Biological function of Dictyocaulus viviparus asparaginyl peptidase legumain-1 and its suitability as a vaccine target

Abstract

The present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35-39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

Trichomonas vaginalis Legumain-2, TvLEGU-2, Is an Immunogenic Cysteine Peptidase Expressed during Trichomonal Infection

“…These data are consistent with the results of T. vaginalis immunoproteome analysis, in which TvLEGU-2 was also detected by antibodies anti-TvLEGU-2 present in the sera from Tv (+) patients [10], suggesting that TvLEGU-2 has a potential role in trichomoniasis and could serve as a biomarker for the disease. These results are consistent with those of other legumains from Opisthorchis viverrini, Clonorchis sinensis, and Angiostrongylus cantonensis, whose legumains have been proposed as biomarkers of disease [72][73][74][75], and in Dictyocaulus viviparus, whose legumain is being tested as a target for a vaccine against this helminth [76].…”

Immunization Trials with Recombinant Major Sperm Protein of the Bovine Lungworm Dictyocaulus viviparus

“…In contrast, IgM values showed large fluctuations both in the vaccinated groups and in the control groups. The same was observed in previous D. viviparus vaccination trials [14,15,35] and might be due to copurified E. coli antigens accidentally present on the ELISA plates or an unspecific antibody binding to rMSP [15]. Due to the lack of a commercially available conjugate against bovine IgG2a, detection of IgG2a as a specific indicator of a Th1 response was not possible.…”