Iris Peelaers

Identification of excretory-secretory products of larval and adult Ostertagia ostertagi by immunoscreening of cDNA libraries

Excretory-secretory (ES) products of Ostertagia ostertagi, an abomasal nematode of cattle, are considered to be important for the development and survival of the parasite within the host. To gain insight in the composition of these ES products of both larval (L3, L4) and adult life stages of Ostertagia cDNA libraries of the parasite were immunoscreened with polyclonal rabbit serum raised against these ES products. This approach led to the identification of 41 proteins, amongst which are structural proteins such as actin, kinesin and vitellogenin, housekeeping proteins such as those involved in protein folding, different metabolic pathways or mitochondrial functioning and proteins associated with stress (heat shock protein) or antioxidantia (thioredoxin peroxidase). A large number of the isolated proteins were similar to hypothetical proteins of the model nematode Caenorhabditis elegans. Because somatic proteins can be non-specifically released during in vitro culturing as nematodes deteriorate, it was checked if the isolated proteins are genuinely secreted. The amino acid sequences of the translated cDNAs were investigated for signal peptides and monospecific antibodies against the isolated proteins were purified and used to develop Western blots of ES and somatic extracts. In this manner it could be proven that 15 cDNAs code for genuine secreted proteins. The identification of these ES antigens allows to select proteins with potential protective capacities, which are targets for vaccine development.

Vaccination with an Ostertagia ostertagi Polyprotein Allergen Protects Calves against Homologous Challenge Infection

ABSTRACT As an alternative to antihelminthic drugs, we are exploiting vaccination to control infections with the abomasal nematode Ostertagia ostertagi in cattle. Our focus for vaccine targets is excretory-secretory (ES) products of this parasite. One of the most abundant antigens in larval and adult Ostertagia ES products is a protein homologous to nematode polyprotein allergens. We found that the Ostertagia polyprotein allergen (OPA) is encoded by a single-copy gene. OPA comprises three or more repeated units, and only the 15-kDa subunits are found in ES products. The native antigen is localized in the intestinal cells of third-stage larvae and in the hypodermis and cuticle of fourth-stage larvae and adult parasites. Vaccination of cattle with native OPA (nOPA) in combination with QuilA resulted in protection against Ostertagia challenge infections. The geometric mean cumulative fecal egg counts in the nOPA-vaccinated animals were reduced by 60% compared to the counts in the control group during the 2-month course of the experiment. Both male and female adult worms in nOPA-vaccinated animals were significantly shorter than the worms in the control animals. In the abomasal mucus of vaccinated animals the nOPA-specific immunoglobulin G1 (IgG1) and IgG2 levels were significantly elevated compared to the levels in the control animals. Reductions in the Ostertagia egg output and the length of the adult parasites were significantly correlated with IgG1 levels. IgG2 titers were only negatively associated with adult worm length. Protected animals showed no accumulation of effector cells (mast cells, globular leukocytes, and eosinophils) in the mucosa. In contrast to the native antigen, recombinant OPA expressed in Escherichia coli did not stimulate any protection.

Structure of Ostertagia ostertagi ASP-1: insights into disulfide-mediated cyclization and dimerization

The cysteine-rich secretory/antigen 5/pathogenesis-related 1 (CAP) protein superfamily is composed of a functionally diverse group of members that are found in both eukaryotes and prokaryotes. The excretome/secretome of numerous helminths (parasitic nematodes) contains abundant amounts of CAP members termed activation-associated secreted proteins (ASPs). Although ASPs are necessary for the parasitic life cycle in the host, the current lack of structural and functional information limits both understanding of their actual role in host-parasite interactions and the development of new routes in controlling parasitic infections and diseases. Alleviating this knowledge gap, a 1.85 Å resolution structure of recombinantly produced Oo-ASP-1 from Ostertagia ostertagi, which is one of the most prevalent gastrointestinal parasites in cattle worldwide, was solved. Overall, Oo-ASP-1 displays the common hallmark architecture shared by all CAP-superfamily members, including the N-terminal CAP and C-terminal cysteine-rich domains, but it also reveals a number of highly peculiar features. In agreement with studies of the natively produced protein, the crystal structure shows that Oo-ASP-1 forms a stable dimer that has been found to be primarily maintained via an intermolecular disulfide bridge, hence the small interaction surface of only 306.8 Å(2). Moreover, unlike any other ASP described to date, an additional intramolecular disulfide bridge links the N- and C-termini of each monomer, thereby yielding a quasi-cyclic molecule. Taken together, the insights presented here form an initial step towards a better understanding of the actual biological role(s) that this ASP plays in host-parasite interactions. The structure is also essential to help to define the key regions of the protein suitable for development of ASP-based vaccines, which would enable the current issues surrounding anthelmintic resistance in the treatment of parasitic infections and diseases to be circumvented.

In-Depth Proteomic and Glycomic Analysis of the Adult-Stage Cooperia oncophora Excretome/Secretome

Cooperia oncophora is one of the most common intestinal parasitic nematodes in cattle worldwide. To date, C. oncophora infections are treated using broad-spectrum anthelmintics. However, during the past decade, reports of anthelmintic resistance in this parasite species have emerged worldwide, necessitating new avenues for its control, possibly through vaccination. In this frame, we analyzed the adult-stage C. oncophora excretome/secretome (ES), covering both the protein and glycan components, since this fraction constitutes the primary interface between parasite and host and may hold potential vaccine candidates. Two-dimensional gel electrophoretic separation of the ES material enabled the MALDI-TOF mass spectrometry (MS)-directed identification of 12 distinct proteins, grouped in three separate molecular weight fractions: (i) a high molecular weight fraction consisting of a double-domain activation-associated secreted protein (ASP), (ii) a midmolecular weight fraction predominantly containing a single-domain ASP, a thioredoxin peroxidase and innexin, and (iii) a low molecular weight protein pool essentially holding two distinct low molecular weight antigens. Further MS-driven glycan analysis mapped a variety of N-glycans to the midmolecular weight single-domain ASP, with Man6GlcNAc2 oligomannosyl glycans as the major species. The predominance of the nonglycosylated double-domain ASP in the high-molecular weight fraction renders it ideal for advancement toward vaccine trials and development.

Identification and characterization of a novel specific secreted protein family for selected members of the subfamily Ostertagiinae (Nematoda)

It has been shown that the bovine abomasal parasite, Ostertagia ostertagi, drastically modulates its microenvironment, causing epithelial cell damage, accumulation of inflammatory cells and pH changes in the stomach. The mechanisms used by the parasite to change the abomasal environment are largely unknown, but an important role has been attributed to excretory-secretory (ES) products from the parasite. In this study we have identified proteins representing a novel ES protein family, characterized by the SCP/Tpx-1/Ag5/PR-1/Sc7 protein motif. These proteins were named Oo-AL1 and Oo-AL2 (O. ostertagi ASP-like protein). Both proteins contain a signal peptide and 1 predicted N-glycosylation site. The transcript for Oo-AL1 was present from the L4 stage onwards in both male and female adult worms, whereas the Oo-AL2 transcript was hardly detectable. Western blots of somatic extracts and ES products from different developmental stages of O. ostertagi, probed with anti-Oo-AL1 antibodies, revealed Oo-AL proteins in the ES products of adult worms. An analysis of the nematode genome and EST databases indicated that these novel ES proteins are unique to O. ostertagi and its relative, Teladorsagia circumcincta, suggesting a key function in these abomasal parasites.

Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity.

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection.

Interleukin-17 receptor A (IL-17RA) as a central regulator of the protective immune response against Giardia

The protozoan parasite Giardia is a highly prevalent intestinal pathogen with a wide host range. Data obtained in mice, cattle and humans revealed the importance of IL-17A in the development of a protective immune response against Giardia. The aim of this study was to further unravel the protective effector mechanisms triggered by IL-17A following G. muris infection in mice, by an RNA-sequencing approach. C57BL/6 WT and C57BL/6 IL-17RA KO mice were orally infected with G. muris cysts. Three weeks post infection, intestinal tissue samples were collected for RNA-sequencing, with samples from uninfected C57BL/6 WT and C57BL/6 IL-17RA KO animals serving as negative controls. Differential expression analysis showed that G. muris infection evoked the transcriptional upregulation of a wide array of genes, mainly in animals with competent IL-17RA signaling. IL-17RA signaling induced the production of various antimicrobial peptides, such as angiogenin 4 and α- and β-defensins and regulated complement activation through mannose-binding lectin 2. The expression of the receptor that regulates the secretion of IgA into the intestinal lumen, the polymeric immunoglobulin receptor, was also dependent on IL-17RA signaling. Interestingly, the transcriptome data showed for the first time the involvement of the circadian clock in the host response following Giardia infection.