Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation

Abstract

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system.

Keywords: Ixodes ricinus; MIP; SIFamide; multiple antigenic peptides; vaccines.

Figure 1

Antibody (IgG1) response to SIFamide (SIFa; (A)) and Myoinhibitory (MIP; (B)) peptides in vaccinated mice and whole-mount immunohistochemistry on the salivary glands of an Ixodes ricinus unfed female (C–F). Antibody titers were determined by ELISA in serum samples collected at different time points from day 0 to day 90 against the specific peptide both in vaccinated mice (plain lines) and control mice (dashed lines) that received only adjuvant, and represented as arbitrary units (AU). Arrows indicate dates for 1st, 2nd, 3rd immunizations (days 0, 14 and 28) and tick infestations (Day 42). (C) and (E) show the reaction of anti-mouse antibody generated by MIP or SIFa multiple antigen peptide (MAP) constructs, respectively. Note that both antibodies recognized specific axon terminals (green, arrows) in acini type II and III of salivary glands. (D) and (F) are the negative controls where only pre-immune serum of mice subsequently immunized by MIP or SIFa was used, respectively. Note that no reaction in axon terminals in salivary gland acini was observed when pre-immune sera were used. Bar is 10 μm.

Figure 2

Antibody (IgG) response to SIFamide (SIFa) (A,B) and Myoinhibitory (MIP) (C,D) peptides in sheep vaccinated with SIFa (A), MIP (C), both SIFa and MIP (B,D) (plain lines), and in control injected with adjuvant (dashed lines). Antibody titers were determined by ELISA in serum samples collected at different time points from day 0 to day 73 against the specific peptide and are represented as arbitrary units (AU). Arrows indicate dates for 1st, 2nd, and 3rd immunizations (Days 0, 15, and 30) and tick infestations (Days 45, 46).

Figure 3

Correlation between antibody titers against SIFamide (SIFa) (A–C, G–I) and myoinhibitory (MIP) (D–F, J–L) neuropeptides in sheep vaccinated against SIFa (A–C), MIP (D–F), both SIFa and MIP (G–L), and I. ricinus larvae mortality, engorgement and molting at Day 45 after immunization. The linear correlation coefficients (R2) and p-values are shown (N = 6). Antibody titers are represented as arbitrary units.

Figure 4

Daily temperature (°C) recorded for groups of sheep vaccinated with SIFamide (SIFa), Myoinhibitory (MIP), and both SIFamide and Myoinhibitory (SIFa + MIP) peptides or injected with adjuvant (Control) from 45 to 54 days after immunisation. Animals were infested with Anaplasma phagocytophilum-infected Ixodes ricinus nymphs on day 46. Note that the dotted line representing the control group has a very large standard deviation due to a single sheep with a high fever of up to 42 °C on day 52. Results are presented as means +/− standard deviation.

Figure 5

Schematic depiction of multiple antigen peptides (MAPs) used in this study. Note that Ixodes ricinus mature neuropeptide B-cell epitops (SIFa or MIP) were fused to a T-helper cell epitop PADRE to construct 4-branched MAPs.