Evasin-3-like anti-chemokine activity in salivary gland extracts of ixodid ticks during blood-feeding: a new target for tick control.

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin-3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti-human CXCL8 and anti-mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood-feeding. Both anti-CXCL8 activity and anti-CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin-3-like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood-feeding. As such, Evasin-3 offers a new target for anti-tick vaccine development.

Chemokine-binding activities of M3 protein encoded by Murine gammaherpesvirus 72.

Murine gammaherpesvirus 68 (MHV-68) contains gene-encoding M3 protein expressed during the acute and persistent phase of infection. This protein features a chemokine-binding activities (Parry et al., 2000; van Berkel et al., 2000). In this study, we demonstrated that the Murine gammaherpesvirus 72 (MHV-72) also contained M3 gene with the codon-changing mutation at the position 920 nt converting amino acid (aa) 307 Asp (GAC) to Gly (GGC). The mutation in the M3 protein was localized near chemokine-binding domain and was able to change the secondary structure of M3 protein. We examined the binding activities of M3 proteins of MHV-72 and MHV-68 to five human chemokines (CCL3, CCL5, CCL11, CCL2, and CXCL8). Binding activity of MHV-72 M3 protein to CCL5 as well as to CXCL8 reached only 11.1% (day 3 p.i.) to 20% (day 4 p.i.) of the activity detected for MHV-68 M3 protein. On the other hand, MHV-72 M3 protein bound to human cytokines CCL11 and CCL2 reached about 90% of the binding detected for MHV-68 M3 protein. The binding activity of both M3 proteins to human CCL3 was similar. These data implied that mutation identified in MHV-72 M3 protein might be involved in attenuation of immune response to infection with MHV-72.

Immunomodulatory arsenal of nymphal ticks.

Ticks have developed their own immunomodulatory mechanisms to inhibit the host inflammatory response. One of them involves the ability to subvert the cytokine network at the site of tick feeding by secreting cytokine binding molecules. Most studies have focused on the immunomodulatory prowess of adult female ticks. Here we describe anti-cytokine activity in salivary gland extracts (SGEs) prepared from 2-day-fed nymphs of Dermacentor reticulatus Fabricius, Ixodes ricinus L., Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius. Anti-CXCL8 activity was detected in nymphs of all species. Relatively high activity against CCL2, CCL3 and CCL11 was observed in SGEs of R. appendiculatus and A. variegatum nymphs, whereas SGEs of I. ricinus nymphs showed comparatively high anti-interleukin-2 (-IL-2) and anti-IL-4 activities. These data show that nymphs, which epidemiologically are usually more important than adults as disease vectors, possess a range of anti-cytokine activities that may facilitate pathogen transmission.

Differential anti-chemokine activity of Amblyomma variegatum adult ticks during blood-feeding.

Ticks secrete a cocktail of immunomodulatory molecules in their saliva during blood-feeding, including chemokine-binding factors that help control the activity of host immunocompetent cells. Here we demonstrate differential dynamics of anti IL-8 (CXCL8), MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5) and eotaxin (CCL11) activities in salivary gland extracts of adult Amblyomma variegatum. Unfed male and female ticks showed activity against all the chemokines except CCL5; anti-CCL11 activity was particularly high. However, during feeding the dynamics of anti-chemokine activity differed significantly between males and females, and varied between chemokines. In males, anti-chemokine activities increased, whereas in females they declined or increased slightly as feeding progressed. The exception was anti-CCL11 activity, which declined and then increased in both males and females. Comparison of salivary gland equivalents of individual ticks prepared at various feeding intervals revealed some differences that were most pronounced between individual females fed for 8 days. These observations reflect the feeding behaviour of male and female A. variegatum. They support the concept of 'mate guarding', in which males help their mates to engorge by controlling their host's immune response, and the possibility that ticks benefit from feeding together by exploiting molecular individuality.

Manipulation of host cytokine network by ticks: a potential gateway for pathogen transmission.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1alpha, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1alpha. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

Vesicular stomatitis virus nucleocapsid protein production in cells treated with selected fast protein liquid chromatography fractions of tick salivary gland extracts.

A salivary gland extract (SGE) prepared from 5-days-fed Dermacentor reticulatus female ticks was fractionated by fast protein liquid chromatography (FPLC). The effect of three FPLC fractions selected on the basis of anti-interleukin 8 (anti-IL-8) activity on vesicular stomatitis virus (VSV) nucleocapsid (N) protein formation in mouse L-cells was determined. Infected 14C-labeled cells treated with the FPLC fractions were analyzed by two-dimensional (2D) electrophoresis. The yields of VSV N protein were evaluated by Imagemaster software analysis. Most noticeable was an increase in the N protein production after treatment with the fraction 39 corresponding to the major peak of the anti-IL-8 activity. The nature of the substance in SGE that was responsible for this effect remains unclear.

The carboxyterminal domains of human IFN-alpha2 and IFN-alpha8 are antigenically homologous.

The antigenic properties of human hybrid IFN-alpha8(60)/alpha1(92)/alpha8 were compared with those of human IFN-alpha1 and IFN-alpha2 using monoclonal antibodies (mAb). Hybrid IFN demonstrated a significantly closer antigenic relationship to the subtype alpha2 than to the subtype alpha1. In particular, high homology was observed between antigenic structures located in the C-terminal domains (93-166) of IFN-alpha8 and IFN-alpha2, whereas the corresponding N-terminal receptor-binding domains (30-53) showed distinct antigenic characteristics. The 100% homology between IFN-alpha8 and IFN-alpha2 in the region 114-131 (helix D) indicated the role of this region in formation of the common antigenic structure. In IFN-alpha8/1/8, this shared antigenic structure was important for antiviral activity and exhibited immunodominant properties, consistent with functional and antigenic properties of the corresponding structure in IFN-alpha2. Based on this antigenic homology, we suggest that IFN-alpha8 and IFN-alpha2 are evolutionarily more closely related to each other than to IFN-alpha1. This study will contribute to a better understanding of evolutionary events in the human IFN-alpha family.

Radioiodination of human interferon-alpha2 interferes with binding of C-terminal specific antibodies.

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-alpha2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-alpha2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-alpha2 with Na(125)I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30-53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues approximately 120-145 approximately ). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-alpha2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

Vaccinia virus protein B18R inhibits the activity and cellular binding of the novel type interferon-delta.

The soluble vaccinia virus-encoded protein B18R inhibits the antiviral activity and cellular binding of the type I interferons (IFN)-alpha, -beta and -omega of different mammalian species. Recently, a novel type I IFN was detected in pigs and classified as a member of a distinct IFN family designated IFN-delta. Our study aimed to determine if the structural properties of this shortest (149 residues long) type I IFN allow its interaction with the type I IFN-binding protein B18R. Experiments using bovine (MDBK) cells demonstrated that B18R neutralized the antiviral activity of porcine IFN-delta with high efficiency. Preincubation of B18R with radiolabelled IFN-delta specifically inhibited binding of IFN to bovine cells. These data indicate that the overall conformation of the novel IFN-delta might be similar to that of other type I IFNs.