Trichuris muris whey acidic protein induces type 2 protective immunity against whipworm

Abstract

Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm's pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.

Fig 1. Amino acid sequences and phylogenetic analyses of the WAP and CAP-1 proteins from T. muris.

The predicted amino acid sequences of (A) Tm-WAP and (C) Tm-CAP-1. The predicted signal peptide of each protein is highlighted in red (SignalP 4.1). The Tm-WAP protein contains ten repeats of a whey acidic protein (WAP)-type four-disulfide core domain that were aligned using CLUSTAL W and prepared for display using BOXSHADE. Identical amino acids are shaded in black and similar amino acids in gray. This 50 amino acid domain contains 6 conserved cysteine residues. The 49 kDa N-terminus sequence was expressed in yeast as rTm-WAP49 (in red box). The repeat fragment #8 was expressed in E. coli as Tm-WAP-F8 (in yellow box) using an Na-GST-1 expression tag. Phylogenetic tree depicting the sequence identify of (B) Tm-WAP and corresponding gene TMUE_s0165000300 and (D) rTm-CAP-1 and corresponding gene TMUE (s0030008500), to their respective homologues in different Trichuris species.

Fig 2. Immunogenicity of rTm-WAP-F8+Na-GST-1 and rTm-CAP-1 proteins in a murine model.

(A) Vaccination schedule to evaluate immunogenicity in male AKR/J mice. Humoral immune response was measured by end-point serum titer for antigen-specific (B) IgG1 and (C) IgG2a by ELISA to calculate the (D) IgG1 to IgG2a ratio for the mice in each group. Mice in groups injected with PBS or Montanide ISA 720 adjuvant were used as negative controls for each coating antigen. Statistical significance: *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. (E) Cellular immune responses in terms of cytokines in the supernatants of splenocytes stimulated with media or cognate antigens, rTm-WAP-F8+Na-GST-1 (10 μg/mL), rTm-CAP-1 (10 μg/mL), or Tm-ES (50 μg/mL), for 72 hours. Values for cells cultured in media only were subtracted as background from those of cells stimulated with cognate antigens in the corresponding groups of mice. Values from duplicate wells for each treatment/stimulation were averaged for individual cytokines for the 5 mice in each group to calculate statistical significance. Individual cytokines and statistics are shown in S1 Fig.

Fig 3. Efficacy and anamnestic responses in mice immunized with rTm-WAP-F8+Na-GST-1 and rTm-CAP-1 proteins.

(A) Vaccine schedule to measure efficacy and post-challenge anamnestic response in male AKR/J mice. (B) Vaccine efficacy was evaluated on day 15 post-infection by counting the numbers of worms in the intestines by microscopy. At time of sacrifice, anamnestic humoral response was measured by end-point serum titer for antigen-specific (C) IgG1 and (D) IgG2a by ELISA to calculate the (E) IgG1 to IgG2a ratio for the mice in each group. Mice in groups injected with Montanide ISA 720 were used as negative controls for each coating antigen. Statistical significance: *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. (F) Cellular immune responses in terms of cytokines in the supernatants of splenocytes stimulated with media or cognate antigen, rTm-WAP-F8+Na-GST-1 (10 μg/mL), rTm-CAP-1 (10 μg/mL), or Tm-ES (50 μg/mL), for 72 hours. Values from duplicate wells for each treatment/stimulation were averaged for individual cytokines for the 13–15 mice in each group to calculate statistical significance. Individual cytokines and statistics are shown in S2 Fig.

Fig 4. Immunogenicity of rTm-WAP-F8+Na-GST-1 and rTm-WAP49 proteins in a murine model.

Humoral immune response was measured by end-point serum titer for antigen-specific (A) IgG1and (B) IgG2a by ELISA to calculate the (C) IgG1 to IgG2a ratio for the mice in each group. Mice in groups injected Montanide ISA 720 adjuvant were used as negative controls for each coating antigen. Values from duplicate wells for each treatment/stimulation were averaged for individual cytokines for the 15 mice in each group to calculate statistical significance. Statistical significance: *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001.

Fig 5. Efficacy and anamnestic responses in mice immunized with rTm-WAP49 or rTm-WAP-F8+Na-GST-1.

(A) Vaccine efficacy was evaluated on day 15 post-infection by counting of worms in the intestines by microscopy and compared to Montanide ISA 720 control. At the time of sacrifice, anamnestic humoral immune response in terms of end-point serum titers for antigen-specific (B) IgG1 and (C) IgG2a was measured by ELISA and shown as an (D) IgG1 to IgG2a ratio. (E) Splenocytes are shown as individual values for each group (n = 15), and (F) MLNs and (G) ILNs are separately pooled as three groups of five mice (n = 3). Mice in groups injected with Montanide ISA 720 were used as negative controls for each stimulant. Cells receiving no stimulus (media only) were subtracted as background from stimulated cells from corresponding mice. Statistical significance: *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. Individual cytokines and statistics are shown in S3 Fig.

Fig 6. Cross-recognition of native and recombinant Tm-WAP and Tm-CAP-1 proteins.

(A) Serum samples from mice immunized with rTm-WAP49, rTm-WAP-F8+Na-GST-1, and rTm-CAP-1, and Tm-ES were tested for their IgG antibody recognition of Tm-ES by ELISA endpoint titers. Statistical significance: *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. (B) Western blot analyses for the serological recognition and cross-recognition of rTm-WAP49, rTm-WAP-F8+Na-GST-1, and Tm-ES. In each panel, the lane numbers on the top represent: (1) SeeBlue pre-stained protein marker; (2) T. muris adult homogenate; (3) T. muris ES; (4) Recombinant Tm-WAP49 expressed in P. pastoris; (5) Recombinant Tm-WAP-F8+Na-GST-1 fusion protein expressed in E. coli; (6) Recombinant Na-GST-1 expressed in P. pastoris. The SDS-PAGE was performed using 4–20% NuPAGE/MES gel. WB was performed using different mouse anti-sera (1:3,000) as shown under each panel. For WB with anti-Tm-ES, the loaded amount of protein was adjusted for lane 2 to 6 as 2.0 μg, 500 ng, 1.0 μg, 400 ng and 400 ng. The nitrocellulose membranes of anti-rTm-WAP-F8+Na-GST-1 and anti-Tm-ES were cut just prior to ECL substrate reaction between lanes 4–5 and 3–4, respectively, to allow for different length of exposure time to detect the signal.

Fig 7. Immunofluorescent staining of Tm-WAP proteins expressed in adult Trichuris muris stichosome.

(A) Cross-sections of murine cecum embedded with multiple adjacent adult T. muris at 20x magnification. The orange box was imaged at 40x to the right. Embedded T. muris revealed Tm-WAP recognition in a ring pattern adjacent to the cuticle of the helminth (orange arrow) with presumptive Tm-WAP secretions into the caecal lumen (white arrow). (B) Sections of embedded T. muris showing Tm-WAP recognition in a distinct granular pattern within a discernable internal structure (red arrow). Primary staining was performed with PBS, naïve sera (1:100), or anti-rTm-WAP49 (1:100). Secondary staining was with FITC-conjugated goat anti-mouse IgG (1:500). DAPI was embedded in the mounting medium. SCID mice were infected with T. muris for 42 days.