Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19

Abstract

Amblyomma americanum tick serine protease inhibitor (serpin, AAS) 19, is a highly conserved protein that is characterized by its functional domain being 100% conserved across tick species. We also reported that AAS19 was an immunogenic tick saliva protein with anti-haemostatic functions and an inhibitor of trypsin-like proteases including five of the eight serine protease factors in the blood clotting cascade. In this study the goal was to validate the importance of AAS19 in A. americanum tick physiology, assess immunogenicity and investigate tick vaccine efficacy of yeast-expressed recombinant (r) AAS19. We confirm that AAS19 is important to A. americanum fitness and blood meal feeding. AAS19 mRNA disruption by RNAi silencing caused ticks to obtain blood meals that were 50% smaller than controls, and treated ticks being morphologically deformed with 100% of the deformed ticks dying in incubation. We show that rAAS19 is highly immunogenic in that two 500 µg inoculations mixed with TiterMax Gold adjuvant provoked antibody titers of more than 1:320000 that specifically reacted with native AAS19 in unfed and partially fed tick tissue. Since AAS19 is injected into animals during tick feeding, we challenge infested immunized rabbits twice to test if tick infestations of immunized rabbits could act as booster. While in the first infestation significantly smaller tick blood meals were observed on one of the two immunized rabbits, smaller blood meals were observed on both rabbits, but 60% of ticks that engorged on immunized rabbits in the second infestation failed to lay eggs. It is notable that ticks fed faster on immunized animals despite obtaining smaller blood meals. We conclude that rAAS19 is a potential component of cocktail tick vaccine.

Field evaluation of poultry red mite ( Dermanyssus gallinae ) native and recombinant prototype vaccines

“…Therefore vaccination with one protein may have the additional benefit of conferring cross-protection immunological cross-reactivity against the other. Likewise, Deg-SRP-1 also had a rational basis for its inclusion into the recombinant antigen cocktail vaccine: in addition to the induced mortality in PRM feeding on sera from hens immunised with recombinant Deg-SRP-1 ( Bartley et al, 2015 ), previous vaccination studies on several tick species have shown that serpin-based vaccines induce a variety of effects including: increased parasite mortality, reduced reproductive parameters and interference with feeding ( Sugino et al, 2003 , Imamura et al, 2005 , Imamura et al, 2006 , Prevot et al, 2007 , Jittapalapong et al, 2010 , Kim et al, 2016 ). In contrast, the final protein included into the recombinant antigen cocktail, Deg-PUF-1, has no ascribed putative function.…”

Heparan sulfate/heparin glycosaminoglycan binding alters inhibitory profile and enhances anticoagulant function of conserved Amblyomma americanum tick saliva serpin 19

“…On the basis of data in study, we conclude that native AAS19 is a likely inhibitor of thrombin and other trypsin-related proteases. Kim et al, (2016a) showed that RNAi silencing of AAS19 caused deformities in ticks suggesting that this serpin regulated an important protease in the tick. There is evidence that invertebrates do produce heparin-like GAGs (Chavante et al, 2014; Pavão, 2014).…”

“…We showed that this protein was injected into the host during tick feeding, and that Pichia pastoris expressed recombinant (r)AAS19 was a broad-spectrum inhibitor of hemostasis and inflammation system proteases with anti-hemostatic functions (Kim et al, 2015). In another study target validation by RNAi silencing and immunization affected tick fitness, prevented successful tick feeding, and female ability to lay eggs (Kim et al, 2016a). Comparative modeling predicted four basic patches on AAS19 tertiary structure (Kim et al, 2015) that were similar to functionally validated GAG binding sites in mammalian serpins (Huntington, 2013; Koide, 1993; Pratt and Church, 1993).…”

Peptidase inhibitors in tick physiology

“…AAS‐19 can bind heparan sulfate/heparin glycosaminoglycans, and this interaction alters the activity of the inhibitor (Radulovic and Mulenga, ). Disruption of AAS‐19 by RNAi or immunization assays leads to tick deformation and a significant decrease in tick attachment and engorgement rates (Kim et al ., ). Similarly to AAS‐19, the homologous serpin RmS‐15 from R. microplus functions as an anticoagulant by inhibiting thrombin (Xu et al ., ).…”

“…Amblyomma americanum tick serine protease inhibitor 19 (AAS‐19) is an immunogenic tick salivary serpin with anti‐hemostatic functions. It suppresses thrombin‐induced platelet aggregation, delays clotting in recalcification and thrombin time assays, and inhibits the activity of five serine protease blood clotting factors (Kim et al ., , ). AAS‐19 can bind heparan sulfate/heparin glycosaminoglycans, and this interaction alters the activity of the inhibitor (Radulovic and Mulenga, ).…”