Evasins: Tick Salivary Proteins that Inhibit Mammalian Chemokines.

Ticks are hematophagous arachnids that parasitize mammals and other hosts, feeding on their blood. Ticks secrete numerous salivary factors that enhance host blood flow or suppress the host inflammatory response. The recruitment of leukocytes, a hallmark of inflammation, is regulated by chemokines, which activate chemokine receptors on the leukocytes. Ticks target this process by secreting glycoproteins called Evasins, which bind to chemokines and prevent leukocyte recruitment. This review describes the recent discovery of numerous Evasins produced by ticks, their classification into two structural and functional classes, and the efficacy of Evasins in animal models of inflammatory diseases. The review also proposes a standard nomenclature system for Evasins and discusses the potential of repurposing or engineering Evasins as therapeutic anti-inflammatory agents.

Anti-bacterial Monoclonal Antibodies.

The failing efficacy of antibiotics and the high mortality rate among high-risk patients calls for new treatment modalities for bacterial infections. Due to the vastly divergent pathogenesis of human pathogens, each microbe requires a tailored approach. The main modes of action of anti-bacterial antibodies are virulence factor neutralization, complement-mediated bacterial lysis and enhancement of opsonophagocytic uptake and killing (OPK). Gram-positive bacteria cannot be lysed by complement and their pathogenesis often involves secreted toxins, therefore typically toxin-neutralization and OPK activity are required to prevent and ameliorate disease. In fact, the success stories in terms of approved products, in the anti-bacterial mAb field are based on toxin neutralization (Bacillus anthracis, Clostridium difficile). In contrast, Gram-negative bacteria are vulnerable to antibody-dependent complement-mediated lysis, while their pathogenesis rarely relies on secreted exotoxins, and involves the pro-inflammatory endotoxin (lipopolysaccharide). Given the complexity of bacterial pathogenesis, antibody therapeutics are expected to be most efficient upon targeting more than one virulence factor and/or combining different modes of action. The improved understanding of bacterial pathogenesis combined with the versatility and maturity of antibody discovery technologies available today are pivotal for the design of novel anti-bacterial therapeutics. The intensified research generating promising proof-of-concept data, and the increasing number of clinical programs with anti-bacterial mAbs, indicate that the field is ready to fulfill its promise in the coming years.

International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors.

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

Identification of the pharmacophore of the CC chemokine-binding proteins Evasin-1 and -4 using phage display.

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.

Targeting chemokines: Pathogens can, why can't we?

Chemoattractant cytokines, or chemokines, are the largest sub-family of cytokines. About 50 distinct chemokines have been identified in humans. Their principal role is to stimulate the directional migration of leukocytes, which they achieve through activation of their receptors, following immobilization on cell surface glycosaminoglycans (GAGs). Chemokine receptors belong to the G protein-coupled 7-transmembrane receptor family, and hence their identification brought great promise to the pharmaceutical industry, since this receptor class is the target for a large percentage of marketed drugs. Unfortunately, the development of potent and efficacious inhibitors of chemokine receptors has not lived up to the early expectations. Several approaches to targeting this system will be described here, which have been instrumental in establishing paradigms in chemokine biology. Whilst drug discovery programs have not yet elucidated how to make successful drugs targeting the chemokine system, it is now known that certain parasites have evolved anti-chemokine strategies in order to remain undetected by their hosts. What can we learn from them?

Serological status of Canadian cattle for brucellosis, anaplasmosis, and bluetongue in 2007-2008.

A national bovine serological survey was conducted to confirm that the prevalence of brucellosis, bluetongue, and anaplasmosis does not exceed 0.02% (95% confidence) in live cattle in Canada. Sampling consisted of a systematic random sample of 15 482 adult cattle slaughtered in federally inspected abattoirs, stratified by province. Samples were tested to detect antibodies for brucellosis, bluetongue, and anaplasmosis. All samples were negative for brucellosis. Three samples were seroreactors to bluetongue, 2 of which originated from the Okanagan Valley in British Columbia and 1 from Ontario, which after follow-up, was considered an atypical result. A total of 244 samples were seroreactors to Anaplasma and follow-up identified infection in Saskatchewan, Manitoba, and Quebec. In conclusion, the Canadian cattle population remains free of brucellosis and free of bluetongue outside the Okanagan Valley. Canada is no longer free of anaplasmosis and will be unable to claim freedom until eradication measures are completed.

RésuméStatut sérologique des bovins canadiens à l'égard de la brucellose, l'anaplasmose et la fièvre catarrhale du mouton en 2007­2008. L'enquête sérologique sur les bovins a été menée à l'échelle nationale afin de confirmer que la brucellose, la fièvre catarrhale du mouton et l'anaplasmose demeurent à une prévalence inférieure à 0,02 % (intervalle de confiance de 95 %) dans le cheptel bovin canadien. Un échantillonnage systématique de 15 482 bovins adultes a été effectué dans les abattoirs sous inspection fédérale, en stratifiant par province. Tous les échantillons se sont avérés négatifs en sérologie pour la brucellose. Une réaction sérologique a été identifiée pour la fièvre catarrhale du mouton chez trois bovins, dont deux provenaient de la vallée de l'Okanagan en Colombie-Britanique. L'autre réacteur provenait d'une ferme d'Ontario, où, après investigation, les résultats ont été considérés atypiques. Une réaction sérologique à l'anaplasmose a été détectée dans 244 échantillons. Les investigations ont permis d'identifier des fermes infectées en Saskatchewan, au Manitoba et au Québec. Le cheptel bovin canadien demeure donc indemne de la brucellose, et de la fièvre catarrhale du mouton à l'exclusion des bovins la vallée de l'Okanagan en Colombie-Britannique. Le Canada n'est plus considéré comme étant indemne de l'anaplasmose et ne pourra réclamer ce statut tant que l'éradication ne sera pas terminée.(Traduit par les auteurs).

A role for CCR4 in development of mature circulating cutaneous T helper memory cell populations.

Expression of the chemokine receptor CCR4 is strongly associated with trafficking of specialized cutaneous memory T helper (Th) lymphocytes to the skin. However, it is unknown whether CCR4 itself participates in the development of cutaneous Th populations. We have addressed this issue via competitive bone marrow (BM) reconstitution assays; equal numbers of BM cells from CCR4(+/+) and CCR4(-/-) donors were allowed to develop side-by-side within RAG-1(-/-) hosts. Cells from both donor types developed equally well into B cells, naive CD8 T cells, naive CD4 T cells, interferon-gamma(+) Th1 cells, and interleukin-4(+) Th2 cells. In marked contrast, circulating cutaneous memory Th cells (i.e., E-selectin ligand(+) [E-lig(+)]) were more than fourfold more likely to be derived from CCR4(+/+) donors than from CCR4(-/-) donors. Most of this effect resides within the CD103(+) subset of the E-lig(+) Th population, in which donor CCR4(+/+) cells can outnumber CCR4(-/-) cells by >12-fold. No similar effect was observed for alpha4beta7(+) intestinal memory Th cells or CD103(+)/E-lig(-) Th cells. We conclude that CCR4 expression provides a competitive advantage to cutaneous Th cells, either by participating in their development from naive Th cells, or by preferentially maintaining them within the memory population over time.

David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments.

Since the licensing of the first monoclonal antibody therapy in 1986, monoclonal antibodies have become the largest class of biopharmaceuticals with over 80 antibodies currently approved for a variety of disease indications. The development of smaller, antigen binding antibody fragments, derived from conventional antibodies or produced recombinantly, has been growing at a fast pace. Antibody fragments can be used on their own or linked to other molecules to generate numerous possibilities for bispecific, multi-specific, multimeric, or multifunctional molecules, and to achieve a variety of biological effects. They offer several advantages over full-length monoclonal antibodies, particularly a lower cost of goods, and because of their small size they can penetrate tissues, access challenging epitopes, and have potentially reduced immunogenicity. In this review, we will discuss the structure, production, and mechanism of action of EMA/FDA-approved fragments and of those in clinical and pre-clinical development. We will also discuss current topics of interest surrounding the potential use of antibody fragments for intracellular targeting and blood-brain barrier (BBB) penetration.

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC in in vitro and in vivo lung infection models using monoclonal antibodies.

Streptococcus pneumoniae isolates express up to three neuraminidases (sialidases), NanA, NanB and NanC, all of which cleave the terminal sialic acid of glycan-structures that decorate host cell surfaces. Most research has focused on the role of NanA with limited investigations evaluating the roles of all three neuraminidases in host-pathogen interactions. We generated two highly potent monoclonal antibodies (mAbs), one that blocks the enzymatic activity of NanA and one cross-neutralizing NanB and NanC. Total neuraminidase activity of clinical S. pneumoniae isolates could be inhibited by this mAb combination in enzymatic assays. To detect desialylation of cell surfaces by pneumococcal neuraminidases, primary human tracheal/bronchial mucocilial epithelial tissues were infected with S. pneumoniae and stained with peanut lectin. Simultaneous targeting of the neuraminidases was required to prevent desialylation, suggesting that inhibition of NanA alone is not sufficient to preserve terminal lung glycans. Importantly, we also found that all three neuraminidases increased the interaction of S. pneumoniae with human airway epithelial cells. Lectin-staining of lung tissues of mice pre-treated with mAbs before intranasal challenge with S. pneumoniae confirmed that both anti-NanA and anti-NanBC mAbs were required to effectively block desialylation of the respiratory epithelium in vivo. Despite this, no effect on survival, reduction in pulmonary bacterial load, or significant changes in cytokine responses were observed. This suggests that neuraminidases have no pivotal role in this murine pneumonia model that is induced by high bacterial challenge inocula and does not progress from colonization as it happens in the human host.

Chemokine blockers--therapeutics in the making?

Chemokines are a family of small chemoattractant cytokines that have an important role in controlling leukocyte migration. The finding that some chemokines and their receptors are upregulated in both acute and chronic inflammatory diseases, and that they are key players in the development of AIDS, has provided the pharmaceutical industry with new targets for therapeutic intervention in these diseases. Although the chemokine system shows apparent redundancy in vitro, target validation is possible largely through expression studies in human disease tissues and the use of transgenic and knockout mice as disease models. Several approaches are being developed to block the effects of chemokines, including small-molecule antagonists of chemokine receptors, modified chemokines and antibodies directed against chemokine receptors. Here, we describe the rationale behind these different approaches, the pitfalls that have been encountered and future perspectives.

Endotoxin neutralization by an O-antigen specific monoclonal antibody: A potential novel therapeutic approach against Klebsiella pneumoniae ST258.

Klebsiella pneumoniae ST258 is a globally distributed multi-drug resistant pathogen responsible for severe invasive infections. In this study, the different virulence potential of K. pneumoniae ST258 isolates in endotoxin susceptible versus resistant animal models was shown. Furthermore, ST258 clinical isolates were found highly sensitive to the bactericidal effect of naive animal and human serum. These observations imply that LPS, released from the rapidly lysed bacteria, may contribute to the high mortality associated with ST258 bacteremia cases. A humanized version (mAb A1102) of a previously described murine mAb specific for the conserved LPS O-antigen, was tested for endotoxin neutralization. A1102 was able to neutralize TLR-4 activation by ST258-derived LPS in vitro with an efficacy exceeding that of polymyxin B by 3 orders of magnitude. Passive immunization with A1102 afforded a significant level of protection in a galactosamine-sensitized mouse model of endotoxemia, induced by ST258-derived LPS, or upon challenge with live bacteria. Efficacy was retained using an aglycosylated IgG, as well as upon complement depletion, suggesting that Fc-independent endotoxin neutralization may be the main protective mechanism in this model, in spite of the complement-dependent bactericidal and opsonic activities additionally observed for A1102 in vitro. Furthermore, rabbits that are naturally highly susceptible to endotoxin, were also significantly protected by low doses of A1102 when challenged with an ST258 strain. Given this unique mode of action and the high protective efficacy of this mAb, passive immunization, as prophylactic or adjunct therapeutic approach for the treatment of infections caused by ST258 isolates should be considered.

Evasins: Therapeutic Potential of a New Family of Chemokine-Binding Proteins from Ticks.

Blood-sucking parasites, such as ticks, remain attached to their hosts for relatively long periods of time in order to obtain their blood meal without eliciting an immune response. One mechanism used to avoid rejection is the inhibition of the recruitment of immune cells, which can be achieved by a class of chemokine-binding proteins (CKBPs) known as Evasins. We have identified three distinct Evasins produced by the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. They display different selectivities for chemokines, the first two identified show a narrow selectivity profile, while the third has a broader binding spectrum. The Evasins showed efficacy in animal models of inflammatory disease. Here, we will discuss the potential of their development for therapeutic use, addressing both the advantages and disadvantages that this entails.

Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4-/- mice.

The role of CC chemokine receptor 4 (CCR4) during the development and maintenance of Th2-type allergic airway disease is controversial. In this study, we examined the role of CCR4 in the chronic allergic airway response to live Aspergillus fumigatus spores, or conidia, in A. fumigatus-sensitized mice. After the conidia challenge, mice lacking CCR4 (CCR4-/- mice) exhibited significantly increased numbers of airway neutrophils and macrophages, and conidia were more rapidly eliminated from these mice compared with control CCR4 wild-type (CCR4+/+) mice. Significant airway hyperresponsiveness to intravenous methacholine was observed at day 3 in CCR4-/- mice, whereas at days 7 and 30, airway hyperresponsiveness was attenuated in these mice compared with control mice. A major reduction in peribronchial and airway eosinophilia was observed in CCR4-/- mice at all times after conidia challenge in contrast to CCR4+/+ mice. Further, whole lung levels of interleukin (IL) 4 and IL-5 were significantly increased in CCR4-/- mice at day 3, whereas these Th2 cytokines and IL-13 were significantly decreased at day 30 in CCR4-/- mice compared with their wild-type counterparts. Peribronchial fibrosis and goblet cell hyperplasia were similar in both groups of mice throughout the course of this model. In summary, CCR4 modulates both innate and acquired immune responses associated with chronic fungal asthma.

Knock out models to dissect chemokine receptor function in vivo.

Chemokines are a family of small proteins involved in numerous biological processes ranging from hematopoiesis, angiogenesis and lymphocyte trafficking to the extravasation and tissue infiltration of leukocytes in response to inflammatory agents, tissue damage and bacterial or viral infection. Chemokines exert their effects by binding to specific G-protein-coupled seven-transmembrane receptors. In vitro studies suggest that the chemokine system is highly redundant in that most chemokines bind to more than one receptor and most receptors bind multiple chemokines. Therefore, targeted deletion of chemokine receptors has proved to be a useful tool for determining the distinct biological role of these molecules in vivo.

Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

Properties of 7ND-CCL2 are modulated upon fusion to Fc.

7ND, a truncated version of the chemokine MCP-1/CCL2 lacking amino acids 2-8, is a potent antagonist of CCR2. In contrast to CCL2, 7ND is an obligate monomer. Similar to other chemokines, the in vivo half-life of 7ND is very short and its use as an antagonist in disease models is thus limited. We therefore constructed a 7ND-Fc fusion protein to extend the half-life of 7ND and overcome its limitations as a potential therapeutic antagonist. When we tested the properties of the fusion molecule in vitro, we found to our surprise that 7ND-Fc, in contrast to 7ND, produced a distinct, albeit small, chemotactic response in THP-1 cells, and a robust chemotactic response in L1.2 cells stably transfected with CCR2. To test whether this unexpected observation might be due to the bivalency of 7ND-Fc stemming from the dimeric nature of Fc fusions, we produced a heterodimeric Fc fusion which displays only one 7ND moiety, using a technology called strand exchange of engineered CH3 domains (SEED). The monovalent construct had properties equivalent to the parent 7ND. Furthermore, partial agonist activity appears to depend on receptor density as well as the signaling pathway examined. However, we were able to show that 7ND-Fc, but not 7ND alone, has antagonistic activity in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis.

Anti-chemokine small molecule drugs: a promising future?

IMPORTANCE OF THE FIELD: Chemokines have principally been associated with inflammation due to their role in the control of leukocyte migration, but just over a decade ago chemokine receptors were also identified as playing a pivotal role in the entry of the HIV virus into cells. Chemokines activate seven transmembrane G protein-coupled receptors, making them extremely attractive therapeutic targets for the pharmaceutical industry. AREAS COVERED IN THIS REVIEW: Although there are now a large number of molecules targeting chemokines and chemokine receptors including neutralizing antibodies in clinical trials for inflammatory diseases, the results to date have not always been positive, which has been disappointing for the field. These failures have often been attributed to redundancy in the chemokine system. However, other difficulties have been encountered in drug discovery processes targeting the chemokine system, and these will be addressed in this review. WHAT THE READER WILL GAIN: In this review, the reader will get an insight into the hurdles that have to be overcome, learn about some of the pitfalls that may explain the lack of success, and get a glimpse of the outlook for the future. TAKE HOME MESSAGE: In 2007, the FDA approved maraviroc, an inhibitor of CCR5 for the prevention of HIV infection, the first triumph for a small-molecule drug acting on the chemokine system. The time to market, 11 years from discovery of CCR5, was fast by industry standards. A second small-molecule drug, a CXCR4 antagonist for hematopoietic stem cell mobilization, was approved by the FDA at the end of 2008. The results of a Phase III trial with a CCR9 inhibitor for Crohn's disease are also promising. This could herald the first success for a chemokine receptor antagonist as an anti-inflammatory therapeutic and confirms the importance of chemokine receptors as a target class for anti-inflammatory and autoimmune diseases.

Structural basis of chemokine sequestration by a tick chemokine binding protein: the crystal structure of the complex between Evasin-1 and CCL3.

BACKGROUND: Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins. METHODOLOGY/PRINCIPAL FINDINGS: We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1:1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent pi-pi interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine. CONCLUSIONS/SIGNIFICANCE: However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the "address" whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity.

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Molecular cloning and characterization of a highly selective chemokine-binding protein from the tick Rhipicephalus sanguineus.

Ticks are blood-feeding parasites that secrete a number of immuno-modulatory factors to evade the host immune response. Saliva isolated from different species of ticks has recently been shown to contain chemokine neutralizing activity. To characterize this activity, we constructed a cDNA library from the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. Pools of cDNA clones from the library were transfected into HEK293 cells, and the conditioned media from the transfected cells were tested for chemokine binding activity by chemical cross-linking to radiolabeled CCL3 followed by SDS-PAGE. By de-convolution of a single positive pool of 270 clones, we identified a full-length cDNA encoding a protein of 114 amino acids, which after signal peptide cleavage was predicted to yield a mature protein of 94 amino acids that we called Evasin-1. Recombinant Evasin-1 was produced in HEK293 cells and in insect cells. Using surface plasmon resonance we were able to show that Evasin-1 was exquisitely selective for 3 CC chemokines, CCL3 and CCL4 and the closely related chemokine CCL18, with K(D) values of 0.16, 0.81, and 3.21 nm, respectively. The affinities for CCL3 and CCL4 were confirmed in competition receptor binding assays. Analysis by size exclusion chromatography demonstrated that Evasin-1 was monomeric and formed a 1:1 complex with CCL3. Thus, unlike the other chemokine-binding proteins identified to date from viruses and from the parasitic worm Schistosoma mansoni, Evasin-1 is highly specific for a subgroup of CC chemokines, which may reflect a specific role for these chemokines in host defense against parasites.