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.

Resistance to Ticks and the Path to Anti-Tick and Transmission Blocking Vaccines

“…Anti-tick vaccine candidates include tick saliva inhibitors of hemostasis [ 203 ]. Three Amblyomma sculptum protease inhibitors were characterized as inhibitors of factor Xa, trypsin, and/or thrombin, assessed as vaccine antigens, and found to induce variable levels of protection against adult female and nymph challenges [ 204 ]. All three proteins inhibited an activation of both the alternative and classical pathways of human complement.…”

“…All three proteins inhibited an activation of both the alternative and classical pathways of human complement. Vaccination efficiency against adult females ranged from 59.4% to 85% and mortality of nymphs fed upon vaccinated mice ranged from 70% to 100% [ 204 ].…”

Host Immune Responses to Salivary Components - A Critical Facet of Tick-Host Interactions

“…A particularly notable role of PIs has been detected in tick–host crosstalk, as the host defense mechanisms are highly regulated by specific endogenous inhibitors subsequently inhibited by tick-secreted PIs ( Martins et al., 2020 ; Jmel et al., 2021 ). The salivary PIs of several ticks have been characterized and found to be differentially expressed throughout the blood-feeding process, inducing local immunosuppression by inhibiting platelet aggregation or blocking cascade elements of intrinsic, extrinsic, and common pathways of blood coagulation, tumor development, and angiogenic factors ( Chmelař et al., 2017 ; Costa et al., 2021 ). The host hemostatic mechanisms are not directly hindered by the tick salivary molecules; instead, the active sites, exocites, and receptors of regulatory factors or components involved in driving mechanisms such as thrombin, plasmin, factor V (fV), factor Xa (fXa), kallikrein, and kallikrein-associated fXIIa–fXIa and fXa-TF-VIIa components are inhibited.…”

“…In addition, TAP derived from O. moubata interfere its tripeptide-containing amino terminal regions (Tyr-Asn-Arg) with both exocites and active site of thrombin. However, AsKunitz inhibition of thrombin’s activity might be due to the interaction of 2 cysteine components in the carboxy terminal region with exosite I only ( Costa et al., 2021 ). The Amblyomma variegatum saliva protein, variegin, displays its C-terminal tail and directly binds thrombin’s active site to exosite I ( Koh et al., 2007 ).…”

“…Haemaphysalin, which binds to fXIIa and kallikrein BmTI-A, performs a crucial role in the inhibition of blood coagulation ( Kato et al., 2005 ). A. sculptum secretes As8.9kDa, AsBasicTail, and AsKunitz; the former two prevent host trypsin and thrombin while inhibiting enzymatic activity derived from fXa, whereas AsKunitz inhibits only thrombin ( Costa et al., 2021 ; Van Oosterwijk and Wikel, 2021 ). IxscS-1E1I of I. scapularis specifically inhibits thrombin, trypsin, cathepsin G, and fXa.…”