Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display

Abstract

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

Figure 1. Enrichment and selection of yeast cells expressing immunogenic I. scapularis salivary proteins.

FACS analysis of yeast cells using nymph-immune rabbit IgG (red) and IgG derived from normal rabbit serum (blue) of transformed yeast cells (sort 0); autoMACS sort (AutoMACS); MidiMACS sort 1, 2 and 3 (MidiMACS1, 2 and 3). (B) Selection for IgG binding clones using 50 µg/ml Alexa-488 conjugated nymph immune rabbit IgG. Sort 0 was used as a negative control and MACS sort 4 as a positive control. Upper panel: mouse anti-Xpress antibody binding to induced yeast cells (PE). Lower panel: Cells binding nymph immune rabbit IgG (IR IgG) shown within the pink gate (Alexa-488). (C) Titration of binding of each unique using 50 µg/ml (green bars), 10 µg/ml (red bars) or 1 µg/ml (blue bars) of immune rabbit IgG. The percentage IgG binding yeast cells were determined by FACS analysis as was shown in the right lower panel of Fig. 1B.

Figure 2. Purified recombinant Ixodes scapularis salivary proteins.

(A) Coomassie blue staining of purified recombinant I. scapularis salivary proteins rP8, rP19 and rP23 electrophoresed on SDS 12% polyacrylamide gel. (B) Western blot analysis of the recombinant proteins probed with nymph-immune rabbit serum (IRS) and normal rabbit serum (NRS). (C) PAS staining of rP8, rP19, rP23 and Salp15 electrophoresed on SDS 12% polyacrylamide gel.

Figure 3. Expression of the genes coding for the four salivary proteins during several life stages of I. scapularis.

Quantitative reverse-transcription polymerase chain reaction performed on RNA isolated from whole larvae, and from salivary glands and midguts I. scapularis from nymphs and adults. Expression profile of: A. p8; B. p19; C. p23; and D. p32 in unfed (grey bars) and fed ticks (black bars). SG; salivary glands, MG; midguts.

Figure 4. Influence of recombinant salivary proteins on human complement system.

Serum sensitive strain Borrelia garinii A87S was incubated with 12.5% NHS in the presence of BSA, Salp15, rP19, rP23 or rP8 for (A) 1.5 hours; or (B) 4.5 hours and the percentage of immotile spirochetes were determined. Control spirochetes were incubated with heat-inactivated NHS (HI). Two hundred spirochetes were counted. Results represent mean ± SEM of values from a representative of 3 replicate experiments.

Figure 5. Influence of recombinant salivary proteins on human coagulation system.

(A) Thrombin generation was initiated in human pooled normal plasma with 1 pM tissue factor (TF) in the presence of rP8 (orange), rP19 (green) or rP23 (red) and thrombin generation was measured using a fluorogenic substrate. (B) Lagtime, (C) time to peak (ttpeak) and (D) Endogenous Thrombin Potential (ETP) were measured. Unpaired t-test was used to determine statistical significance. Representatives of three experiments are shown. Results described represent the mean ± SEM.

Figure 6. Nymph feeding after rabbit immunization with recombinant salivary proteins.

(A) Nymph salivary gland extract probed with rP8/rP19/rP23 immune rabbit serum (panel 1), rP8 or rP19 or rP23 immune rabbit serum (panel 2), and with serum from the control (OVA) rabbit. (B) Nymph weights recovered from the control and the rP8/rP19/rP23 immunized rabbits. Tick weights of the heavy group of nymphs (C) and light group of nymphs (D) fed on rP8/rP19/rP23 immunized rabbits compared to the control rabbits. Weights of the heavy group of nymphs (E) and light group (F) of nymphs fed on rabbits immunized singly with OVA, rP19, rP8 or rP23 respectively. The horizontal bars represent the means of the respective groups. Unpaired t-test was used to determine statistical significance.