Amblyomma sculptum Salivary Protease Inhibitors as Potential Anti-Tick Vaccines

Abstract

Amblyomma sculptum is the main tick associated with human bites in Brazil and the main vector of Rickettsia rickettsii, the causative agent of the most severe form of Brazilian spotted fever. Molecules produced in the salivary glands are directly related to feeding success and vector competence. In the present study, we identified sequences of A. sculptum salivary proteins that may be involved in hematophagy and selected three proteins that underwent functional characterization and evaluation as vaccine antigens. Among the three proteins selected, one contained a Kunitz_bovine pancreatic trypsin inhibitor domain (named AsKunitz) and the other two belonged to the 8.9 kDa and basic tail families of tick salivary proteins (named As8.9kDa and AsBasicTail). Expression of the messenger RNA (mRNA) encoding all three proteins was detected in the larvae, nymphs, and females at basal levels in unfed ticks and the expression levels increased after the start of feeding. Recombinant proteins rAs8.9kDa and rAsBasicTail inhibited the enzymatic activity of factor Xa, thrombin, and trypsin, whereas rAsKunitz inhibited only thrombin activity. All three recombinant proteins inhibited the hemolysis of both the classical and alternative pathways; this is the first description of tick members of the Kunitz and 8.9kDa families being inhibitors of the classical complement pathway. Mice immunization with recombinant proteins caused efficacies against A. sculptum females from 59.4% with rAsBasicTail immunization to more than 85% by immunization with rAsKunitz and rAs8.9kDa. The mortality of nymphs fed on immunized mice reached 70-100%. Therefore, all three proteins are potential antigens with the possibility of becoming a new tool in the control of A. sculptum.

Keywords: Amblyomma sculptum; antihemostatic; complement inhibitors; saliva; vaccine.

Figure 1

Relative messenger RNA (mRNA) levels of AsKunitz, As8.9kDa, and AsBasicTail in Amblyomma sculptum instars during feeding. Measurements were performed using quantitative polymerase chain reaction in whole body for larvae (A–C) and nymphs (D–F) and in the salivary glands for females (G–I). Feeding = 3 days of feeding. Statistical analysis: (A–F)—t-test, (G–I)—ANOVA–Dunnett, *p < 0.05, **p < 0.01, and ***p < 0.001 indicate difference from the fasting group. Data is shown as mean ± standard deviation (SD).

Figure 2

Expression of rAsKunitz, rAs8.9kDa, and rAsBasicTail and their effect on the coagulation cascade. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (silver staining) and western blotting (developed using anti-His-tag antibodies) of each recombinant protein (A). Effect of 0.25 µM of each recombinant on the human coagulation cascade (B). One unit (U) of anticoagulant activity indicates that the coagulation time was doubled. Statistical analysis: ANOVA–Tukey, ns = not significant (p>0.05). Data is shown as mean ± SD.

Figure 3

Effect of the recombinant proteins on the activity of factor Xa (A–C), thrombin (D–F), and trypsin (G–I). Data is shown as mean ± standard error (SE). Statistical analysis: ANOVA–Dunnett, *p < 0.05, **p < 0.01, and ***p < 0.001 indicate difference from the sample without recombinants (0 µM).

Figure 4

Effect of rAsKunitz, rAs8.9kDa, and rAsBasicTail on the classical (A–C) and alternative (D–F) pathways of the human complement system. Recombinant proteins were incubated with the respective mice antiserum before being added to the classical (G, H) or the alternative (I) pathway assays. Data is shown as mean ± SD. Statistical analysis: ANOVA–Dunnett, *p < 0.05 and ***p < 0.001. In (A–F), asterisks indicate statistical difference from controls, i.e., 0 µM in (A–F) and the sample without specific antiserum in (G–I).

Figure 5

Profile of specific serum IgG levels in mice vaccinated with rAsKunitz (A), rAs8.9kDa (B), and rAsBasicTail (C). Serum IgG levels against each of the recombinant were accessed by ELISA. Arrows indicate the three injections used for immunization and the cross indicates the challenge with ticks. Statistical analysis between control and vaccinated groups: Two-way ANOVA–Bonferroni, *p < 0.05; **P < 0.01; ***p < 0.001.

Figure 6

Feeding (A, B) and reproductive (C, D) parameters of Amblyomma sculptum females fed on mice previously immunized with rAsKunitz, rAs8.9kDa, or rAsBasicTail. Data is shown as mean ± SD. Statistical analysis: Kruskal-Wallis–Dunn’s, *p < 0.05 and **p < 0.01 indicate significant difference from controls.

Figure 7

Feeding parameters of Amblyomma sculptum nymphs fed on mice previously immunized with rAsKunitz. Duration of blood feeding (A) and tick weight after detachment (B). Data is shown as mean ± SD. Statistical analysis: Mann-Whitney, *p < 0.05.