TRICHURIS MURIS WHEY ACIDIC PROTEIN INDUCES TYPE 2 PROTECTIVE IMMUNITY AGAINST WHIPWORM

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 near full-length 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 immunization 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. While both rTm-WAP49 or rTm-WAP-F8+Na-GST-1 have high purity and stability properties by analytical analysis, rTm-WAP49 has a more complex biophysical profile, with oligomerization to dimers and trimers through intermolecular disulfide bond formation. Recognition of rTm-WAP49 by endemic patient serum samples suggests that conserved epitopes may exist between T. muris and T. trichiura (or possibly non-Trichuris nematodes) derived WAPs. To our knowledge, this is the first study identifying a promising immunogen for further investigation of its vaccine potential against T. muris and eventually against T. trichiura

The Hygiene Hypothesis and Its Inconvenient Truths about Helminth Infections

Current iterations of the hygiene hypothesis suggest an adaptive role for helminth parasites in shaping the proper maturation of the immune system. However, aspects of this hypothesis are based on assumptions that may not fully account for realities about human helminth infections. Such realities include evidence of causal associations between helminth infections and asthma or inflammatory bowel disease as well as the fact that helminth infections remain widespread in the United States, especially among populations at greatest risk for inflammatory and autoimmune diseases

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

<div><p>Human whipworm (<i>Trichuris trichiura</i>) 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 <i>T</i>. <i>muris</i> infection model. The recombinant WAP protein (r<i>Tm</i>-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an <i>Na-</i>GST-1 fusion protein (r<i>Tm</i>-WAP-F8+<i>Na</i>-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 <i>T</i>. <i>muris</i> infective eggs following vaccination led to a significant reduction in worm burden of 48% by r<i>Tm</i>-WAP49 and 33% by r<i>Tm</i>-WAP-F8+<i>Na</i>-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 r<i>Tm</i>-WAP49 or r<i>Tm</i>-WAP-F8+<i>Na</i>-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 <i>T</i>. <i>muris</i> with WAP antisera identified the worm’s pathogenic stichosome organ as the site of secretion of native <i>Tm</i>-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from <i>T</i>. <i>muris</i> and <i>T</i>. <i>trichiura</i>, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.</p></div

Properties of recombinant As14 and As16 proteins expressed in <i>P</i>. <i>pastoris</i> X-33.

<p>(<b>A</b>) SDS-PAGE of purified rAs14 and rAs16 proteins (1 μg each). (<b>B</b>) Western blot probed with sera from mice infected with <i>A</i>. <i>suum</i> eggs (diluted 1:3,000) shows that only rAs16 was recognized, but not rAs14 (all antigens 50 ng). (<b>C</b>) Western blot with anti-rAs16 mouse sera (1:3,000). (<b>D</b>) Western blot with anti-rAs14 mouse sera (1:3,000) (all antigens 50 ng). (<b>E</b>) Sequence comparison between As16 and As14 proteins shows 47% sequence identity and 66% similarity.</p

Lung larva reduction (A) and stunted development (B) in mice immunized with rAs16 formulated with ISA720 on Day 8 after being challenged with 2,500 <i>A</i>. <i>suum</i> eggs.

<p>Lung larvae are presented as the mean ± S.D (N = 15). The asterisks indicate statistically significant differences in larval reduction compared to the PBS or adjuvant control groups (*<i>p</i>< 0.05, ***<i>p</i>< 0.001). The larvae collected from lung Baermann culture were observed under 4x objective lens.</p

Mouse immune responses to the immunization of rAs16 and rAs14.

<p><b>(A)</b> Anti-As16 and anti-As14 IgG1and IgG2a titers (Log10) in sera of BALB/c mice immunized with rAs16 and rAs14 formulated with ISA720, as measured by ELISA. The IgG1/IgG2a titer ratio for rAs16 immunization is higher than for the rAs14 immunization (2662:1 and 206:1, respectively). <b>(B)</b> Cytokine profiles (IFN-γ, IL-2, IL-4, IL-5 and IL-10) of BALB/c mice immunized with rAs16 and rAs14 formulated with ISA720. Cytokine levels were determined in supernatants of splenocytes after being re-stimulated with rAs16 or rAs14 (25 μg/ml) for 48 hours. Results are shown as means ± standard deviation (SD) and individual data points for each group (n = 5), **<i>p</i><0.01, NS, non-significant.</p

Protective immunity induced by immunization of mice with rAs16 formulated with different adjuvants (Alhydrogel, MPLA and Addavax).

<p>(<b>A</b>) Lung larval count on Day 8 after challenge with 2,500 <i>A</i>. <i>suum</i> eggs. Values are presented as the mean ± S.D. The asterisks indicate statistically significant differences (*<i>p</i> < 0.05) in lung larval reduction compared to the PBS and adjuvant control groups (n = 15). (<b>B</b>) Anti-As16 IgG1 and IgG2a titers (Log₁₀) in sera from BALB/c mice immunized with rAs16 formulated with different adjuvants as measured by ELISA. Values are shown as means ± S.D and individual data points (n = 15). (<b>C</b>) Cytokine profiles (IL-2, IL-4, IL-5 and IL-10, IL-12, GM-CSF, IFN-γ and TNF-α) of BALB/c mice immunized with rAs16 formulated with different adjuvants. Cytokines detected in supernatants of splenocytes after stimulation with rAs16 (25 μg/ml) for 48 hours. Data are presented as means ± S.D and individual values for each group (n = 5). *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001.</p

Native As16 and As14 expressed at the lung larval stage of <i>A</i>. <i>suum</i>.

<p>Western blots with anti-As16 or anti-As14 mouse serum (1:3,000) demonstrate that native As16 and As14 are expressed in L3 larvae collected from the lungs of infected mice, but not in infective eggs of <i>A</i>. <i>suum</i>. The unrelated <i>T</i>. <i>cruzi</i> Tc24 protein (50 ng) was used as a negative control.</p