Abstract
ackground: Dictyocaulus viviparus is a parasitic nematode causing bronchitis in cattle worldwide. In general, infections do not cause high mortality, but the morbidity can be high with concomitant loss of production. Parasitic nematode infections are treated in general with anthelmintic drugs, but resistance against these drugs is widespread and still
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increasing. Because calves infected with lungworm eventually develop a protective immune response, a vaccine will be more efficient to prevent disease. D. viviparus is the only nematode for which there is a commercial vaccine available, but this vaccine, consisting of infective larvae attenuated by radiation, has important drawbacks like limited shelf-life and the inability to induce immunological memory. A defined, animal friendly produced vaccine lacking these limitations is therefore in demand. Because passive immunisation proved that antibodies can harness protection, we analysed the antibody responses after infection and vaccination against several antigen extracts in order to identify candidates for a defined synthetic/recombinant vaccine. Results: Excretory/secretory (ES) products of adult worms contain glycoproteins and are well recognized by all immunogloblin isotypes present in sera of infected and vaccinated calves. The glycoproteins only contain N-linked glycans and the immunodominant part resides on the glycan. Booster responses were observed only after re-infection, not after vaccination, and these booster responses were directed against the protein backbone. IgG1 and IgE are the only isotypes that were boosted, suggesting that these isotypes are components of protective memory response after re-infection. The most immunodominant protein is a high molecular glycoprotein (GP300). This antigen is well recognized by IgG1 and IgE. Characterization of GP300 demonstrated that N-glycans of GP300 bind to the lectin wheat germ agglutinin (WGA) and are substituted with phosphorylcholine (PC). Both properties depend on the presence of N-acetylglucosamine (GlcNAc) on the antenna of the N-glycan. Within D. viviparus this was unique for GP300. PC was the immunodominant epitope of GP300 and antibodies against GP300 cross-react with the pro-inflammatory mediator platelet-activating factor (PAF) via the PC moiety. Analysis by mass spectrometry identified GP300 as the homologue of thrombospondin from Haemonchus contortus. H. contortus thrombospondine is a component of an experimental, partly protective vaccine. The finding that thrombospondin-like proteins contain PC-substituted glycans is novel for D. viviparus, H. contortus and other nematodes. Conclusions: The most immunogenic antigen of D. viviparus is the PC-containing glycoprotein GP300. IgE titers directed against GP300 correlates with protection, while PC-specific IgG cross-react with PAF from the host. The immunising potential of GP300 has to be evaluated in a vaccination trial with deglycosylated and untreated GP300. Challenge infection will demonstrate whether there is (IgE mediated) protection or not. Inflammation parameters (eosinophilia, respiration frequency), but also possibly protective responses (IgG2) can be downregulated by anti-PAF antibodies in the case of vaccination with the untreated GP300. In that case, a vaccine containing GP300 will reduce disease, but not the number of worms. Because PC has many other immunomodulatory properties and GP300 homologues with PC substitutions are also present in other nematodes, GP300 is an interesting and important antigen in nematode-immunology.
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