Targeting Secreted Protease/Anti-Protease Balance as a Vaccine Strategy against the Helminth Fasciola hepatica

Abstract

The liver fluke Fasciola hepatica is an economically important global pathogen of humans and their livestock. To facilitate host invasion and migration, F. hepatica secretes an abundance of cathepsin peptidases but prevents excessive damage to both parasite and host tissues by co-secreting regulatory peptidase inhibitors, cystatins/stefins and Kunitz-type inhibitors. Here, we report a vaccine strategy aimed at disrupting the parasite’s protease/anti-protease balance by targeting these key inhibitors. Our vaccine cocktail containing three recombinant stefins (rFhStf-1, rFhStf-2, rFhStf-3) and a Kunitz-type inhibitor (rFhKT1) formulated in adjuvant Montanide 61VG was assessed in two independent sheep trials. While fluke burden was not reduced in either trial, in Trial 1 the vaccinated animals showed significantly greater weight gain (p < 0.05) relative to the non-vaccinated control group. In both trials we observed a significant reduction in egg viability (36–42%). Multivariate regression analyses showed vaccination and increased levels of IgG2 antibodies specific for the F. hepatica peptidase inhibitors were positive indicators for increased weight gain and levels of haemoglobin within the normal range at 16 weeks post-infection (wpi; p < 0.05). These studies point to the potential of targeting peptidase inhibitors as vaccine cocktails for fasciolosis control in sheep.

Exploiting Comparative Omics to Understand the Pathogenic and Virulence-Associated Protease: Anti-Protease Relationships in the Zoonotic Parasites Fasciola hepatica and Fasciola gigantica

“…Previously characterised F. hepatica sequences identified as cathepsin L peptidases, cathepsin B peptidases, legumain, Kunitz-type inhibitors, serine protease inhibitors (serpins) and stefins/cystatins from the following studies [ 4 , 5 , 7 , 9 , 12 ] were used as reference sequences for BLASTp analysis to (a) confirm the sequences within the F. hepatica genome, and (b) identify homologous sequences within the stage-specific F. gigantica transcriptome datasets (PRJNA350370). The in silico descriptive annotations of the gene transcripts in the specific datasets were also screened.…”

“…Proteomic studies of the molecules released by the F. hepatica stages in the mammalian host, termed excreted-secreted (ES) products, found that a large proportion of these host interacting proteins were comprised of highly proteolytic cathepsin cysteine peptidases (reviewed by [ 4 ]). Further studies revealed that the activity of these peptidases is strictly regulated by the co-release of a variety of cognate peptidase inhibitors, namely Kunitz-type inhibitors, and stefins/cystatins [ 5 , 6 , 7 ]. In addition to regulating the parasite cathepsin peptidases, we have shown that the peptidase inhibitors also play an important role in regulating host lysosomal-like cathepsin peptidases involved in the immune response to parasite infection [ 5 , 6 , 7 ].…”

“…Further studies revealed that the activity of these peptidases is strictly regulated by the co-release of a variety of cognate peptidase inhibitors, namely Kunitz-type inhibitors, and stefins/cystatins [ 5 , 6 , 7 ]. In addition to regulating the parasite cathepsin peptidases, we have shown that the peptidase inhibitors also play an important role in regulating host lysosomal-like cathepsin peptidases involved in the immune response to parasite infection [ 5 , 6 , 7 ]. Besides cysteine peptidase inhibitors, we discovered that F. hepatica also secretes a range of serine peptidase inhibitors (serpins), which have no parasite peptidase target but are exclusively applied to block host serine peptidases, for example Mannose Binding Serine Proteases (MASP) that are critical to complement activation via the lectin pathway [ 8 , 9 ].…”

“…Genes encoding the cathepsin peptidases and the inhibitors of serine and cysteine peptidases belong to multi-membered gene families, thought to have evolved by gene duplication followed by structural/functional diversification [ 4 , 5 , 7 , 9 , 10 , 11 ]. Deciphering the number and structure of genes contributing to these peptidase families is reliant on robust genome assemblies.…”

Draft genome of the bluefin tuna blood fluke, Cardicola forsteri

“…Cathepsins B and L (cysteine proteases) are secreted in high concentrations and often elicit humoral immune responses. These cathepsins, particularly F. hepatica cathepsin L1 (Q7JNQ9), have been the target of vaccination of ruminants against fascioliases [82]. A putative C. forsteri protease was classified as CfCL (Table 4) from sequence identity (44.68%) and a conserved active site (Cys 134 , His 281 , and Asn 301 ), which clusters with cathepsin L1 of schistosomes (Fig 4A).…”