Tick feeding or vaccination with tick antigens elicits immunity to the Ixodes scapularis exoproteome in guinea pigs and humans

Abstract

Ixodes scapularis is a primary vector of tick-borne pathogens in North America. Repeated exposure to these ticks can induce a humoral response to tick antigens and acquired tick resistance. However, identifying antigens contributing to this resistance is challenging because of the vast number of I. scapularis proteins secreted during feeding. To address this, we developed I. scapularis rapid extracellular antigen monitoring (IscREAM), a technique to detect antibody responses to more than 3000 tick antigens. We validated IscREAM with immunoglobulin G (IgG) from guinea pigs vaccinated with tick antigens, including a cement antigen cocktail that induced tick resistance. Furthermore, we explored the natural response to tick bites by profiling antigens recognized by IgG isolated from a tick-resistant individual, as well as from others with Lyme disease and tick-bitten guinea pigs and mice, to identify 199 recognized antigens. We observed that several antigens contained histamine-binding domains. This work enhances our understanding of the host immune response to I. scapularis and defines immunogen candidates for future antitick vaccines.

Fig. 1.. IscREAM performed at least as well as ELISA in validations with serum samples from vaccinated guinea pigs.

IgG isolated from luciferase (yellow) or 19ISP-vaccinated (blue) guinea pigs (n = 20 per group) was screened against the IscREAM library. (A) Heatmap illustrating antigen binding. Each row represents an individual antigen, and each column is the composite of one sample run in duplicate. A technical negative control of no IgG is presented as (−). Cells are colored by IscREAM score according to the legend to the right. Only cells with score ≥1.5 are colored. Antigens are sorted by homology to 19ISP antigens and are listed in data file S2 with scores. (B) Comparisons of reactivity of 19ISP-vaccinated guinea pigs to 19ISP antigens in IscREAM and in published ELISA results. SG09 and SG10 are closely related, so the published ELISA results grouped these two antigens together (23). N/A, not applicable. (C to E) Shown is the reactivity of sera from 19ISP-vaccinated and luciferase-vaccinated guinea pigs to 19ISP antigens or homologs (C), Salp14/ TSLPI homologs (D), and antigens that are not 19ISP homologs (E). The reactivity of a sample is represented by the number of hits per sample, and bars represent the means ± standard deviation. Data in (C) to (E) were analyzed by the Mann-Whitney U test; ***P < 0.001 and ****P < 0.0001.

Fig. 2.. An mRNA-LNP vaccine cocktail of 25 I. scapularis cement antigens induced acquired tick resistance in guinea pigs and elicited an IgG response detectable by IscREAM.

(A and B) Erythema was scored for I. scapularis nymph bites on the backs of Cement-25 (green; n = 3 guinea pigs)– or luciferase (Luc, gray; n = 2 guinea pigs)–vaccinated guinea pigs at 24 (n = 57 ticks on Luc guinea pigs and 82 ticks on Cement-25 guinea pigs) (A) or 48 (n = 57 ticks on Luc guinea pigs and 78 ticks on Cement-25 guinea pigs) (B) hours post–tick attachment. Data were analyzed by the linear mixed model; 48 hours: β = 1.0060, SE = 0.1904, P = 0.0126. (C) The percentage of tick attachment was recorded every 24 hours; data were analyzed by the logistic mixed model; 96 hours: β = −2.8381, SE = 0.6725, P < 0.0001; 120 hours: β = −2.7318, SE = 0.6491, P < 0.0001. (D) The weight of ticks fed on Cement-25– and luciferase-vaccinated guinea pigs was measured the day they dropped off the guinea pigs (n = 51 Luc and 68 Cement-25 ticks recovered); data were analyzed by the linear mixed model; β = −0.7945, SE = 0.3836, P = 0.1352. (E) A heatmap illustrates antigen binding. Each row represents an I. scapularis protein sorted by reactivity and Cement-25 relation, and each column represents IgG of one guinea pig (n = 2 Luc and 3 Cement-25 guinea pigs). Cells are colored by IscREAM score. Data in (A) to (D) are presented as the means ± SD. *P < 0.05 and ****P < 0.0001.

Fig. 3.. IscREAM identified antigens recognized by IgG of individuals with active Lyme disease.

IgG isolated from healthy blood donors (“HBD”; gray; n = 9) or individuals with active Lyme disease (“Lyme”; orange; n = 52) was screened. (A) A heatmap is shown. Each row represents one antigen sorted by reactivity and predicted antigen source, and each column represents one donor sorted by reactivity. Cells are colored by IscREAM score according to the legend. Antigens and scores are in data file S4. (B) Antigen recognition was measured in samples from individuals with active Lyme disease and HBDs; data were analyzed by the Mann-Whitney U test. (C) ELISAs to validate IscREAM reactivity were run against three antigens (“P32,”“XP_042150701.1,” and “XP_040072495.1”) using IgG isolated from four unreactive HBDs (1:50 dilution; gray) or four individuals with active Lyme disease (1:50 dilution: blue; 1:500 dilution; green). BSA (bovine serum albumin) was probed as a negative control. Data were analyzed by the Mann-Whitney U test. OD₄₅₀, optical density at 450 nm. (D) Reactivity to antigens known to be produced in tick tissues exposed to a tick-bitten person (“Exposed”: “Saliva,”“Cement,” or “Cuticle”) or other antigens (“Midgut,”“Synganglion,” and “Unmapped”) was measured in IgG from individuals with active Lyme disease; data were analyzed by Fisher’s exact test. Data in (B) and (C) are shown as the means ± standard deviation. *P < 0.05 and **P < 0.01.

Fig. 4.. Antigens associated with confirmed tick resistance were profiled by IscREAM.

(A) Serum (1.5 ml) isolated from the resistant individual (“Resistant”; red) or a naïve individual (“Naïve”; gray) was transferred to two guinea pigs each, and tick attachment was recorded every 24 hours. Data were analyzed by the logistic mixed model; 72 hours: β = −1.4113, SE = 0.6079, P = 0.0202; 96 hours: β = −1.9649, SE = 0.6976, P = 0.0049; 120 hours: β = −1.4219, SE = 0.6821, P = 0.0371. Shown are the means ± standard deviation. *P < 0.05 and **P < 0.01. (B) Ticks were weighed after detachment from guinea pigs from (A) (n = 42 naïve and 47 resistant ticks recovered). Data were analyzed by the linear mixed model; β = −1.8168, SE = 0.4889, P = 0.0695. Bars represent the means ± standard deviation. (C) IscREAM was run with IgG isolated from the human tick-resistant and naïve individuals, six tick-resistant or naïve guinea pigs, or five repeatedly tick-exposed or naïve mice. Recognized antigens and antigen sources are listed to the left, and cells are colored according to the legend to the right. I. scapularis antigens containing histamine-binding domains are highlighted in green. Antigens and scores are in data file S5. The attachment and weights of ticks fed on each guinea pig are shown in fig. S3.