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) [
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 [
Phylogenetically, TLR2 belongs to a TLR family that includes TLR1, TLR6, TLR10, TLR14, and possibly the avian TLR15 [
Like other TLRs, TLR2 evolved under strong selection pressure, being preserved in all the vertebrate species tested so far [
Species-specific variations in TLR2 have been reported, mainly at the extracellular domains, possibly reflecting adaptation to different microbial environments [
TLR2 expression has been reported in antigen presenting cells (APCs), namely, macrophages, monocytes, and dendritic cells (DCs), including CD8
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 [
(a) TLR2 expression; (b) specificities reported for veterinary species.
Human and mouse | Reference |
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Antigen presenting cells: macrophages, monocytes, and DCs (CD8 |
[ |
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Lymphocytes: B cells, CD4+, and CD8+ T cells, Treg cells, |
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Granulocytes: neutrophils, basophils | [ |
Some epithelial cells |
Species | Cells and tissues reported to express TLR2 | Methoda | Information on the level of expression | Reference |
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Bovine | Monocytes | RT-PCR/FC | Strong | [ |
Monocyte derived-macrophages | RT-PCR/FC | |||
Alveolar macrophages | RT-PCR/FC | Intermediate | ||
Monocyte derived-DCs | RT-PCR/FC | Weak | ||
CD172+ DCs | RT-PCR/FC | |||
CD172− DCs | RT-PCR/FC | |||
CD21+ B cells | RT-PCR | No signal | [ | |
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Ovine | CD14+ monocytes from PBMCs | FC | [ | |
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Nilgai and Buffalo | PBMCs, monocytes, DCs, testes, skin | RT-PCR | Higher in Nilgai than Buffalo |
[ |
Buffalo | Kidney, endometrium, bone marrow, trachea | RT-PCR | Higher in endometrium and bone marrow | |
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Swine | Mesenteric lymph nodes and Peyer's patches | RT-PCR, IHC, FC | Higher than in spleen by RT-PCR |
[ |
Heart, thymus, lung, kidney, skeletal muscle, small intestine | RT-PCR | Lower than in spleen | ||
M cells | IHC, FC | |||
T and B cells | FC | Higher in T cells than in B cells | ||
Monocytes, macrophage, and granulocytes, but not on peripheral blood lymphocytes | FC |
[ | ||
Epithelial cells lining body entries (Lung, jejunum, kidney, liver) | IHC | |||
Alveolar Macrophages | WB | [ | ||
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Equine | PBMCs | RT-PCR | [ | |
Alveolar macrophages | RT-PCR | [ | ||
Respiratory epithelial tissues | RT-PCR | [ | ||
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Chicken | Heart, liver, gizzard, muscle | RT-PCR, WB | Strong | [ |
Spleen, caecal tonsil, bursa, liver | RT-PCR | Strong | [ | |
Heterophils, monocytes, macrophages, B and T cells | RT-PCR | [ | ||
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Canine | Blood mononuclear cells, lymph node, lung, liver, spleen, bladder, pancreas, small intestine, large intestine, and skin | RT-PCR | [ | |
Blood neutrophils | RT-PCR |
[ | ||
Blood neutrophils, monocytes | FC | Higher levels | ||
Lymphocytes | FC | Lower levels | ||
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Feline | Spleen, thymus | RT-PCR |
[ | |
CD4+ T cells, CD8+ T cells, CD21+ B cells | RT-PCR | Higher in B cells than in T cells | ||
BM-DCs | RT-PCR | [ | ||
Palatoglossal mucosa | RT-PCR | [ |
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 [
Studying the expression of TLR2 in gut-associated lymphoid tissues from adult swine, Tohno et al. [
After raising a panel of monoclonal antibodies for porcine TLR2, Alvarez et al. [
Expression of chicken TLR2a and TLR2b was detected in high levels by Western blot in heart, liver, gizzard, and muscle [
Ishii et al. [
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) [
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.,
Lipoproteins are membrane structural components of bacteria with diverse molecular structure but with a common lipidic modification at an N-terminal cysteine [
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 [
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. [
Irvine et al. [
Different studies addressed the ligand recognition by both types of chicken TLR2 and TLR1 [
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
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
The first plasmidic vectors for the expression of proteins in fusion with bacterial lipoproteins in
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 [
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., [
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 [
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 [
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
Immunomodulation by TLR2 ligands in mouse and human models.
TLR2 liganda | Species and cells/observationsb | Reference |
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Recruitment of leukocytes | ||
Pam3CSK4 |
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[ |
Transient reduction of DC motility at inflammatory sites | ||
Pam3CSK |
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[ |
Increase in DC antigen internalization | ||
Pam3CSK4; Pam3CSK |
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[ |
Mouse anti-human TLR2 mAb |
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[ |
Pam3CSK4 |
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[ |
Promotion of DC migration to regional lymph nodes | ||
Pam3CSK4 |
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[ |
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Enhanced presentation on MHC class II | ||
Pam3CSK4 |
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[ |
MALP-2 |
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[ |
Upregulation of costimulatory molecules and MHC class II | ||
rLipo-D1E3; OprI BLP; PGN, LTA; MALP-2 |
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[ |
[Th]-K(P2CSS)-[B] |
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[ |
Pam3CSK4; 19-kDa and Tp47 LPs |
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[ |
[Th]-K(P2CSS)-[Tc] |
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[ |
Pam3C |
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[ |
BPPcysMPEG |
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[ |
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[ |
Pam3CSK4 |
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[ |
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Induction of Th2 responses | ||
Pam3CSK4 |
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[ |
FSL-1 |
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[ |
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[ |
Pam3CSK4 |
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[ |
PGN, Pam3C, and zymosan |
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[ |
Induction of Th1 responses | ||
K(Pam)-[Th] versus K(Chol)-[Th] |
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[ |
OprI-COOHgp63 BLP |
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[ |
[Th]-K(P2CSS)-[Tc] |
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[ |
Pam2CSK4 and Pam3CSK4 |
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[ |
Pam3CSK4 |
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[ |
Pam3C, 19-kDa, and Tp47 LPs |
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[ |
PGN, Pam3C, Pam3CSK4 |
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[ |
rlipo-E7m |
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[ |
19- and 38-kD BLPs |
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[ |
19 kDa BLP; Tp47, OspA, and 19 kDa LPs; Pam3CSK4 |
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[ |
Pam3CSK4 and MALP-2 |
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[ |
Induction of Th17 responses | ||
PGN, Pam3CSK4, MALP-2 |
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[ |
Induction of Th1/Treg differentiation and inhibition of Th2 responses | ||
OprI BLP |
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[ |
LP40 |
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[ |
Pam3CSK4 |
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[ |
Regulatory role | ||
Zymosan |
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[ |
Zymosan |
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[ |
Zymosan |
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[ |
FSL-1 |
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[ |
Pam2 lipopeptides |
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[ |
Antiregulatory role | ||
Pam3CSK4 |
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[ |
Pam3CSK4 |
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[ |
Pam3CSK4 |
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[ |
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Pam3CSK4-[Tc] |
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[ |
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[ |
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[ |
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[ |
Pam3CSS- |
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[ |
Hda- |
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[ |
K(Pam)- |
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[ |
Pam3CSK4- |
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[ |
MALP-2 |
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[ |
(K(Pam))1,2,or 3- |
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[ |
BPPcysMPEG |
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[ |
FSL-1 |
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[ |
rlipo-E7m |
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[ |
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Stimulation of NK cell activity | ||
Pam2C lipopeptides |
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[ |
Pam2CSK4 versus MALP-2 |
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[ |
Induction of ADCC by NK | ||
FSL-1 |
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[ |
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Induction of antibody secreting cells (ASC) differentiation | ||
Natterins |
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[ |
Pam2CSK4; Pam3CSK4 |
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[ |
Increasing of antibody responses | ||
Fusion lipoproteins |
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[ |
Synthetic lipopeptides |
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[ |
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Immunization via mucosal surfaces | ||
((Pam)K)3- |
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[ |
MAP-Pam3C |
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[ |
LT-IIa-B5 |
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[ |
Pam3CSK4 |
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[ |
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[ |
Mucosal imprinting of specific immunity | ||
Pam3CSK4 |
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[ |
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 [
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 [
The above-mentioned transitory reduction in DC motility and the enhancement of antigen internalization was observed upon stimulation of different TLRs including TLR2 [
Although still controversial [
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 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 [
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 [
In other experimental conditions, the induction of Th17 polarization by IL-1
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
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 [
Monoacylated lipopeptides, lipidated through the covalent binding of palmitic acid to the lateral chain of lysine, induce CD8+ T cell responses [
TLR2-dependent NK cell activation was demonstrated to play a role in the immune response against different virus and bacteria [
The role of TLR-mediated activation of NK cells in immune responses against infectious and tumor disease is now emerging [
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 [
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) [
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 [
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 ovine, stimulation of bone marrow-derived DCs by LTA resulted in upregulation of MHC class II in the
Nelson et al. [
Investigating the potential of
Aiming at optimizing the protective efficacy of
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 [
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-
Stimulation of equine monocytes with Pam3CSK4 induced the production of TNF-
Immunizing pigs against mouse IgG, an increase in the anti-mouse IgG titres was observed by targeting the antibody to TLR2 [
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 [
Stimulation of chicken splenocytes with Pam3CSK4 upregulated not only Th1-associated cytokines IFN-
Based on previous demonstration of the immunostimulatory properties of protozoan HSP70 through TLR2 and TLR4, Zhang et al. [
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.
Antibody dependent cellular cytotoxicity
Antibody secreting cells
Antigen presenting cell
Cytotoxic T lymphocyte
Dendritic cell
Equine infectious anemia virus
Foot-and-mouth disease virus
Lipopolysaccharide
Lipoteichoic acid
Monophosphoryl lipid A
Nitric oxide
Natural killer
Outer membrane lipoprotein I
Ovalbumin
Di-palmitoyl-S-glyceryl cysteine
Tri-palmitoyl-S-glyceryl cysteine
Peptidoglycan
Pattern recognition receptor
Toll-like receptor.
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).