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. 2016 Apr;7(3):405-14.
doi: 10.1016/j.ttbdis.2015年12月01日7. Epub 2015 Dec 29.

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

Affiliations

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

Tae K Kim et al. Ticks Tick Borne Dis. 2016 Apr.

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 1:320,000 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.

Keywords: A. americanum serpin 19; Amblyomma americanum; Tick vaccine antigens candidate.

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Figures

Figure 1
Figure 1. Validating the disruption of AAS19 mRNA in AAS19 double stranded (ds) RNA injected ticks
Fifteen ticks were microinjected with 0.5–1 μL (~3 μg/μL) of AAS19- or EGFP- (control) dsRNA in nuclease free water. At 48 h post-attachment, three ticks per treatment of EGFP-dsRNA injected control and AAS19-dsRNA injected ticks, were manually detached. Tick organs including salivary glands (SG), midguts (MG), synganglion (SYN), Malpighian tubules (MT), ovaries (OV) and carcass (CA, tick remnants after removal of SG, MG, OV, and MT) were dissected and individually processed for mRNA extraction and then subjected to two-step quantitative (q) reverse transcriptase (RT)-PCR using AAS19 primers described in materials and methods section 2.4. Relative expression (RQ) of AAS19 mRNA was determined using the Comparative CT Method (ΔΔCT). Relative suppression of AAS19 mRNA was determined using the following formula, S = 100-(RQT/RQC X 100) where S = mRNA suppression, RQT and RQC = RQ of tissues of AAS19-dsRNA and EGFP-dsRNA injected ticks respectively.
Fig. 2
Fig. 2. Phenotype and engorgement weights of AAS19 dsRNA injected ticks
(A) Spontaneously detached ticks were photographed to document phenotypic changes of AAS19-dsRNA injected ticks compared to control EGFP-dsRNA injected ticks. Deformed ticks are asterisks marked (*). (B) After spontaneously detaching from the host, ticks were individually weighed to determine engorgement weights (EWs) as indices for amounts of blood imbibed by ticks. EWs were subjected to unpaired student t-test and Mann-Whitney analysis to determine statistical significance. Data is reported as mean (M) EWs (M ± SEM).
Figure 3
Figure 3. Western blotting analyses
Yeast and bacteria expressed rAAS19 (Fig. 3A) and crude tick saliva protein (Figs. 3B, C, and D) extracts were subjected to western blotting analysis using the rabbit antibody to yeast expressed rAAS19 as described in materials and methods section 2.6. Figure 3A, rB = E. coli expressed rAA19, rY+ and rY− = deglycosylated and glycosylated, respectively, Pichia pastoris expressed rAAS19. Crude protein extracts of dissected salivary glands (SG), midgut (MG), synganglion (SYN), Malpighian tubules (MT), ovaries (OVR) and the remnants as carcass (CA) were subjected to western blotting analyses using antibodies to yeast expressed rAAS19 (Fig. 3B), control PBS and adjuvant injected antibody (Fig. 3C), and rabbit pre-immune serum (Fig. 3D). Lanes 1, 2, 3, and 4 = Protein extracts of unfed, 24, 72, and 120 h fed ticks respectively. L = molecular weight ladder, Asterisks marked (*) indicate potential glycosylated native AAS19 form.
Figure 4
Figure 4. Effect of repeated tick infestation on antibody levels in immunized rabbits
Affinity purified recombinant (r) AAS19 was subjected to routine ELISA as described in section 2.6. Data points are represented by (1) pre-immune (2) immune sera collected at two weeks after the booster, (3) two and (4) four weeks post-first tick infestation, and (5) two weeks after second tick infestation. Filled circle (●くろまる) = control rabbit inoculated with TiterMax Gold in PBS, filled triangle (▲さんかく) and square (■しかく) = immunized rabbits one and two.
Figure 5
Figure 5. Effect of rAAS19 immunization on completion of feeding
Ticks challenged on rAAS19 rabbits were observed through 3 weeks of feeding until detachment. (A) Detached ticks were recorded every 24 hours for 20 days for the first tick challenge infestation on control rabbit (broken lines) and rabbit (R)1 (solid lines) and R2 (dotted lines) that were immunized with rAAS19. (B) Detached ticks from second tick challenge infestation on control, R1, and R2 for 22 days. Each node represents the number of detached ticks. Data is reported as mean of number of detached ticks (M ± SEM).
Figure 6
Figure 6. Effect of rAAS19 immunization on blood meal size and egg laying
Ticks were allowed to feed on control and immunized rabbits (R)1 and 2 to repletion. Engorgement weights (EW) of detached ticks that fed on control and immunized rabbit groups were recorded from first (A) and second (B) infestations as indices for amounts of blood imbibed by ticks. Engorged ticks were incubated at 25°C at 85–90% relative humidity until oviposition. Eggs were collected and weighed from the first (C) and second (D) infestations. To determine the egg mass conversion ratio (EMCR), as a measure of the tick’s ability to utilize the blood meal to produce eggs, from first (E) and second (F) infestations, weights of egg masses were divided by engorgement mass. EW, egg weights and EMCRs were subjected to One-Way ANOVA and Tukey’s analysis to determine statistical significance. Data is reported as mean of EW, egg weights or EMCR (M ± SEM).

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