Yves Meyvis

Vaccine testing of a recombinant activation-associated secreted protein (ASP1) from Ostertagia ostertagi

Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation‐associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo‐asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challenge infection in cattle. The full coding sequence of Oo‐asp1 was cloned in a baculovirus expression vector in frame with a carboxy‐terminal Histidine tag and recombinant virus was used to infect an insect cell culture. Western blot analysis with anti‐His and anti‐Oo‐ASP1 antibodies showed the production of recombinant Oo‐ASP1. The cell pellet containing the recombinant was subsequently used to immunize seven calves three times intramuscularly with QuilA as adjuvant. Control animals were solely injected with the QuilA adjuvant. The challenge infection with O. ostertagi consisted of 30 000 L3 larvae per animal given over 30 days (1000 larvae/day, 5 days/week) and started the same day as the final immunization. Immunization with the recombinant Oo‐ASP1 did not result in any level of protection against the challenge infection. There was no reduction in faecal egg output or in worm burdens. Moreover, Western blot analyses and ELISA indicated that, although the animals raised an antibody response against the recombinant Oo‐ASP1, there was hardly a response against the native Oo‐ASP1, suggesting that the baculovirus expressed recombinant was wrongly folded or lacked essential secondary modifications. Further analysis of the structure of the native ASPs and their glycosylations is being done.

Analysis of the translationally controlled tumour protein in the nematodes Ostertagia ostertagi and Caenorhabditis elegans suggests a pivotal role in egg production.

The translationally controlled tumour protein (TCTP) is a conserved protein which has been described for a wide range of eukaryotic organisms including protozoa, yeasts, plants, nematodes and mammals. Several parasitic organisms have been shown to actively secrete TCTP during host infection as part of their immuno-evasive strategy. In this study, we have studied TCTP in Ostertagia ostertagi, a parasitic nematode of cattle, and in the free-living nematode Caenorhabditis elegans. An analysis of the transcription and expression patterns showed that TCTP was present in the eggs of both species. This localisation is consistent for some other Strongylida such as Teladorsagia circumcincta, Cooperia oncophora and Haemonchus contortus. TCTP was also detected at low levels in excretory-secretory material from adult O. ostertagi worms. The role of TCTP in nematode biology was also investigated by RNA interference in C. elegans. Knock-down of C. elegans tctp (tct-1) transcription reduced the numbers of eggs laid by the hermaphrodite in the F(0) and F(1) generations by 90% and 72%, respectively, indicating a pivotal role of TCTP in reproduction.

Hybrid N-glycans on the host protective activation-associated secreted proteins of Ostertagia ostertagi and their importance in immunogenicity.

Previous vaccination trials with calves have shown that intramuscular immunization with natively purified activation-associated secreted proteins (ASPs) of Ostertagia ostertagi induces protection against a homologous challenge infection with a 74% reduction in cumulative faecal egg counts and a significant reduction in worm length. Recently, O. ostertagi ASP1 was recombinantly expressed using a baculovirus system and tested in a vaccination trial. However, immunized calves failed to recognize native ASPs and no protection was observed. These results suggest an important structural difference between the baculo r-ASP1 and its native counterpart. Therefore, we investigated whether glycans and/or structural epitopes are key features in the induction of a protective immune response. The results show that ASPs carry two hybrid N-glycosylations with a complex alpha-1,3-arm, an unprocessed alpha-1,6-arm and an alpha-1,6-fucose core. While removal of these glycans had little effect on antibody recognition by vaccinated animals, denaturing and reducing the proteins dramatically reduced recognition, suggesting the importance of conformational protein backbone epitopes.