Potential of Nucleic Acid Receptor Ligands to Improve Vaccination Efficacy against the Filarial Nematode Litomosoides sigmodontis

Abstract

More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.

Keywords: 3pRNA; Litomosoides sigmodontis; filariae; helminth; nucleic acid receptor; poly(I:C); vaccination.

Figure 1

Enhanced local immune activation after injection of poly(I:C) or 3pRNA. (A–G) Mice were injected subcutaneously with attenuated (att.) L. sigmodontis L3 larvae with or without 3pRNA and poly(I:C) and the skin was analyzed four hours after injection. (B–G) Black circles indicate naïve animals, black squares animals receiving att. L. sigmodontis L3 larvae, black triangles animals receiving att. L. sigmodontis L3 larvae plus poly(I:C), black diamonds animals receiving att. L. sigmodontis L3 larvae plus 3pRNA. (A) Experimental setup. (B–E) Skin cells were analyzed by flow cytometry. Frequency of (B) neutrophils (CD45⁺CD11b⁺Ly6G⁺), (C) monocytes (CD45⁺CD11b⁺Ly6C⁺Ly6G⁻), (D) CD11b⁺ DCs (CD45⁺CD11b⁺Ly6C⁻Ly6G⁻CD11c⁺SiglecF⁻), and (E) eosinophils (CD45⁺CD11b⁺SiglecF⁺) among CD45⁺ cells. (F,G) Skin samples were analyzed by RT-PCR. The Δct values of (F) IFN−β expression and (G) IP−10/CXCL10 expression compared to β−actin levels in the corresponding sample. (B–G) Error bars show the median with IQR. Data were statistically analyzed by Kruskal–Wallis with Dunn’s post hoc test. (B–E) Data from one experiment, n = 8. (F,G) representative for three experiments with n = 5–6.

Figure 2

Immunization with adjuvants enhances functional parasite-specific antibody responses. (A–D) Mice were immunized three times in two−week intervals by subcutaneous injection of attenuated (att.) L. sigmodontis L3 larvae in combination with poly(I:C) or 3pRNA. (A) Experimental setup. (B–D) L. sigmodontis-specific (B) IgE, (C) IgG1, and (D) IgG2a/b serum antibody levels two weeks after the last injection and before challenge infection. Black circles indicate naïve animals, as well as animals that received either poly(I:C) or 3pRNA alone, black squares indicate animals receiving att. L. sigmodontis L3 larvae, black triangles animals receiving att. L. sigmodontis L3 larvae plus poly(I:C), black diamonds animals receiving att. L. sigmodontis L3 larvae plus 3pRNA. (E) Experimental setup of an ADCC assay that was performed using cocultures with a serum of immunized BALB/c mice as well as L. sigmodontis L3 larvae and naïve peritoneal exudate cells. The motility of individual larvae shown in (F) was assessed for three days by the following score: 4: fast and continuous movement, 3: slower but continuous movement, 2: slower and discontinuous movement, 1: sporadic movement limited to the ends, 0: no movement. (G–I) Immunized mice were naturally infected with L. sigmodontis two weeks after the last immunization injection. Serum was collected 37, 50, 57, and 63 days after infection and analyzed by ELISA for L. sigmodontis-specific (G) IgE, (H) IgG1, and (I) IgG2a/b antibodies. (B–D) Data shown as median with IQR. Statistical analysis using Kruskal–Wallis with Dunn’s post hoc test with n = 8 − 28. Data from untreated mice and groups receiving att. L3 larvae with or without agonist were pooled from 3 individual experiments. Data from groups that only received agonist are from one experiment. (F) Data presented as mean ± SEM and was statistically analyzed by a 2-way ANOVA with Bonferroni’s multiple comparison test. The ^$$$ p < 0.001, ^$$$$ p < 0.0001: comparison of L3 + att. L3 serum to L3 + ctrl. serum. * p < 0.05, **** p < 0.0001: comparison of groups including agonist treatment to L3 + att. L3 serum. Data from L3 + cells + FCS pooled from two independent experiments. Other data were pooled from three individual experiments. For all groups, n = 49–72 larvae. (G–I) Data from one experiment presented as mean ± SEM (n = 6–10) and was analyzed by a 2-way ANOVA with Tukey’s multiple-comparison test for differences at each time point.

Figure 3

Immunization with poly(I:C) or 3pRNA as an adjuvant significantly reduces the worm burden following challenge infection in mice. (A–M) Mice were immunized three times in two-week intervals by subcutaneous injection of attenuated (att.) L. sigmodontis L3 larvae in combination with poly(I:C) or 3pRNA. Two weeks after the last injection, the mice were naturally infected with L. sigmodontis for 63 days. (A–I) Black circles indicate naïve animals, black squares animals receiving att. L. sigmodontis L3 larvae, black triangles animals receiving att. L. sigmodontis L3 larvae plus poly(I:C), black diamonds animals receiving att. L. sigmodontis L3 larvae plus 3pRNA. (A) Quantification of adult worms and (B) total cell count in the pleural cavity. Quantification of (C) Eotaxin-1/CCL11, (D) RANTES/CCL5 and (E) IFN−γ in the pleura lavage by ELISA. Frequency of (F) Th1 (CD3⁺CD4⁺CD8⁻FOXP3⁻T−bet⁺), (G) Th2 (CD3⁺CD4⁺CD8⁻FOXP3⁻T−bet⁻GATA3⁺), (H) Th17 (CD3⁺CD4⁺CD8⁻FOXP3⁻T−bet⁻GATA3⁻RORγt⁺) and (I) Treg cells (CD3⁺CD4⁺CD8⁻FOXP3⁺) in the spleen quantified by flow cytometry analysis. (J) IL−5, (K) IP−10, (L) IFN−γ, (M) RANTES/CCL5 levels determined via ELISA of splenocytes that were restimulated with Concanavalin A (ConA, grey bars), L. sigmodontis adult-worm extract (LsAg, black bars) or left unstimulated (white bars). (A–I) Data shown as median with IQR. (J–M) Data shown as box and whiskers blot. (A–M) Data were statistically analyzed by Kruskal–Wallis with Dunn’s post hoc test. (A) Grey number indicates p value as assessed by direct comparison (Mann-Whitney U test). (C) Grey number indicates p value between 0.05 and 0.1. (A) Pooled data from two individual experiments. (B) Data from one experiment (C–M) and representative for two individual experiments with n = 6–20.