Type 2 immune-inducing helminth vaccination maintains protective efficacy in the setting of repeated parasite exposures

Abstract

Animal studies have demonstrated that helminth vaccines which induce type 2 immune responses can be protective. To date, however, such vaccines have not been tested against repeated parasite challenges. Since repeated antigenic challenge of patients with allergic disease results in immunologic tolerance, we hypothesized that a helminth vaccine which induces type 2 immune responses may lose its protective efficacy in the setting of repeated parasite exposures (RPEs). To test this hypothesis, we examined whether RPEs induce immunological tolerance and reduce the effectiveness of a type 2 immune-inducing vaccine. BALB/c mice vaccinated against Litomosoides sigmodontis, a filarial nematode of rodents, were repeatedly exposed to irradiated larvae for 2 or 8 weeks or to non-irradiated infectious larvae for three months. Vaccination-induced parasite-specific IgE levels, parasite antigen-driven basophil interleukin 4 (IL-4) release, and Th2 skewing of the cellular immune response remained stable in the face of RPEs. Furthermore, RPEs in vaccinated mice did not augment immunoregulatory responses, as parasite antigen-driven cellular proliferation, production of IL-10, and frequencies of CD4(+)CD25(+)FoxP3(+) regulatory T-cells were not altered by RPEs. Challenge infections with infectious L3-stage larvae resulted in lower worm burdens in vaccinated mice given RPEs than in vaccinated controls. These results demonstrate that vaccines which induce type 2 immune responses can maintain their efficacy in the setting of repeated parasite exposures.

Fig. 1

Comparisons of mice that were vaccinated against L. sigmodontis and non-vaccinated controls. (A) Plasma levels of L. sigmodontis-specific IgE (OD). (B) Percentages of basophils that stain positively for IL-4 by multicolor flow cytometry when cultured with media alone or with parasite antigen (LsAg). Shown are mice four or ten weeks after vaccination and the corresponding controls. (C) Splenic IL-4 and (D) IFNγ cytokine production in response to parasite antigen and (E) IL-4/IFNγ ratio in response to anti-CD3/anti-CD28 stimulation. (F) Worm recovery 8 weeks after challenge with 40 infectious L. sigmodontis larvae. Significant differences between unpaired groups were analyzed by Mann-Whitney-U-test and between paired groups by Wilcoxon matched pairs test (*p<0.05). Horizontal lines represent median values obtained from all experiments.

Fig. 2

(A) L. sigmodontis-specific plasma IgE levels (OD) from mice that were vaccinated against L. sigmodontis and/or given repeated parasite exposures (RPE) with irradiated larvae for two (○) or eight weeks (●). OD levels from unvaccinated, media-exposed controls were set to zero. (B) Percentages of IL-4 positive basophils in response to parasite antigen from mice that were vaccinated and/or repeatedly parasite exposed with irradiate larvae for 2 or 8 weeks, as determined by multicolor flow cytometry. Significant differences between groups were analyzed by the Kruskal-Wallis test, followed by Dunn’s post-hoc multiple comparisons (*p<0.05). Timepoints listed are duration of RPEs. As RPEs started 2 weeks after final vaccination, mice in the 2 week group were tested 4 weeks after final vaccination and those in the 8 week group tested 10 weeks after final vaccination. Shown are medians from two independent experiments (2 week timepoint: RPE n=5, Vaccination n=8, Vaccination + RPE n=8; 8 week timepoint: RPE n=4, Vaccination n=7, Vaccination + RPE n=8).

Fig. 3

Proliferation of murine spleen and lymph node cells to L. sigmodontis antigen (LsAg) and anti-CD3/anti-CD28. Shown is the stimulation index (OD of stimulated cells/baseline)*100. Mice were vaccinated with irradiated L. sigmodontis L3s and/or given repeated parasite exposures (RPE) with irradiated larvae for two (open bars) or eight weeks (grey bars). Control groups were vaccinated with media and then given repeated media injections. Significant differences between groups were analyzed by the Kruskal-Wallis test, followed by Dunn’s post-hoc multiple comparisons (*p<0.05). Timepoints listed are duration of RPEs. Shown are results from two independent experiments (2 week timepoint: RPE n=5, Vaccination n=8, Vaccination + RPE n=8; 8 week timepoint: RPE n=4, Vaccination n=7, Vaccination + RPE n=8).

Fig. 4

IL-4, IL-5, and IFNγ cytokine production from mice given L. sigmodontis vaccination, RPEs with irradiated larvae for two (open bars) or eight weeks (grey bars), both, or sham-vaccination and sham-exposures (Controls). (A) Supernatant measurements of splenocyte production of IL-4, IL-5, and IFNγ in response to parasite antigen (LsAg) or (B) anti-CD3/anti-CD28. (C) Percentages of CD4⁺IL-4⁺ and CD4⁺IFNγ⁺ spleen cells in response to anti-CD3/anti-CD28. Statistical significant differences between groups were analyzed by the Kruskal-Wallis test, followed by Dunn’s post-hoc multiple comparisons. Only statistical significant differences between vaccinated and vaccinated + RPE mice are shown (*p<0.05). Timepoints listed are duration of RPEs. Shown are results from two independent experiments (2 week timepoint: RPE n=5, Vaccination n=8, Vaccination + RPE n=8; 8 week timepoint: RPE n=4, Vaccination n=7, Vaccination + RPE n=8).

Fig. 5

(A) Gating strategy for identification of CD4⁺CD25⁺FoxP3⁺ regulatory T-cells by flow cytometry. Lymphocytes were gated by forward (FSC) and sidescatter (SSC) characteristics (left panel). CD4 PerCP positive lymphocytes were then gated (middle panel) and analyzed for CD25 APC-AF750 and FoxP3 FITC positivity (right panel). (B) Percentages of splenic CD4⁺ cells that are CD4⁺CD25⁺FoxP3⁺ after in vitro stimulation of splenocytes with parasite antigen (LsAg) or anti-CD3/anti-CD28. (C) IL-10 cytokine release from spleen cell culture supernatants in response to LsAg or anti-CD3/anti-CD28. (D) Percentages of splenic CD4⁺ cells that are CD4⁺IL-10⁺ in response to anti-CD3/anti-CD28. Shown are cells from mice that were vaccinated with L. sigmodontis L3s (Vaccination) and/or given repeated parasite exposures (RPE) with irradiated larvae for two (open bars) or eight weeks (grey bars). Control groups were vaccinated with media and then given repeated media injections. Significant differences between groups were analyzed by the Kruskal-Wallis test, followed by Dunn’s post-hoc multiple comparisons (*p<0.05). Timepoints listed are duration of RPEs. Shown are results from two independent experiments (2 week timepoint: RPE n=5, Vaccination n=8, Vaccination + RPE n=8; 8 week timepoint: RPE n=4, Vaccination n=7, Vaccination + RPE n=8).

Fig. 6

(A) Worm recovery 56 days after challenge infection of mice with 40 infectious L. sigmodontis larvae. Mice were vaccinated against L. sigmodontis and/or given repeated parasite exposures (RPE) with irradiated larvae for two (○) or eight weeks (●) or were given sham vaccination and sham exposures (Control). Significant differences between groups were analyzed by the Kruskal-Wallis test, followed by Dunn’s post-hoc multiple comparisons, single stars show significant differences compared to the Control group (*p<0.05). (B) Worm recovery 20 days after challenge infection with 40 infectious L3 larvae. Mice were vaccinated against L. sigmodontis and given RPEs with infectious L3 larvae or RPMI as control for three months. Significant differences were analyzed by Mann-Whitney-U-test (*p<0.05).

Fig. 7

Immunological studies from BALB/c mice that were vaccinated against parasite antigen (LsAg) and repeatedly injected with LsAg or PBS for two (○) or eight weeks (●). (A) LsAg-specific and total IgE levels after 2 and 8 weeks of repeated LsAg/PBS injections. (B) Frequency of IL-4 positive basophils in response to LsAg after 2 or 8 weeks of repeated injections. (C) Splenic IL-4, IL-5, and IFNγ production in response to LsAg and (D) anti-CD3/anti-CD28 after eight weeks of repeated exposures to LsAg or PBS. (E) Spleen cell proliferation in response to LsAg or anti-CD3/anti-CD28 after eight weeks of repeated injections. (F) Spontaneous frequency of CD4⁺CD25⁺FoxP3⁺ splenic T cells after eight weeks of repeated exposures. (G) Splenic IL-10 production in response to LsAg and anti-CD3/anti-CD28 from eight week repeatedly injected mice. Timepoints listed are duration of RPEs. Shown are medians. Significant differences were analyzed by Mann-Whitney-U-test (*p<0.05, **p<0.01, ***p<0.001).