome LMP2, LMP7, and MECL and an increasing in the proteolytic activity and thus the antigenic processing, suggesting that lipopeptides may indirectly increase the responses restricted to MHC class I .
Monoacylated lipopeptides, lipidated through the covalent binding of palmitic acid to the lateral chain of lysine, induce CD8+ T cell responses [180, 182] and, although monoacylation do not correspond to the native structure of bacterial lipoproteins, Zhu et al.  found that these molecules enhance internalization and DC maturation through TLR2. Zhang et al.  also demonstrated the induction of herpes simplex virus (HSV)-2-specific memory CD8+ CTL both locally and systemically after intravaginal immunization with a peptide extended by a lipid moiety with three palmitic acids and showed that this response was significantly lower in TLR2−/− and MyD88−/− mice. The inoculation of ovalbumin (Ova) peptides together with BPP, a synthetic derivative of the MALP-2, induced CTL responses which were much higher when the peptides were directly linked to the TLR2 ligand comparing to admixed antigens and adjuvants .
4.5. Induction of NK Cells Activity
TLR2-dependent NK cell activation was demonstrated to play a role in the immune response against different virus and bacteria [197–201] suggesting the possibility to explore TLR2 ligands as inducers of NK cell activity in therapeutic or prophylactic immunomodulation. The activation of NK cells by different TLR2 agonists has been demonstrated, although the requirement for accessory cells, namely DCs, and accessory cytokines is still debatable [202–204]. Also, variations in the capacity to stimulate NK cells were found among different TLR2 ligands. For example, the activation of NK cells by MALP-2 through stimulation of TLR2 on bone marrow-derived DCs is much less effective than Pam2CSK4  and the peptide primary sequence in synthetic Pam2C lipopeptides has been demonstrated to influence the capacity for NK activation, both directly through TLR2 on NK cells and via DCs .
In vivo, the subcutaneous injection of Pam2CSK4 around NK sensitive B16D8 tumor cells led to tumor retardation and this activity was abrogated by injection of asialoGM-1 antibodies, while this antitumor activity was not observed for MALP-2 ligand which was less effective in inducing NK activity . However, in another study, the administration of diacylated lipopeptides (MALP-2 and different Pam2 peptides) in mice induced IL-10 and Treg cells that prevented effective antitumor therapeutic responses . The authors also reported the production of IL-10 by NK cells stimulated by the TLR2 agonists in vitro. In an antitumor prophylactic immunization, Kiura et al.  reported that coadminisration of tumor associated antigens with the diacylated lipopeptide FSL-1 could induce antitumor antibody dependent cellular cytotoxicity (ADCC) by NK cells.
The role of TLR-mediated activation of NK cells in immune responses against infectious and tumor disease is now emerging . TLR2-mediated activation of NK activity for the shaping of the adaptive response, as well as the activation of NK activity in the context of a recall response, will certainly be an interesting research field for the development of future prophylactic vaccines.
4.6. Induction of Antibody Responses
Antibody titres, affinity, avidity, and neutralizing capacity are for many diseases the best correlative of protection and to elicit a proper and long-lasting antibody response is thus frequently a desirable achievement in vaccination.
The role of TLR activation in antibody responses and its longevity is a theme of actual research. Pasare and Medzhitov  showed that the generation of T-dependent antigen-specific antibody responses requires activation of TLRs in B cells. Kasturi et al.  have shown that administration of Ova in synthetic nanoparticles together with the TLR4 ligand monophosphoryl lipid A (MPL) and, simultaneously, with the TLR7 ligand R837 leads to synergistic increases in the anti-Ova antibody response and provides a sustained memory for 1.5 years. This was dependent on DCs, Th cells, and the direct TLR stimulation on B cells.
The specific role of TLR2 in the development of antibody responses is now emerging. TLR2 has been recently demonstrated to be involved in the generation and longevity of antibody secreting cells (ASC)  and the addition of CD40 signalling to TLR1/2 and TLR2/6 agonists have been shown to stimulate differentiation of B cells into ASC . Moreover, lipidated formulations targeting TLR2 are widely described as good inducers of antibody responses (e.g., [99, 117, 143, 146, 154, 155, 159]).
4.7. Mucosal Immune Responses
Many of the most relevant diseases of humans and veterinary animals are caused by infectious and parasitic agents entering the target hosts through mucosae. Immunization via different mucosal surfaces using TLR2-targeting formulations has been demonstrated to induce strong immune responses, including mucosal IgA and serum IgG as well as local and systemic CD8+ CTL [124, 146, 187–189]. Efficient induction of mucosal immune responses is not usually achieved by parenteral administration of vaccines, requiring the presentation of antigens directly at the mucosal surfaces. However, TLR2/1 signals, but not signals from other TLRs, have been shown to be capable of educating extraintestinal DCs with gut-specific imprinting properties . Considering that an immune response elicited apart from the strongly regulatory mucosal environment may be more freely modulated, this capacity of localizing immune responses at the mucosal level through nonmucosal immunization may open the possibility to better tailor the adaptive mucosal immunity. For this purpose, TLR2-targeting formulations are particularly interesting tools.
5. Immunomodulation through TLR2 for Veterinary Vaccines
For the veterinary species, the modulatory effect of TLR2 activation on immune response is much less characterized than in humans or in the mouse model. However, in vitro activation of APCs or PBMCs by TLR2 ligands was demonstrated for different species as it was the adjuvanticity of TLR2-targeting formulations when inoculated in vivo. Evidences on the capacity to elicit CTL responses were also obtained but information on the immune response profiles is sparse. Here, we compile some of the most relevant studies available regarding immunomodulation through TLR2 in veterinary species.
In ovine, stimulation of bone marrow-derived DCs by LTA resulted in upregulation of MHC class II in the subset, which acquired strong capacity of stimulating CD4+ T cells in allogenic assays . In bovine, Pam3CSK4 stimulation of monocyte-derived DC also induced DC maturation, with upregulation of MHC class I, MHC class II, CD40, CD80, CD86, and CD1b molecules, and lead to the production of IL-12 and TNF-α. In addition, stimulated DCs promoted IFN-γ secretion when cocultured with allogeneic PBMCs . In contrast, the stimulation of macrophages lead to the downregulation of MHC expression and to an almost null effect on IL-12 and TNF-α production and on IFN-γ secretion in mixed leukocyte reaction . However, Franchini et al.  reported that bovine macrophages produce nitric oxide (NO) and TNF-α in response to TLR2 activation and that this production is strongly increased by costimulation with IFN-γ.
Nelson et al.  have shown the possibility to induce active vitamin D3 (1α,25-dihydroxyvitamin D3) in bovine monocytes through TLR4 and TLR2 activation. Notably, in the mouse, the induction of vitamin D3 in APCs was shown to be related with the imprinting of skin-tropism by APCs in T cells . How this could be exploited for the induction of skin-tropic responses in farm animals remains to be studied. More recently, it was reported that bovineγδ T cells directly respond to TLR2 ligands with increased proliferation and cytokine production in a TCR-independent manner .
Investigating the potential of Mycobacterium bovis antigens to stimulate delayed-type hypersensitivity (DTH) response in cattle, Whelan et al.  have shown that the combination of Pam3CSK4 lipopeptide with the ESAT-6 antigen allowed the induction of DTH in experimentally infected calves which did not occur when the antigen was used alone. In vitro, the authors showed the induction of TNF-α production by bovine DCs stimulated by Pam3CSK4 and pointed this induction of proinflammatory cytokines as a possible explanation for what was observed in vivo.
Aiming at optimizing the protective efficacy of Mycobacterium bovis BCG, Wedlock et al.  tested combinations of BCG with culture filtrate proteins formulated with a depot adjuvant and mixed with different stimulatory molecules: MPL, a synthetic mycobacterial phosphatidylinositol mannoside-2 (PIM2) and Pam3CSK4. Evaluating different pathological and microbiological disease parameters, such as the proportion of animals with tuberculous lesions in the lungs and lymph nodes and the number of M. bovis culture-positive lymph nodes, the inclusion of Pam3CSK4 in the vaccine formulation was shown to induce the best protection.
The potential to use lipopeptides for vaccination against foot-and-mouth disease was tested using seven peptides containing FMDV-specific B-cell linear epitopes from structural and nonstructural proteins, synthesized with a Pam3C moiety, and delivered intramuscularly emulsified with Montanide ISA 9 . Twenty days after a single immunization, the animals were challenged and four of the seven immunized animals were protected. No correlation was found between protection and antibody titre or virus-specific proliferation but all protected animals showed a strong T-cell response against at least one of the peptides used for immunization.
Also in cattle, lipopeptides with a palmitic acid coupled to the NH2-terminal amino acid and delivered in Freund’s adjuvant were used to boost an anti-Neospora caninum SRS2 (NcSRS2) DNA immunization . Lipopeptide boosting induced strong immune response, characterized by increased NcSRS2-specific lymphocyte proliferation, IFN-γ-secreting cells, and levels of specific IgG1 and IgG2a antibodies. Regarding these parameters, this immunization strategy reproduced the immune response observed against N. caninum infection in cattle.
Stimulation of equine monocytes with Pam3CSK4 induced the production of TNF-α, IL-6, IL-1β, and IL-10 , and in a whole blood assay TNF-α, IL-6, and IL-1β were also induced by PGN and LTA . Using equine infectious anemia virus (EIAV) CTL epitopes synthesized on multiple antigenic peptide (MAP) system linked to Pam3C, Ridgely and McGuire  demonstrated the capacity to stimulate CTL activity in vitro on PBMCs obtained from horses of different ELA-A haplotypes chronically infected. The stimulated cells were able to specifically lyse EIAV-infected target cells. In addition, immunization of horses with a Pam3C-MAP-CTL epitope induced transitory peptide and virus-specific CTL and, although it neither prevented infection nor affected viral load, it induced a protective effect against development of clinical disease following virus challenge, in which vaccinated horses showed less severe fever and thrombocytopenia and did not develop anemia during the first 2 months after infection . In another approach for anti-EIAV immunization with lipopeptides, Fraser et al.  inoculated horses with a pool of peptides containing Th and CTL epitopes extended by a palmitic acid molecule to each of the free NH2 groups. The immunized horses showed significant postimmunization proliferative responses to Th peptides but no evident CTL response. After challenge, the immunized group also had a significant increase in the proliferative response to the Th peptides and PBMCs from four of five immunized horses showed CTL activity when stimulated 2 weeks later. Nonetheless, no significant protection was observed considering level and course of viral load, the platelet counts, or fever.
Immunizing pigs against mouse IgG, an increase in the anti-mouse IgG titres was observed by targeting the antibody to TLR2 . In this work, it was also shown that the in vitro proliferative response of PBMCs obtained from pigs immunized with mouse IgG was enhanced when restimulation was performed using an anti-TLR2 mouse monoclonal antibody comparing with restimulation with an isotype-matched control.
Using outer membrane preparations from bacteria expressing African swine fever virus (ASFV) antigens in fusion with the OprI lipoprotein, the entering of the antigens in the class I pathway of antigen presentation and the possibility to identify ASFV epitopes specifically recognised by porcine CTL  as well as to stimulate specific CTL activity in vitro  were demonstrated. In a different study , with the purpose to test OprI as an adjuvant for a subunit vaccine against classical swine fever (CSF), it was shown that this lipoprotein activated porcine monocyte-derived DCs, upregulating CD80/86 and MHC class II expression, as well as proinflammatory cytokines. The antigenic restimulation of lymphocytes obtained from CSFV-immune pigs cocultured with autologous monocyte-derived dendritic cells was also enhanced by OprI, as measured by proliferation and IFN-γ production. In vivo, a subunit vaccine adjuvanted with OprI induced partial protection against CSFV infection but less effective than a water-oil-water adjuvanted vaccine tested in parallel.
Stimulation of chicken splenocytes with Pam3CSK4 upregulated not only Th1-associated cytokines IFN-γ and IL-12 but also the Th2-associated cytokine IL-4 . The direct stimulation of chicken CD4+ T cells by Pam3CSK4 also significantly upregulated IFN-γ but not IL-4, IL-13, and IL-10 . In a study comparing the effect of three different TLR2 ligands on chicken splenocytes, the results observed suggest different kinetics in the production of proinflammatory cytokines. Pam3CSK4 induced high IL-1β response, while FSL-1 induced an early and prolonged expression of IL-8. The three TLR2 ligands, Pam3CSK4, FSL-1, and lipomannan, induced a mixed Th profile with upregulation of IFN-γ, IL-12, IL-4, and IL-13 . Stimulating chicken monocytes, He et al.  demonstrated the induction of iNOS mRNA and of NO production by LTA but not by Pam3CSK4. Erhard et al.  tested the adjuvant effect of Pam3CSK4 and Pam3CS linked to Th epitopes when administered together with different antigens. Although varying with the antigen, the antibody responses were enhanced and in certain cases the combination of more than one adjuvant induced even better responses. Testing two lipoproteins from Pasteurella multocida as vaccine antigens, Wu et al.  report high protection rates in chicken immunized with E. coli expressed lipoprotein E after inoculation in double emulsion adjuvant. OprI lipoprotein was shown to bind in vitro and in vivo to epithelial cells of the trachea and the small intestine of chickens suggesting its potential use as a carrier for antigen delivery at mucosal surfaces .
Based on previous demonstration of the immunostimulatory properties of protozoan HSP70 through TLR2 and TLR4, Zhang et al.  investigated if Eimeria tenella HSP70 could enhance the immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. EtHSP70 induced the production of IL-12 and IFN-γ in chicken embryo fibroblasts and when inoculated in vivo together with EtMIC2 resulted in increased body weight gains, decreased oocyst shedding, and increased antibody responses. Levels of IL-12, IFN-γ, and IL-17 were also higher compared with the inoculation of the antigen alone. Chickens immunized with EtHSP70 alone also revealed a protective effect against E. tenella infection.
6. Concluding Remarks
Vaccination in veterinary animals is a cost-effective strategy to promote animal health and may have an important impact on public health by contributing in reducing the use of antibiotics and controlling zoonotic diseases. The development of new vaccines largely relies on the understanding of how activation of innate immunity through PRRs shapes the subsequent adaptive immune response. The possibility of enhancing antigen presentation by covalently linking TLR2 ligands to the antigen and the particular TLR2 properties at influencing the type and localisation of specific immunity are interesting features that can help at solving some of the present vaccine challenges. However, considering the inconsistencies in results regarding the profile of immune responses, it is of major relevance to address how the specific immune mechanisms elicited upon immunization targeting TLR2 are affected by different factors, such as type of ligand, route of administration, doses, and synergies with other innate stimuli. To extend these studies to the field of veterinary vaccinology further implies to address species-specificities. Clarifying these aspects will allow us in the future to make the innate stimulus adequate for a particular challenge in a given species.
|ADCC:||Antibody dependent cellular cytotoxicity|
|ASC:||Antibody secreting cells|
|APC:||Antigen presenting cell|
|CTL:||Cytotoxic T lymphocyte|
|EIAV:||Equine infectious anemia virus|
|FMDV:||Foot-and-mouth disease virus|
|MPL:||Monophosphoryl lipid A|
|OprI:||Outer membrane lipoprotein I|
|PRR:||Pattern recognition receptor|
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors acknowledge the financial support of Fundação para a Ciência e a Tecnologia (project Grants PTDC/CVT/113889/2009 and PTDC/CVT/65674/2006).
- T. Kawai and S. Akira, “The roles of TLRs, RLRs and NLRs in pathogen recognition,” International Immunology, vol. 21, no. 4, pp. 317–337, 2009.
- N. W. Palm and R. Medzhitov, “Pattern recognition receptors and control of adaptive immunity,” Immunological Reviews, vol. 227, no. 1, pp. 221–233, 2009.
- C. Watts, M. A. West, and R. Zaru, “TLR signalling regulated antigen presentation in dendritic cells,” Current Opinion in Immunology, vol. 22, no. 1, pp. 124–130, 2010.
- D. Van Duin, R. Medzhitov, and A. C. Shaw, “Triggering TLR signaling in vaccination,” Trends in Immunology, vol. 27, no. 1, pp. 49–55, 2006.
- K. J. Ishii and S. Akira, “Toll or toll-free adjuvant path toward the optimal vaccine development,” Journal of Clinical Immunology, vol. 27, no. 4, pp. 363–371, 2007.
- H. J. Warshakoon, J. D. Hood, M. R. Kimbrell et al., “Potential adjuvantic properties of innate immune stimuli,” Human Vaccines, vol. 5, no. 6, pp. 381–394, 2009.
- R. L. Coffman, A. Sher, and R. A. Seder, “Vaccine adjuvants: putting innate immunity to work,” Immunity, vol. 33, no. 4, pp. 492–503, 2010.
- H. Kumar, T. Kawai, and S. Akira, “Pathogen recognition in the innate immune response,” Biochemical Journal, vol. 420, no. 1, pp. 1–16, 2009.
- H. Kumar, T. Kawai, and S. Akira, “Toll-like receptors and innate immunity,” Biochemical and Biophysical Research Communications, vol. 388, no. 4, pp. 621–625, 2009.
- J. C. Roach, G. Glusman, L. Rowen et al., “The evolution of vertebrate Toll-like receptors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 27, pp. 9577–9582, 2005.
- S. Akira, “Pathogen recognition by innate immunity and its signaling,” Proceedings of the Japan Academy B, Physical and Biological Sciences, vol. 85, no. 4, pp. 143–156, 2009.
- T. Kawai and S. Akira, “The role of pattern-recognition receptors in innate immunity: update on toll-like receptors,” Nature Immunology, vol. 11, no. 5, pp. 373–384, 2010.
- R. Brownlie and B. Allan, “Avian toll-like receptors,” Cell and Tissue Research, vol. 343, no. 1, pp. 121–130, 2011.
- T. W. Jungi, K. Farhat, I. A. Burgener, and D. Werling, “Toll-like receptors in domestic animals,” Cell and Tissue Research, vol. 343, no. 1, pp. 107–120, 2011.
- D. Werling and T. J. Coffey, “Pattern recognition receptors in companion and farm animals—the key to unlocking the door to animal disease?” Veterinary Journal, vol. 174, no. 2, pp. 240–251, 2007.
- H. Uenishi and H. Shinkai, “Porcine Toll-like receptors: the front line of pathogen monitoring and possible implications for disease resistance,” Developmental and Comparative Immunology, vol. 33, no. 3, pp. 353–361, 2009.
- Y. Muneta, H. Uenishi, R. Kikuma et al., “Porcine TLR2 and TLR6: identification and their involvement in Mycoplasma hyopneumoniae infection,” Journal of Interferon and Cytokine Research, vol. 23, no. 10, pp. 583–590, 2003.
- M. Ishii, M. Hashimoto, K. Oguma, R. Kano, T. Moritomo, and A. Hasegawa, “Molecular cloning and tissue expression of canine Toll-like receptor 2 (TLR2),” Veterinary Immunology and Immunopathology, vol. 110, no. 1-2, pp. 87–95, 2006.
- D. Werling, O. C. Jann, V. Offord, E. J. Glass, and T. J. Coffey, “Variation matters: TLR structure and species-specific pathogen recognition,” Trends in Immunology, vol. 30, no. 3, pp. 124–130, 2009.
- B. Pulendran, “Modulating vaccine responses with dendritic cells and toll-like receptors,” Immunological Reviews, vol. 199, pp. 227–250, 2004.
- M. S. Duthie, H. P. Windish, C. B. Fox, and S. G. Reed, “Use of defined TLR ligands as adjuvants within human vaccines,” Immunological Reviews, vol. 239, no. 1, pp. 178–196, 2011.
- J. Gibson, N. Gow, and S. Y. C. Wong, “Expression and functions of innate pattern recognition receptors in T and B cells,” Immunology, Endocrine and Metabolic Agents in Medicinal Chemistry, vol. 10, no. 1, pp. 11–20, 2010.
- L. S. Kwong, R. Parsons, R. Patterson et al., “Characterisation of antibodies to bovine toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2,” Veterinary Immunology and Immunopathology, vol. 139, no. 2-4, pp. 313–318, 2011.
- B. Alvarez, C. Revilla, N. Doménech et al., “Expression of toll-like receptor 2 (TLR2) in porcine leukocyte subsets and tissues,” Veterinary Research, vol. 39, no. 2, 2008.
- M. Tohno, H. Kitazawa, T. Shimosato et al., “A swine toll-like receptor 2-expressing transfectant as a potential primary screening system for immunobiotic microorganisms,” FEMS Immunology and Medical Microbiology, vol. 44, no. 3, pp. 283–288, 2005.
- A. Fukui, N. Inoue, M. Matsumoto et al., “Molecular cloning and functional characterization of chicken Toll-like receptors: a single chicken Toll covers multiple molecular patterns,” The Journal of Biological Chemistry, vol. 276, no. 50, pp. 47143–47149, 2001.
- C. Bazzocchi, M. Mortarino, S. Comazzi, C. Bandi, A. Franceschi, and C. Genchi, “Expression and function of Toll-like receptor 2 in canine blood phagocytes,” Veterinary Immunology and Immunopathology, vol. 104, no. 1-2, pp. 15–19, 2005.
- D. Werling, J. Piercy, and T. J. Coffey, “Expression of TOLL-like receptors (TLR) by bovine antigen-presenting cells-Potential role in pathogen discrimination?” Veterinary Immunology and Immunopathology, vol. 112, no. 1-2, pp. 2–11, 2006.
- D. K. Das, M. Saini, A. Dhara, D. Swarup, S. Kumar, and P. K. Gupta, “Molecular characterization of toll-like receptor 2 of ruminants and comparative expression in nilgai (Boselaphus tragocamelus) vs. buffalo (Bubalus bubalis),” International Journal of Immunogenetics, vol. 35, no. 3, pp. 227–234, 2008.
- M. Tohno, T. Shimosato, H. Kitazawa et al., “Toll-like receptor 2 is expressed on the intestinal M cells in swine,” Biochemical and Biophysical Research Communications, vol. 330, no. 2, pp. 547–554, 2005.
- I. Waldschmidt, D. Pirottin, T. Art et al., “Experimental model of equine alveolar macrophage stimulation with TLR ligands,” Veterinary Immunology and Immunopathology, vol. 155, no. 1-2, pp. 30–37, 2013.
- A. M. Quintana, G. A. Landolt, K. M. Annis, and G. S. Hussey, “Immunological characterization of the equine airway epithelium and of a primary equine airway epithelial cell culture model,” Veterinary Immunology and Immunopathology, vol. 140, no. 3-4, pp. 226–236, 2011.
- J. C. Vendrig, L. E. Coffeng, and J. Fink-Gremmels, “Effects of separate and concomitant TLR-2 and TLR-4 activation in peripheral blood mononuclear cells of newborn and adult horses,” PLoS ONE, vol. 8, no. 6, Article ID e66897, 2013.
- M. Iqbal, V. J. Philbin, and A. L. Smith, “Expression patterns of chicken Toll-like receptor mRNA in tissues, immune cell subsets and cell lines,” Veterinary Immunology and Immunopathology, vol. 104, no. 1-2, pp. 117–127, 2005.
- H. He, K. J. Genovese, D. J. Nisbet, and M. H. Kogut, “Profile of Toll-like receptor expressions and induction of nitric oxide synthesis by Toll-like receptor agonists in chicken monocytes,” Molecular Immunology, vol. 43, no. 7, pp. 783–789, 2006.
- G. Ignacio, S. Nordone, K. E. Howard, and G. A. Dean, “Toll-like receptor expression in feline lymphoid tissues,” Veterinary Immunology and Immunopathology, vol. 106, no. 3-4, pp. 229–237, 2005.
- T. L. Lehman, K. P. O'Halloran, S. A. Fallon et al., “Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection,” Immunology, vol. 126, no. 3, pp. 405–412, 2009.
- S. M. J. Dolieslager, D. F. Lappin, D. Bennett, L. Graham, N. Johnston, and M. P. Riggio, “The influence of oral bacteria on tissue levels of Toll-like receptor and cytokine mRNAs in feline chronic gingivostomatitis and oral health,” Veterinary Immunology and Immunopathology, vol. 151, no. 3-4, pp. 263–274, 2013.
- M. Hirschfeld, J. J. Weis, V. Toshchakov et al., “Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages,” Infection and Immunity, vol. 69, no. 3, pp. 1477–1482, 2001.
- U. Zähringer, B. Lindner, S. Inamura, H. Heine, and C. Alexander, “TLR2-promiscuous or specific? A critical re-evaluation of a receptor expressing apparent broad specificity,” Immunobiology, vol. 213, no. 3-4, pp. 205–224, 2008.
- L. H. Travassos, S. E. Girardin, D. J. Philpott et al., “Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition,” EMBO Reports, vol. 5, no. 10, pp. 1000–1006, 2004.
- T. Ogawa, Y. Asai, Y. Makimura, and R. Tamai, “Chemical structure and immunobiological activity of Porphyromonas gingivalis lipid A,” Frontiers in Bioscience, vol. 12, no. 10, pp. 3795–3812, 2007.
- K. Sankaran and H. C. Wu, “Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol,” The Journal of Biological Chemistry, vol. 269, no. 31, pp. 19701–19706, 1994.
- A. Kovacs-Simon, R. W. Titball, and S. L. Michell, “Lipoproteins of bacterial pathogens,” Infection and Immunity, vol. 79, no. 2, pp. 548–561, 2011.
- P. F. Mühlradt, M. Kieß, H. Meyer, R. Süßmuth, and G. Jung, “Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration,” Journal of Experimental Medicine, vol. 185, no. 11, pp. 1951–1958, 1997.
- M. Nishiguchi, M. Matsumoto, T. Takao et al., “Mycoplasma fermentans lipoprotein M161Ag-induced cell activation is mediated by Toll-like receptor 2: role of N-terminal hydrophobic portion in its multiple functions,” Journal of Immunology, vol. 166, no. 4, pp. 2610–2616, 2001.
- K.-I. Shibata, A. Hasebe, T. Into, M. Yamada, and T. Watanabe, “The N-terminal lipopeptide of a 44-kda membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts,” Journal of Immunology, vol. 165, no. 11, pp. 6538–6544, 2000.
- A. O. Aliprantis, R.-B. Yang, M. R. Mark et al., “Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2,” Science, vol. 285, no. 5428, pp. 736–739, 1999.
- W. G. Bessler, R. B. Johnson, K. Wiesmüller, and G. Jung, “B-lymphocyte mitogenicity in vitro of a synthetic lipopeptide fragment derived from bacterial lipoprotein,” Hoppe-Seyler's Zeitschrift fur Physiologische Chemie, vol. 363, no. 7, pp. 767–770, 1982.
- V. Braun, K. Hantke, and U. Henning, “Characterization of the free form of murein lipoprotein from the outer membrane of Escherichia coli B/r,” FEBS Letters, vol. 60, no. 1, pp. 26–28, 1975.
- M. E. Brandt, B. S. Riley, J. D. Radolf, and M. V. Norgard, “Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins,” Infection and Immunity, vol. 58, no. 4, pp. 983–991, 1990.
- S. Akira, “Mammalian Toll-like receptors,” Current Opinion in Immunology, vol. 15, no. 1, pp. 5–11, 2003.
- A. Tschumi, C. Nai, Y. Auchli et al., “Identification of apolipoprotein N-acyltransferase (Lnt) in mycobacteria,” The Journal of Biological Chemistry, vol. 284, no. 40, pp. 27146–27156, 2009.
- M. Hirschfeld, G. J. Kirschning, R. Schwandner et al., “Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2,” Journal of Immunology, vol. 163, no. 5, pp. 2382–2386, 1999.
- O. Takeuchi, A. Kaufmann, K. Grote et al., “Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway,” Journal of Immunology, vol. 164, no. 2, pp. 554–557, 2000.
- K. Takeda, O. Takeuchi, and S. Akira, “Recognition of lipopeptides by Toll-like receptors,” Journal of Endotoxin Research, vol. 8, no. 6, pp. 459–463, 2002.
- O. Takeuchi, T. Kawai, P. F. Mühlradt et al., “Discrimination of bacterial lipoproteins by Toll-like recepttor 6,” International Immunology, vol. 13, no. 7, pp. 933–940, 2001.
- O. Takeuchi, S. Sato, T. Horiuchi et al., “Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins,” Journal of Immunology, vol. 169, no. 1, pp. 10–14, 2002.
- U. Buwitt-Beckmann, H. Heine, K.-H. Wiesmüller et al., “TLR1- and TLR6-independent recognition of bacterial lipopeptides,” The Journal of Biological Chemistry, vol. 281, no. 14, pp. 9049–9057, 2006.
- M. S. Jin, S. E. Kim, J. Y. Heo et al., “Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide,” Cell, vol. 130, no. 6, pp. 1071–1082, 2007.
- J. Y. Kang, X. Nan, M. S. Jin et al., “Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer,” Immunity, vol. 31, no. 6, pp. 873–884, 2009.
- Y. Guan, D. R. E. Ranoa, S. Jiang et al., “Human TLRs 10 and 1 share common mechanisms of innate immune sensing but not signaling,” Journal of Immunology, vol. 184, no. 9, pp. 5094–5103, 2010.
- K. Farhat, S. Riekenberg, G. Jung, K.-H. Wiesmüller, T. W. Jungi, and A. J. Ulmer, “Identification of full length bovine TLR1 and functional characterization of lipopeptide recognition by bovine TLR2/1 heterodimer,” Veterinary Research, vol. 41, no. 3, article 34, 2010.
- S. Willcocks, V. Offord, H.-M. Seyfert, T. J. Coffey, and D. Werling, “Species-specific PAMP recognition by TLR2 and evidence for species-restricted interaction with Dectin-1,” Journal of Leukocyte Biology, vol. 94, no. 3, pp. 449–458, 2013.
- K. L. Irvine, L. J. Hopkins, M. Gangloff, and C. E. Bryant, “The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6,” Veterinary Research, vol. 44, no. 1, article 50, 2013.
- A. M. Keestra, M. R. De Zoete, R. A. M. H. Van Aubel, and J. P. M. Van Putten, “The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2,” Journal of Immunology, vol. 178, no. 11, pp. 7110–7119, 2007.
- M. Higuchi, A. Matsuo, M. Shingai et al., “Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily,” Developmental and Comparative Immunology, vol. 32, no. 2, pp. 147–155, 2008.
- V. Bosch and V. Braun, “Distribution of murein lipoprotein between the cytoplasmic and outer membrane of Escherichia coli,” FEBS Letters, vol. 34, no. 2, pp. 307–310, 1973.
- V. Braun, “Covalent lipoprotein from the outer membrane of Escherichia coli,” Biochimica et Biophysica Acta, vol. 415, no. 3, pp. 335–377, 1975.
- V. Braun and V. Bosch, “Sequence of the murein-lipoprotein and the attachment site of the lipid,” European Journal of Biochemistry, vol. 28, no. 1, pp. 51–69, 1972.
- V. Braun and V. Bosch, “In vivo biosynthesis of murein lipoprotein of the outer membrane of E. coli,” FEBS Letters, vol. 34, no. 2, pp. 302–306, 1973.
- V. Braun and K. Rehn, “Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall. The specific effect of trypsin on the membrane structure,” European Journal of Biochemistry, vol. 10, no. 3, pp. 426–438, 1969.
- K. Hantke and V. Braun, “Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane,” European Journal of Biochemistry, vol. 34, no. 2, pp. 284–296, 1973.
- A. Hirashima, H. C. Wu, P. S. Venkateswaran, and M. Inouye, “Two forms of a structural lipoprotein in the envelope of Escherichia coli. Further characterization of the free form,” The Journal of Biological Chemistry, vol. 248, no. 16, pp. 5654–5659, 1973.
- M. Inouye, J. Shaw, and C. Shen, “The assembly of a structural lipoprotein in the envelope of Escherichia coli,” The Journal of Biological Chemistry, vol. 247, no. 24, pp. 8154–8159, 1972.
- F. Melchers, V. Braun, and C. Galanos, “The lipoprotein of the outer membrane of Escherichia coli: a B lymphocyte mitogen,” Journal of Experimental Medicine, vol. 142, no. 2, pp. 473–482, 1975.
- W. Bessler, K. Resch, E. Hancock, and K. Hantke, “Induction of lymphocyte proliferation and membrane changes by lipopeptide derivatives of the lipoprotein from the outer membrane of Escherichia coli,” Zeitschrift fur Immunitatsforschung, vol. 153, no. 1, pp. 11–22, 1977.
- W. G. Bessler, B. Suhr, H.-J. Buhring et al., “Specific antibodies elicited by antigen covalently linked to a synthetic adjuvant,” Immunobiology, vol. 170, no. 3, pp. 239–244, 1985.
- K.-H. Wiesmuller, G. Jung, and G. Hess, “Novel low-molecular-weight synthetic vaccine against foot-and-mouth disease containing a potent B-cell and macrophage activator,” Vaccine, vol. 7, no. 1, pp. 29–33, 1989.
- P. Hoffmann, S. Heinle, U. F. Schade et al., “Stimulation of human and murine adherent cells by bacterial lipoprotein and synthetic lipopeptide analogues,” Immunobiology, vol. 177, no. 2, pp. 158–170, 1988.
- K. Deres, H. Schild, K.-H. Wiesmuller, G. Jung, and H.-G. Rammensee, “In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine,” Nature, vol. 342, no. 6249, pp. 561–564, 1989.
- T. P. Hopp, “Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier,” Molecular Immunology, vol. 21, no. 1, pp. 13–16, 1984.
- R. Champlin and R. L. Hunter, “Studies on the composition of adjuvants which selectively enhance delayed type hypersensitivity to lipid conjugated protein antigens,” Journal of Immunology, vol. 114, no. 1, part 1, pp. 76–80, 1975.
- J. Coon and R. Hunter, “Selective induction of delayed hypersensitivity by a lipid conjugated protein antigen which is localized in thymus dependent lymphoid tissue,” Journal of Immunology, vol. 110, no. 1, pp. 183–190, 1973.
- J. Coon and R. Hunter, “Properties of conjugated protein immunogens which selectively stimulate delayed type hypersensitivity,” Journal of Immunology, vol. 114, no. 5, pp. 1518–1522, 1975.
- R. Medzhitov, P. Preston-Hurlburt, and C. A. Janeway Jr., “A human homologue of the Drosophila toll protein signals activation of adaptive immunity,” Nature, vol. 388, no. 6640, pp. 394–397, 1997.
- H. D. Brightbill, D. H. Libraty, S. R. Krutzik et al., “Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors,” Science, vol. 285, no. 5428, pp. 732–736, 1999.
- E. Lien, T. J. Sellati, A. Yoshimura et al., “Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products,” The Journal of Biological Chemistry, vol. 274, no. 47, pp. 33419–33425, 1999.
- L. M. Wetzler, “Innate immune function of the neisserial porins and the relationship to vaccine adjuvant activity,” Future Microbiology, vol. 5, no. 5, pp. 749–758, 2010.
- L. Zhang, L. Ma, R. Liu et al., “Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge,” Veterinary Parasitology, vol. 188, no. 3-4, pp. 239–246, 2012.
- J. L. Harrison, I. M. Taylor, and C. D. O'Connor, “Presentation of foreign antigenic determinants at the bacterial cell surface using the TraT lipoprotein,” Research in Microbiology, vol. 141, no. 7-8, pp. 1009–1012, 1990.
- I. M. Taylor, J. L. Harrison, K. N. Timmis, and C. D. O'Connor, “The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure-function relationships in the TraT protein,” Molecular Microbiology, vol. 4, no. 8, pp. 1259–1268, 1990.
- P. Cornells, J. C. Sierra, A. Lim Jr., A. Malur, A. Leitâ, and C. V. Martins, “Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli,” Bio/Technology, vol. 14, no. 2, pp. 203–208, 1996.
- C. R. Rioux, H. Bergeron, L. Lin, S. Grothe, M. O'Connor-McCourt, and P. C. K. Lau, “A fusion plasmid for the synthesis of lipopeptide-antigen chimeras in Escherichia coli,” Gene, vol. 116, no. 1, pp. 13–20, 1992.
- P. A. Cullen, M. Lo, D. M. Bulach, S. J. Cordwell, and B. Adler, “Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli,” Plasmid, vol. 49, no. 1, pp. 18–29, 2003.
- S. Kamalakkannan, V. Murugan, M. V. Jagannadham, R. Nagaraj, and K. Sankaran, “Bacterial lipid modification of proteins for novel protein engineering applications,” Protein Engineering, Design and Selection, vol. 17, no. 10, pp. 721–729, 2004.
- B. Wang, M. Henao-Tamayo, M. Harton et al., “A toll-like receptor-2-directed fusion protein vaccine against tuberculosis,” Clinical and Vaccine Immunology, vol. 14, no. 7, pp. 902–906, 2007.
- Z. Yang, Q. Liu, Q. Wang, and Y. Zhang, “Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum,” Applied and Environmental Microbiology, vol. 74, no. 14, pp. 4359–4365, 2008.
- H.-W. Chen, S.-J. Liu, H.-H. Liu et al., “A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design,” Vaccine, vol. 27, no. 9, pp. 1400–1409, 2009.
- J. V. Sabio, F. Bigi, O. Rossetti, and E. Campos, “Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice,” Microbes and Infection, vol. 12, no. 14-15, pp. 1236–1243, 2010.
- H. Tokuda, “Biogenesis of outer membrenes in gram-negative bacteria,” Bioscience, Biotechnology and Biochemistry, vol. 73, no. 3, pp. 465–473, 2009.
- K. Remans, K. Pauwels, P. van Ulsen et al., “Hydrophobic Surface Patches on LolA of Pseudomonas aeruginosa are Essential for Lipoprotein Binding,” Journal of Molecular Biology, vol. 401, no. 5, pp. 921–930, 2010.
- A. P. Basto, J. Piedade, R. Ramalho et al., “A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations,” Journal of Biotechnology, vol. 157, no. 1, pp. 50–63, 2012.
- C.-Y. Chiang, M.-H. Huang, C.-H. Pan et al., “Induction of robust immunity by the emulsification of recombinant lipidated dengue-1 envelope protein domain III,” Microbes and Infection, vol. 15, no. 10-11, pp. 719–728, 2013.
- Y. Kwok, W.-C. Sung, A. L.-H. Lin et al., “Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis,” Proteomics, vol. 11, no. 13, pp. 2620–2627, 2011.
- A. Leitão, A. Malur, C. Cartaxeiro et al., “Bacterial lipoprotein based expression vectors as tools for the characterisation of African swine fever virus (ASFV) antigens,” Archives of Virology, vol. 145, no. 8, pp. 1639–1657, 2000.
- C.-H. Leng, H.-W. Chen, L.-S. Chang et al., “A recombinant lipoprotein containing an unsaturated fatty acid activates NF-κB through the TLR2 signaling pathway and induces a differential gene profile from a synthetic lipopeptide,” Molecular Immunology, vol. 47, no. 11-12, pp. 2015–2021, 2010.
- A. A. Lugade, A. Bianchi-Smiraglia, V. Pradhan, G. Elkin, T. F. Murphy, and Y. Thanavala, “Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling,” PLoS ONE, vol. 6, no. 5, Article ID e19781, 2011.
- J.-R. Wu, J.-H. Shien, H. K. Shieh, C.-F. Chen, and P.-C. Chang, “Protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE),” Vaccine, vol. 25, no. 21, pp. 4140–4148, 2007.
- M. E. Embers and S. Narasimhan, “Vaccination against Lyme disease: past, present, and future,” Frontiers in Cellular and Infection Microbiology, vol. 3, article 6, 2013.
- W. Zeng, E. M. Eriksson, A. Lew, and D. C. Jackson, “Lipidation of intact proteins produces highly immunogenic vaccine candidates,” Molecular Immunology, vol. 48, no. 4, pp. 490–496, 2011.
- P. M. Moyle, J. Hartas, A. Henningham, M. R. Batzloff, M. F. Good, and I. Toth, “An efficient, chemically-defined semisynthetic lipid-adjuvanted nanoparticulate vaccine development system,” Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 9, no. 7, pp. 935–944, 2013.
- P. M. Moyle and I. Toth, “Self-adjuvanting lipopeptide vaccines,” Current Medicinal Chemistry, vol. 15, no. 5, pp. 506–516, 2008.
- M. Zaman and I. Toth, “Immunostimulation by synthetic lipopeptide-based vaccine candidates: structure-activity relationships,” Frontiers in Immunology, vol. 4, 2013.
- K. S. M. Phillipps, M. N. Wykes, X. Q. Liu, M. Brown, J. Blanchfield, and I. Toth, “A novel synthetic adjuvant enhances dendritic cell function,” Immunology, vol. 128, no. 1, supplement, pp. e582–e588, 2009.
- M. Zaman, A.-B. M. Abdel-Aal, Y. Fujita et al., “Immunological evaluation of lipopeptide group A streptococcus (GAS) vaccine: structure-activity relationship,” PLoS ONE, vol. 7, no. 1, Article ID e30146, 2012.
- X. Zhu, T. V. Ramos, H. Gras-Masse, B. E. Kaplan, and L. BenMohamed, “Lipopeptide epitopes extended by an NE-palmitoyl-lysine moiety increase uptake and maturation of dendritic cells through a Toll-like receptor-2 pathway and trigger a Th1-dependent protective immunity,” European Journal of Immunology, vol. 34, no. 11, pp. 3102–3114, 2004.
- E. Caproni, E. Tritto, M. Cortese et al., “MF59 and Pam3CSK4 boost adaptive responses to influenza subunit vaccine through an IFN type I-independent mechanism of action,” Journal of Immunology, vol. 188, no. 7, pp. 3088–3098, 2012.
- G. Faure-André, P. Vargas, M.-I. Yuseff et al., “Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain,” Science, vol. 322, no. 5908, pp. 1705–1710, 2008.
- M. A. West, A. R. Prescott, M. C. Kui et al., “TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent,” Journal of Cell Biology, vol. 182, no. 5, pp. 993–1005, 2008.
- A. Iwasaki and R. Medzhitov, “Toll-like receptor control of the adaptive immune responses,” Nature Immunology, vol. 5, no. 10, pp. 987–995, 2004.
- S. Sozzani, P. Allavena, G. D'Amico et al., “Cutting edge: differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties,” Journal of Immunology, vol. 161, no. 3, pp. 1083–1086, 1998.
- F. Sallusto and A. Lanzavecchia, “Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression,” Immunological Reviews, vol. 177, pp. 134–140, 2000.
- C. H. Lee, P. Masso-Welch, G. Hajishengallis, and T. D. Connell, “TLR2-dependent modulation of dendritic cells by LT-IIa-B 5, a novel mucosal adjuvant derived from a type II heat-labile enterotoxin,” Journal of Leukocyte Biology, vol. 90, no. 5, pp. 911–921, 2011.
- C. S. McKimmie, M. Moore, A. R. Fraser et al., “ATLR2 ligand suppresses inflammation by modulation of chemokine receptors and redirection of leukocyte migration,” Blood, vol. 113, no. 18, pp. 4224–4231, 2009.
- S. K. Datta, V. Redecke, K. R. Prilliman et al., “A subset of toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells,” Journal of Immunology, vol. 170, no. 8, pp. 4102–4110, 2003.
- M. M. Weck, F. Grünebach, D. Werth, C. Sinzger, A. Bringmann, and P. Brossart, “TLR ligands differentially affect uptake and presentation of cellular antigens,” Blood, vol. 109, no. 9, pp. 3890–3894, 2007.
- B. C. Gil-Torregrosa, A. M. Lennon-Duménil, B. Kessler et al., “Control of cross-presentation during dendritic cell maturation,” European Journal of Immunology, vol. 34, no. 2, pp. 398–407, 2004.
- M. A. West, R. P. A. Wallin, S. P. Matthews et al., “Enhanced dendritic cell antigen capture via Toll-like receptor-induced actin remodeling,” Science, vol. 305, no. 5687, pp. 1153–1157, 2004.
- K. W. Schjetne, K. M. Thompson, N. Nilsen et al., “Cutting edge: link between innate and adaptive immunity: toll-like receptor 2 internalizes antigen for presentation to CD4+ T cells and could be an efficient vaccine target,” Journal of Immunology, vol. 171, no. 1, pp. 32–36, 2003.
- D. G. Russell and R. M. Yates, “TLR signalling and phagosome maturation: an alternative viewpoint,” Cellular Microbiology, vol. 9, no. 4, pp. 849–850, 2007.
- D. G. Russell and R. M. Yates, “Toll-like receptors and phagosome maturation,” Nature Immunology, vol. 8, no. 3, p. 217, 2007.
- R. M. Yates and D. G. Russell, “Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4,” Immunity, vol. 23, no. 4, pp. 409–717, 2005.
- J. M. Blander, “Phagocytosis and antigen presentation: a partnership initiated by Toll-like receptors,” Annals of the Rheumatic Diseases, vol. 67, no. 3, pp. iii44–iii49, 2008.
- J. M. Blander and R. Medzhitov, “Regulation of phagosome maturation by signals from Toll-like receptors,” Science, vol. 304, no. 5673, pp. 1014–1018, 2004.
- J. Magarian Blander and R. Medzhitov, “Toll-dependent selection of microbial antigens for presentation by dendritic cells,” Nature, vol. 440, no. 7085, pp. 808–812, 2006.
- A. De Gassart, V. Camosseto, J. Thibodeau et al., “MHC class II stabilization at the surface of human dendritic cells is the result of maturation-dependent MARCH I down-regulation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 9, pp. 3491–3496, 2008.
- U. Wille-Reece, C.-Y. Wu, B. J. Flynn, R. M. Kedl, and R. A. Seder, “Immunization with HIV-1 gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 gag-specific Th1 and CD8+ T cell responses,” Journal of Immunology, vol. 174, no. 12, pp. 7676–7683, 2005.
- J. W. Huleatt, A. R. Jacobs, J. Tang et al., “Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity,” Vaccine, vol. 25, no. 4, pp. 763–775, 2007.
- S. Khan, M. S. Bijker, J. J. Weterings et al., “Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells,” The Journal of Biological Chemistry, vol. 282, no. 29, pp. 21145–21159, 2007.
- E. Schlosser, M. Mueller, S. Fischer et al., “TLR ligands and antigen need to be coencapsulated into the same biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses,” Vaccine, vol. 26, no. 13, pp. 1626–1637, 2008.
- W. Zeng, S. Ghosh, Y. F. Lau, L. E. Brown, and D. C. Jackson, “Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines,” Journal of Immunology, vol. 169, no. 9, pp. 4905–4912, 2002.
- J. Cote-Sierra, A. Bredan, C. M. Toldos et al., “Bacterial lipoprotein-based vaccines induce tumor necrosis factor-dependent type 1 protective immunity against Leishmania major,” Infection and Immunity, vol. 70, no. 1, pp. 240–248, 2002.
- M. L. Kapsenberg, “Dendritic-cell control of pathogen-driven T-cell polarization,” Nature Reviews Immunology, vol. 3, no. 12, pp. 984–993, 2003.
- C. J. Hertz, S. M. Kiertscher, P. J. Godowski et al., “Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2,” Journal of Immunology, vol. 166, no. 4, pp. 2444–2450, 2001.
- D. C. Jackson, F. L. Yuk, T. Le et al., “A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 43, pp. 15440–15445, 2004.
- H. Revets, G. Pynaert, J. Grooten, and P. De Baetselier, “Lipoprotein I, a TLR2/4 ligand modulates Th2-driven allergic immune responses,” Journal of Immunology, vol. 174, no. 2, pp. 1097–1103, 2005.
- K. S. Michelsen, A. Aicher, M. Mohaupt et al., “The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCs): peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2,” The Journal of Biological Chemistry, vol. 276, no. 28, pp. 25680–25686, 2001.
- C. Link, R. Gavioli, T. Ebensen, A. Canella, E. Reinhard, and C. A. Guzmán, “The Toll-like receptor ligand MALP-2 stimulates dendritic cell maturation and modulates proteasome composition and activity,” European Journal of Immunology, vol. 34, no. 3, pp. 899–907, 2004.
- C. K. Prajeeth, A. C. Jirmo, J. K. Krishnaswamy et al., “The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross-priming against co-administered and linked antigens,” European Journal of Immunology, vol. 40, no. 5, pp. 1272–1283, 2010.
- S. Dillon, A. Agrawal, T. Van Dyke et al., “A Toll-Like Receptor 2 Ligand Stimulates Th2 Responses In Vivo, via Induction of Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase and c-Fos in Dendritic Cells,” Journal of Immunology, vol. 172, no. 8, pp. 4733–4743, 2004.
- B. Pulendran, P. Kumar, C. W. Cutler, M. Mohamadzadeh, T. Van Dyke, and J. Banchereau, “Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo,” Journal of Immunology, vol. 167, no. 9, pp. 5067–5076, 2001.
- S. Agrawal, A. Agrawal, B. Doughty et al., “Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos,” Journal of Immunology, vol. 171, no. 10, pp. 4984–4989, 2003.
- K. Kiura, H. Kataoka, M. Yasuda, N. Inoue, and K.-I. Shibata, “The diacylated lipopeptide FSL-1 induces TLR2-mediated Th2 responses,” FEMS Immunology and Medical Microbiology, vol. 48, no. 1, pp. 44–55, 2006.
- V. Redecke, H. Häcker, S. K. Datta et al., “Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma,” Journal of Immunology, vol. 172, no. 5, pp. 2739–2743, 2004.
- G. Gautier, M. Humbert, F. Deauvieau et al., “A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells,” Journal of Experimental Medicine, vol. 201, no. 9, pp. 1435–1446, 2005.
- B. Y. Chua, E. M. Eriksson, L. E. Brown et al., “A self-adjuvanting lipopeptide-based vaccine candidate for the treatment of hepatitis C virus infection,” Vaccine, vol. 26, no. 37, pp. 4866–4875, 2008.
- M. Ghielmetti, M. Zwicker, T. Ghielmetti, M. M. Simon, P. M. Villiger, and E. Padovan, “Synthetic bacterial lipopeptide analogs facilitate naive CD4+ T cell differentiation and enhance antigen-specific HLA-II-restricted responses,” European Journal of Immunology, vol. 35, no. 8, pp. 2434–2442, 2005.
- M. Huber, W. Baier, W. G. Bessler, and L. Heinevetter, “Modulation of the Th1/Th2 bias by lipopeptide and saponin adjuvants in orally immunized mice,” Immunobiology, vol. 205, no. 1, pp. 61–73, 2002.
- P. A. Sieling, W. Chung, B. T. Duong, P. J. Godowski, and R. L. Modlin, “Toll-like receptor 2 ligands as adjuvants for human Th1 responses,” Journal of Immunology, vol. 170, no. 1, pp. 194–200, 2003.
- T. Watanabe, A. Kitani, P. J. Murray, and W. Strober, “NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses,” Nature Immunology, vol. 5, no. 8, pp. 800–808, 2004.
- C.-Y. Huang, J. J. W. Chen, K.-Y. Shen et al., “Recombinant lipidated HPV E7 induces a Th-1-biased immune response and protective immunity against cervical cancer in a mouse model,” PLoS ONE, vol. 7, no. 7, Article ID e40970, 2012.
- S. Thoma-Uszynski, S. M. Kiertscher, M. T. Ochoa et al., “Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10,” Journal of Immunology, vol. 165, no. 7, pp. 3804–3810, 2000.
- T. Imanishi, H. Hara, S. Suzuki, N. Suzuki, S. Akira, and T. Saito, “Cutting edge: TLR2 directly triggers Th1 effector functions,” Journal of Immunology, vol. 178, no. 11, pp. 6715–6719, 2007.
- E. Aliahmadi, R. Gramlich, A. Grützkau et al., “TLR2-activated human langerhans cells promote Th17 polarization via IL-1β, TGF-β and IL-23,” European Journal of Immunology, vol. 39, no. 5, pp. 1221–1230, 2009.
- C. A. Akdis, F. Kussebi, B. Pulendran et al., “Inhibition of T helper 2-type responses, IgE production and eosinophilia by synthetic lipopeptides,” European Journal of Immunology, vol. 33, no. 10, pp. 2717–2726, 2003.
- V. Lombardi, L. Van Overtvelt, S. Horiot et al., “Toll-like receptor 2 agonist Pam3CSK4 enhances the induction of antigen-specific tolerance via the sublingual route,” Clinical and Experimental Allergy, vol. 38, no. 11, pp. 1819–1829, 2008.
- S. Dillon, S. Agrawal, K. Banerjee et al., “Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance,” Journal of Clinical Investigation, vol. 116, no. 4, pp. 916–928, 2006.
- S. Manicassamy, R. Ravindran, J. Deng et al., “Toll-like receptor 2-dependent induction of vitamin A-metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity,” Nature Medicine, vol. 15, no. 4, pp. 401–409, 2009.
- S. Karumuthil-Melethil, N. Perez, R. Li, and C. Vasu, “Induction of innate immune response through TLR2 and dectin 1 prevents type 1 diabetes,” Journal of Immunology, vol. 181, no. 12, pp. 8323–8334, 2008.
- K. Kiura, A. Hasebe, A. Saeki et al., “In vivo anti- and pro-tumour activities of the TLR2 ligand FSL-1,” Immunobiology, vol. 216, no. 8, pp. 891–900, 2011.
- S. Yamazaki, K. Okada, A. Maruyama, M. Matsumoto, H. Yagita, and T. Seya, “Tlr2-dependent induction of il-10 and foxp3+cd25+cd4+ regulatory t cells prevents effective anti-tumor immunity induced by pam2 lipopeptides in vivo,” PLoS ONE, vol. 6, no. 4, Article ID e18833, 2011.
- H. Liu, M. Komai-Koma, D. Xu, and F. Y. Liew, “Toll-like receptor 2 signaling modulates the functions of CD4+CD25+ regulatory T cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 18, pp. 7048–7053, 2006.
- R. P. M. Sutmuller, M. H. M. G. M. Den Brok, M. Kramer et al., “Toll-like receptor 2 controls expansion and function of regulatory T cells,” Journal of Clinical Investigation, vol. 116, no. 2, pp. 485–494, 2006.
- Y. Zhang, F. Luo, Y. Cai et al., “TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells,” Journal of Immunology, vol. 186, no. 4, pp. 1963–1969, 2011.
- L. Amiset, L. Fend, T. Gatard-Scheikl et al., “TLR2 ligation protects effector T cells from regulatory T-cell mediated suppression and repolarizes T helper responses following MVA-based cancer immunotherapy,” Oncoimmunology, vol. 1, no. 8, pp. 1271–1280, 2012.
- A. Baz, K. Buttigieg, W. Zeng et al., “Branched and linear lipopeptide vaccines have different effects on primary CD4+ and CD8+ T-cell activation but induce similar tumor-protective memory CD8+ T-cell responses,” Vaccine, vol. 26, no. 21, pp. 2570–2579, 2008.
- Y. F. Lau, G. Deliyannis, W. Zeng, A. Mansell, D. C. Jackson, and L. E. Brown, “Lipid-containing mimetics of natural triggers of innate immunity as CTL-inducing influenza vaccines,” International Immunology, vol. 18, no. 12, pp. 1801–1813, 2006.
- F. Martinon, H. Gras-Masse, C. Boutillon et al., “Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant: immune response of BALB/c mice to human immunodeficiency virus envelope glycoprotein,” Journal of Immunology, vol. 149, no. 10, pp. 3416–3422, 1992.
- M. Andrieu, E. Loing, J. F. Desoutter et al., “Endocytosis of an HIV-derived lipopeptide into human dendritic cells followed by class I-restricted CD8(+) T lymphocyte activation,” European Journal of Immunology, vol. 30, no. 11, pp. 3256–3265, 2000.
- S. Khan, J. J. Weterings, C. M. Britten et al., “Chirality of TLR-2 ligand Pam3CysSK4 in fully synthetic peptide conjugates critically influences the induction of specific CD8+ T-cells,” Molecular Immunology, vol. 46, no. 6, pp. 1084–1091, 2009.
- X. Zhang, A. Issagholian, E. A. Berg, J. B. Fishman, A. B. Nesburn, and L. BenMohamed, “Th-cytotoxic T-lymphocyte chimeric epitopes extended by Nε-palmitoyl lysines induce herpes simplex virus type 1-specific effector CD8+ Tc1 responses and protect against ocular infection,” Journal of Virology, vol. 79, no. 24, pp. 15289–15301, 2005.
- M. Azuma, R. Sawahata, Y. Akao et al., “The peptide sequence of diacyl lipopeptides determines dendritic cell TLR2-mediated NK activation,” PLoS ONE, vol. 5, no. 9, Article ID e12550, pp. 1–12, 2010.
- R. Sawahata, H. Shime, S. Yamazaki et al., “Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2,” Microbes and Infection, vol. 13, no. 4, pp. 350–358, 2011.
- E. N. Komegae, L. Z. Grund, M. Lopes-Ferreira, and C. Lima, “TLR2, TLR4 and the MyD88 signaling are crucial for the in vivo generation and the longevity of long-lived antibody-secreting cells,” PLoS ONE, vol. 8, no. 8, Article ID e71185, 2013.
- E. Boeglin, C. R. Smulski, S. Brun, S. Milosevic, P. Schneider, and S. Fournel, “Toll-Like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells,” PLoS ONE, vol. 6, no. 10, Article ID e25542, 2011.
- X. Zhang, A. A. Chentoufi, G. Dasgupta et al., “A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8+ T cells and protects against herpes simplex virus type 2 challenge,” Mucosal Immunology, vol. 2, no. 2, pp. 129–143, 2009.
- B. Nardelli, P. B. Haser, and J. P. Tam, “Oral administration of an antigenic synthetic lipopeptide (MAP-P3C) evokes salivary antibodies and systemic humoral and cellular responses,” Vaccine, vol. 12, no. 14, pp. 1335–1339, 1994.
- W. Baier, N. Masihi, M. Huber, P. Hoffmann, and W. G. Bessler, “Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization,” Immunobiology, vol. 201, no. 3-4, pp. 391–405, 2000.
- S. Wang, E. J. Villablanca, J. De Calisto et al., “MyD88-dependent TLR1/2 signals educate dendritic cells with gut-specific imprinting properties,” Journal of Immunology, vol. 187, no. 1, pp. 141–150, 2011.
- B. Pulendran, H. Tang, and S. Manicassamy, “Programming dendritic cells to induce T H 2 and tolerogenic responses,” Nature Immunology, vol. 11, no. 8, pp. 647–655, 2010.
- T. Mele and J. Madrenas, “TLR2 signalling: at the crossroads of commensalism, invasive infections and toxic shock syndrome by Staphylococcus aureus,” International Journal of Biochemistry and Cell Biology, vol. 42, no. 7, pp. 1066–1071, 2010.
- S. Burgdorf, C. Schölz, A. Kautz, R. Tampé, and C. Kurts, “Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation,” Nature Immunology, vol. 9, no. 5, pp. 558–566, 2008.
- V. Durand, S. Y. C. Wong, D. F. Tough, and A. Le Bon, “Shaping of adaptive immune responses to soluble proteins by TLR agonists: a role for IFN-α/β,” Immunology and Cell Biology, vol. 82, no. 6, pp. 596–602, 2004.
- V. Durand, S. Y. C. Wong, D. F. Tough, and A. Le Bon, “IFN-α/β-dependent cross-priming induced by specific toll-like receptor agonists,” Vaccine, vol. 24, supplement 2, pp. S2/22–S2/23, 2006.
- A. Leitão, A. Malur, P. Cornelis, and C. L. V. Martins, “Identification of a 25-aminoacid sequence from the major African swine fever virus structural protein VP72 recognised by porcine cytotoxic T lymphocytes using a lipoprotein based expression system,” Journal of Virological Methods, vol. 75, no. 1, pp. 113–119, 1998.
- M. Kim, N. R. Osborne, W. Zeng et al., “Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes,” Journal of Immunology, vol. 188, no. 9, pp. 4158–4170, 2012.
- Å. Lindgren, V. Pavlovic, C.-F. Flach, Å. Sjöling, and S. Lundin, “Interferon-gamma secretion is induced in IL-12 stimulated human NK cells by recognition of Helicobacter pylori or TLR2 ligands,” Innate Immunity, vol. 17, no. 2, pp. 191–203, 2011.
- E. Marcenaro, B. Ferranti, M. Falco, L. Moretta, and A. Moretta, “Human NK cells directly recognize Mycobacterium bovis via TLR2 and acquire the ability to kill monocyte-derived DC,” International Immunology, vol. 20, no. 9, pp. 1155–1167, 2008.
- J. Martinez, X. Huang, and Y. Yang, “Direct TLR2 signaling is critical for NK cell activation and function in response to vaccinia viral infection,” PLoS Pathogens, vol. 6, no. 3, Article ID e1000811, 2010.
- E. Szomolanyi-Tsuda, X. Liang, R. M. Welsh, E. A. Kurt-Jones, and R. W. Finberg, “Role for TLR2 in NK cell-mediated control of murine cytomegaloviras in vivo,” Journal of Virology, vol. 80, no. 9, pp. 4286–4291, 2006.
- M. Eriksson, S. K. Meadows, S. Basu, T. F. Mselle, C. R. Wira, and C. L. Sentman, “TLRs mediate IFN-γ production by human uterine NK cells in endometrium,” Journal of Immunology, vol. 176, no. 10, pp. 6219–6224, 2006.
- N. M. Lauzon, F. Mian, R. MacKenzie, and A. A. Ashkar, “The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity,” Cellular Immunology, vol. 241, no. 2, pp. 102–112, 2006.
- J. Sawaki, H. Tsutsui, N. Hayashi et al., “Type 1 cytokine/chemokine production by mouse NK cells following activation of their TLR/MyD88-mediated pathways,” International Immunology, vol. 19, no. 3, pp. 311–320, 2007.
- M. Adib-Conquy, D. Scott-Algara, J. M. Cavaillon, and F. Souza-Fonseca-Guimaraes, “TLR-mediated activation of NK cells and their role in bacterial/viral immune responses in mammals,” Immunology and Cell Biology, vol. 92, no. 3, pp. 256–262, 2014.
- C. Pasare and R. Medzhitov, “Control of B-cell responses by Toll-like receptors,” Nature, vol. 438, no. 7066, pp. 364–368, 2005.
- S. P. Kasturi, I. Skountzou, R. A. Albrecht et al., “Programming the magnitude and persistence of antibody responses with innate immunity,” Nature, vol. 470, no. 7335, pp. 543–550, 2011.
- E. Foulon and G. Foucras, “Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression,” Journal of Immunological Methods, vol. 339, no. 1, pp. 1–10, 2008.
- J. C. Hope, A. O. Whelan, R. G. Hewinson, M. Vordermeier, and C. J. Howard, “Maturation of bovine dendritic cells by lipopeptides,” Veterinary Immunology and Immunopathology, vol. 95, no. 1-2, pp. 21–31, 2003.
- M. Franchini, M. Schweizer, P. Mätzener et al., “Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages,” Veterinary Immunology and Immunopathology, vol. 110, no. 1-2, pp. 37–49, 2006.
- C. D. Nelson, T. A. Reinhardt, T. C. Thacker, D. C. Beitz, and J. D. Lippolis, “Modulation of the bovine innate immune response by production of 1α,25-dihydroxyvitamin D3 in bovine monocytes,” Journal of Dairy Science, vol. 93, no. 3, pp. 1041–1049, 2010.
- H. Sigmundsdottir and E. C. Butcher, “Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking,” Nature Immunology, vol. 9, no. 9, pp. 981–987, 2008.
- D. Wesch, C. Peters, H.-H. Oberg, K. Pietschmann, and D. Kabelitz, “Modulation of γδ T cell responses by TLR ligands,” Cellular and Molecular Life Sciences, vol. 68, no. 14, pp. 2357–2370, 2011.
- A. O. Whelan, J. C. Hope, C. J. Howard, D. Clifford, R. G. Hewinson, and H. M. Vordermeier, “Modulation of the bovine delayed-type hypersensitivity responses to defined mycobacterial antigens by a synthetic bacterial lipopeptide,” Infection and Immunity, vol. 71, no. 11, pp. 6420–6425, 2003.
- D. N. Wedlock, M. Denis, G. F. Painter et al., “Enhanced protection against bovine tuberculosis after coadministration of Mycobacterium bovis BCG with a mycobacterial protein vaccine-adjuvant combination but not after coadministration of adjuvant alone,” Clinical and Vaccine Immunology, vol. 15, no. 5, pp. 765–772, 2008.
- B.-J. Höhlich, K.-H. Wiesmüller, B. Haas et al., “Induction of an antigen-specific immune response and partial protection of cattle against challenge infection with foot-and-mouth disease virus (FMDV) after lipopeptide vaccination with FMDV-specific B-cell epitopes,” Journal of General Virology, vol. 84, no. 12, pp. 3315–3324, 2003.
- T. V. Baszler, V. Shkap, W. Mwangi et al., “Bovine immune response to inoculation with Neospora caninum surface antigen SRS2 lipopeptides mimics immune response to infection with live parasites,” Clinical and Vaccine Immunology, vol. 15, no. 4, pp. 659–667, 2008.
- M. D. Figueiredo, M. L. Vandenplas, D. J. Hurley, and J. N. Moore, “Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists,” Veterinary Immunology and Immunopathology, vol. 127, no. 1-2, pp. 125–134, 2009.
- A. E. DeClue, P. J. Johnson, J. L. Day, J. R. Amorim, and A. R. Honaker, “Pathogen associated molecular pattern motifs from Gram-positive and Gram-negative bacteria induce different inflammatory mediator profiles in equine blood,” Veterinary Journal, vol. 192, no. 3, pp. 455–460, 2012.
- S. L. Ridgely and T. C. McGuire, “Lipopeptide stimulation of MHC class I-restricted memory cytotoxic T lymphocytes from equine infectious anemia virus-infected horses,” Vaccine, vol. 20, no. 13-14, pp. 1809–1819, 2002.
- S. L. Ridgely, B. Zhang, and T. C. McGuire, “Response of ELA-A1 horses immunized with lipopeptide containing an equine infectious anemia virus ELA-A1-restricted CTL epitope to virus challenge,” Vaccine, vol. 21, no. 5-6, pp. 491–506, 2003.
- D. G. Fraser, S. R. Leib, B. S. Zhang, R. H. Mealey, W. C. Brown, and T. C. McGuire, “Lymphocyte proliferation responses induced to broadly reactive Th peptides did not protect against equine infectious anemia virus challenge,” Clinical and diagnostic laboratory immunology, vol. 12, no. 8, pp. 983–993, 2005.
- B. Alvarez, T. Poderoso, F. Alonso, A. Ezquerra, J. Domínguez, and C. Revilla, “Antigen targeting to APC: from mice to veterinary species,” Developmental and Comparative Immunology, vol. 41, no. 2, pp. 153–163, 2013.
- H. Rau, H. Revets, C. Balmelli, K. C. McCullough, and A. Summerfield, “Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo,” Veterinary Research, vol. 37, no. 1, pp. 155–168, 2006.
- M. St. Paul, S. Paolucci, and S. Sharif, “Treatment with ligands for toll-like receptors 2 and 5 induces a mixed T-helper 1- and 2-like response in chicken splenocytes,” Journal of Interferon and Cytokine Research, vol. 32, no. 12, pp. 592–598, 2012.
- M. St Paul, N. Barjesteh, S. Paolucci, Y. Pei, and S. Sharif, “Toll-like receptor ligands induce the expression of interferon-gamma and interleukin-17 in chicken CD4+ T cells,” BMC Research Notes, vol. 5, article 616, 2012.
- M. St. Paul, S. Paolucci, and S. Sharif, “Characterization of responses initiated by different Toll-like receptor 2 ligands in chicken spleen cells,” Research in Veterinary Science, vol. 95, no. 3, pp. 919–923, 2013.
- M. H. Erhard, P. Schmidt, P. Zinsmeister et al., “Adjuvant effects of various lipopeptides and interferon-γ on the humoral immune response of chickens,” Poultry Science, vol. 79, no. 9, pp. 1264–1270, 2000.
- K. Loots, H. Revets, and B. M. Goddeeris, “Attachment of the outer membrane lipoprotein (OprI) of Pseudomonas aeruginosa to the mucosal surfaces of the respiratory and digestive tract of chickens,” Vaccine, vol. 26, no. 4, pp. 546–551, 2008.
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