Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection

Abstract

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

Fig 1. HES vaccination elicits sterile immunity to challenge and blocks parasite maturation.

A. Schematic protocol for immunization. B. Parasite recoveries d 28 post-challenge following vaccination of C57BL/6 females with HES or somatic extract (HEx). Data shown are combined from two experiments each of 4–5 mice per group. Significance determined by ANOVA. C. Faecal egg counts (d 14, 21, 28 post-challenge) from (B). Significance determined by ANOVA. D. Parasite recoveries at d 7 (small intestinal wall) and 14 post-challenge (gut lumen). Representative of two independent experiments. Significance determined by t-test. E. H&E sections of duodenum in control and vaccinated animals at d9 post challenge; parasites in control animals are observed in gut lumen, but remain trapped in submucosa of vaccinated mice. Black circles indicate positions of parasite. Scale bar represents 100 μm.

Fig 2. HES vaccination promotes extensive myeloid cell extravasation and accumulation around site of larval invasion.

A. H&E staining of duodenal sections from naïve and d 7 post-challenge PBS control or HES-immunized C57BL/6 mice. Arrows indicate position of parasites. B. Immunohistology showing recruitment of CD11b⁺ (green) and Gr1⁺ (red) myeloid cells. Representative of 3–4 mice per group. Scale bar in A-B represents 100 μm. C. Rolling velocities of LysM-GFP⁺ cells along vessels imaged by two-photon microscopy of duodenum from the serosal aspect. Data accumulated from two mice per group, 3–7 vessels per mouse. Vessels were selected on basis of showing rolling behaviour. Significance determined by Kruskal-Wallis test. D. Tissue infiltration of LysM-GFP⁺ cells surrounding vessels in (C). Significance determined by t-test. E, F. Examples of two-photon microscopy from (D) showing tissue-infiltrating LysM-GFP⁺ cells in PBS- and HES-vaccinated mice. Scale bar represents 50μm. See also Supplemental Movie S1.

Fig 3. Immunity is dependent on cognate B cells and partially transferrable by antibody.

A. Gated CD19⁺ MLN cell expression of cell surface IgM and IgG1 in naïve and d 7 and d 14 post-challenge PBS and HES B6 mice. B. Gated CD19⁺ MLN cell expression of cell surface CD21/CD35 (CR2/CR1) and CD23 (FcεRII) in naïve and d 7 post-challenge PBS and HES B6 mice. Numbers represent geoMFI of CD23 (blue) and CD21/CD35 (red) expression. A-B representative of two independent experiments with 3–5 mice per group. C. Pre-challenge anti-HES IgM, IgG1, IgG2c, IgA and IgE titers in PBS and HES vaccinated B6 mice with 5 mice per group. D. Day 28 post-challenge worm burdens from C57BL/6 and μMT mice following HES immunization or PBS control. Data pooled from two experiments. Significance determined by ANOVA as indicated. E, F. Day 28 post-challenge worm and fecal egg burdens (d 14, 21, 28) in naïve C57BL/6 mice receiving IgG from vaccinated or primary infected mice, or PBS, as detailed in materials and methods. Data are pooled from two experiments, with significance determined by ANOVA Vs C57BL/6 PBS. G. Day 21 post-challenge adult worm burdens in C57BL/6 and FcRγ^–/–x C3^–/– mice following HES immunization or PBS control. Significance determined by t-test as indicated. See also S2 Fig.

Fig 4. Vaccine-induced immunity requires IL-4R-mediated signaling but not eosinophils, mast cells, basophils or CCR2+ monocytes.

A-F. Day 21–28 worm counts in control and vaccinated (A) C57BL/6 and IL-4Rα^–/– mice, (B-C) BALB/c and GATA-1 Δdbl mice, (D) ACK-2-treated and control C57BL/6 mice, (E) C57BL/6 4get/Mcpt8 cre and 4get controls and (F) CCR2^–/– and C57BL/6 controls. n.b. PBS/alum immunized CCR2^–/– mice died or were culled between 7–9 days post-challenge, with significant intestinal bleeding observed. Luminal worms in (A, B, D, E, and F), whereas counts in (C) represent live worms recovered from the intestinal wall at day 21 (i.e. presumed non-migratory parasites). Data in (A, B, C, E and F) pooled from 2 independent experiments. Significance in (A), (C) and (D) determined by ANOVA, in (B) by Kruskal-Wallis test, and (E-F) by unpaired t-test. See also S3 Fig.

Fig 5. Vaccine-induced immunity requires IL-25.

A. Anti-HES pre-challenge IgG1 titres in control and vaccinated C57BL/6 and IL-17RA-deficient mice. B, C. Day 28 worm counts and fecal egg burdens (d 14, 21 and 28) in control and vaccinated C57BL/6 and IL-17RA-deficient mice. D. Intracellular IL-4 production by naïve and d 7 post-challenge CD45⁺CD4⁺ lamina propria cells from C57BL/6 and IL-17RA^–/– mice. Pooled from two experiments. E. Proportion of lamina propria CD45⁺CD11b⁺Ly6G⁺ in naïve, d 3 and d 7 challenged C57BL/6 and IL-17RA^–/– mice. F. Adult worm burdens (d 16) in control and vaccinated C57BL/6 mice treated with anti-Ly6G (clone 1A8; left) or anti-Gr-1 (clone RB6-3C5; right) as detailed in materials and methods. G. Adult worm burdens (d 28) in primary infected BALB/c mice treated with anti-Ly6G, anti-Gr-1, or rat IgG control, as in (F). H. Day 28 worm counts in control and vaccinated C57BL/6 and IL-25-deficient mice. Significance in (A-E, G-H) determined by unpaired t-test or Mann-Whitney test, significance in (F) determined by ANOVA Vs PBS control. Data from A-G pooled from 2 independent experiments. See also S4 Fig.

Fig 6. Identification of protective antigens following HES immunization.

A-B. Day 28 post-challenge worm and fecal egg burdens (d 14, 21, 28) in C57BL/6 mice immunized with native or heat-treated HES. Data pooled from two experiments. Significance determined by ANOVA as indicated. C. Protective antibody targets revealed by immunoprecipitation of biotin-labeled HES by serum antibodies from immunized mice. Immunoprecipitated proteins were separated by pI (range 3–10) and molecular weights (indicated in kDa) and visualized with streptavidin HRP. Blue, red, green and brown circles represent VAL-1, 2, 3 and 4, respectively. Unknown antigens circled black. Sera pooled from 5 HES-vaccinated C57BL/6 mice pre-challenge and representative of two independent experiments. D. Day 28 post-challenge worm burdens from C57BL/6 mice immunized with a combination of VAL-1,-2 and -3, or with HES depleted of these 3 antigens. Data pooled from two experiments. Significance determined by ANOVA Vs PBS/alum. E-F. Immunoprecipitation of biotin-labeled HES antigens with vaccination sera as (C) from mice immunized with VAL-1/2/3 (E), or VAL-1/2/3 depleted HES (F). G. LC-MS/MS identification of antibody targets in mice immunized with VAL-1/2/3-depleted HES, performed on samples of unlabeled HES immunoprecipitated with serum antibodies from these mice. Proteins ranked according to spectral count (“count”). “Unique” represents number of unique peptide sequences in identification, “score” is Mascot score, and emPAI estimated abundance also shown. Highest scoring BLAST homolog with expect values indicated. H. Day 28 post-challenge worm burdens from C57BL/6 mice immunized with HES or HES depleted of VAL-1, -2, -3 and -4. See also S5 Fig.