Immunomics-Guided Antigen Discovery for Praziquantel-Induced Vaccination in Urogenital Human Schistosomiasis

Abstract

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P<0.0001) and intestinal egg burdens (50-54%, P<0.0003) were achieved in mice vaccinated with cystatin in two independent trials. This study has revealed numerous antigens that are targets of DIV antibodies in urogenital schistosomiasis and offer promise as subunit vaccine targets for a drug-linked vaccination approach to controlling schistosomiasis.

Keywords: cystatin; immunomics; praziquantel; proteome microarray; urogenital schistosomiasis; vaccine.

Figure 1

Quality control probe of the S. haematobium protein microarray. Arrays were probed with (A) anti-hemagglutinin (HA) antibody to detect expression of the N-terminal HA tag and determine the percentage of protein expression from all arrayed antigens, and (B) anti-6-HIS antibody to detect expression of the C-terminal 6-HIS tag and determine the percentage of full-length protein expression from all arrayed antigens. No DNA refers to control spots where plasmid DNA (encoding genes to be expressed) is omitted from the reverse transcription-translational products printed at those locations. Sh Adult ES, S. haematobium adult fluke Excretory/Secretory products; Sh Adult PBS extract, S. haematobium adult fluke PBS-soluble somatic extract; Sh egg ES, S. haematobium egg ES products; Sh Adult triton extract, S. haematobium adult fluke TritonX-114-soluble extract; Sh SEA, S. haematobium soluble egg antigen.

Figure 2

Approach to identification of antigenic targets of DIV. Antigens which were targets of IgG responses that were significantly elevated 18 months after PZQ treatment compared to before treatment in DIV subjects (1, upper left panel), and significantly elevated in DIV subjects compared to CI subjects 18 months after PZQ treatment (2, upper right panel) were considered potential targets of DIV. The Venn diagram shows nine antigens that were shared between the two datasets.

Figure 3

Antibody signatures to arrayed antigens differ in S. haematobium-infected subjects before and after PZQ treatment. Graph showing antigens which are targets of the top 15 significant (sorted by signal intensity) IgG responses in the DIV cohort before and after PZQ treatment. The dashed line represents the reactivity cut-off, determined as the mean SI + 1.5SD of the IgG response to all negative control (empty vector) spots. Significance was determined using a Mann-Whitney test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4

IgG profiles to arrayed antigens differ between S. haematobium-infected subjects who do and do not acquire resistance 18 months after PZQ treatment. Average signal intensities depicting IgG responses to each antigen are shown for the CI and DIV cohorts after PZQ treatment. Black arrows denote antigens which are also the target of significantly higher IgG responses in the DIV cohort after PZQ treatment. The dashed line represents the reactivity cut-off, determined as the average + 3SD of the IgG response to all arrayed antigens from the non-endemic negative cohort. Significance determined by student’s t-test. *P ≤ 0.05, **P ≤ 0.01.

Figure 5

Vaccination of mice with recombinant S. mansoni cystatin confers protection in the form of significantly reduced liver and intestinal S. mansoni egg burdens but not adult fluke burdens across two independent trials. (A) liver egg reduction trial 1, (B) liver egg reduction trial 2, (C) intestinal egg reduction trial 1 and (D) intestinal egg reduction trial 2. The percentage of reductions in parasite burden are above each dataset. Differences between cystatin (smp_034420) vaccinated group and the control group vaccinated with recombinant thioredoxin (TrX) were analysed with a student’s t-test. ***P < 0.001. Adult fluke burdens did not significantly differ between vaccinated and control groups in either trial (E, F).