ABSTRACT
The need for alternative ways to control helminth parasites has in recent years led to a boost in vaccination experiments with recombinant antigens. Despite the use of different expression systems, only a few recombinants induced high levels of protection against helminths. This is often attributed to the limitations of the current expression systems. Therefore, the need for new systems that can modify and glycosylate the expressed antigens has been advocated. However, analysis of over 100 published vaccine trials with recombinant helminth antigens indicates that it is often not known whether the native parasite antigen itself can induce protection or, if it does, which epitopes are important. This information is vital for a well-thought-out strategy for recombinant production. So, in addition to testing more expression systems, it should be considered that prior evaluation and characterization of the native antigens might help the development of recombinant vaccines against helminths in the long term.
Subject(s)
Antigens, Helminth/immunology , Helminthiasis/immunology , Helminths/immunology , Vaccines, Synthetic/immunology , Animals , Antigens, Helminth/biosynthesis , Antigens, Helminth/metabolism , Glycosylation , Helminthiasis/prevention & control , Humans , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/geneticsABSTRACT
In this study, we isolated and analyzed a small heat shock protein (HSP) of Ostertagia ostertagi (Oo-HSP18). Oo-hsp18 is encoded by a single-copy gene and the full-length cDNA represents an 18-kDa protein. The expression of Oo-hsp18 is highly stage specific and restricted to the adult stage. The protein is synthesized in a tissue-specific manner and localized in the body muscle layer. The levels of Oo-hsp18 mRNAs are sharply induced by heat shock but not by other stressors such as levamisole and H2O2. A vaccination trial with recombinant Oo-HSP18 failed to protect calves against a challenge infection.
Subject(s)
Heat-Shock Proteins, Small/immunology , Ostertagia/immunology , Amino Acid Sequence , Animals , Blotting, Western , Cattle , Cattle Diseases/prevention & control , Cells, Cultured , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , DNA, Helminth/chemistry , DNA, Helminth/isolation & purification , Gene Expression , Heat-Shock Proteins, Small/biosynthesis , Heat-Shock Proteins, Small/chemistry , Heat-Shock Proteins, Small/genetics , Hot Temperature , Male , Molecular Sequence Data , Ostertagia/genetics , Ostertagiasis/prevention & control , Ostertagiasis/veterinary , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , Spodoptera , Transcription, GeneticABSTRACT
Protease inhibitors are thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. In a previous study, screening of Ostertagia ostertagi cDNA libraries with local antibody probes of the abomasal lymph nodes and mucus revealed a (28 kDa) aspartyl protease inhibitor (API), which was exclusively recognised by antibodies from immune calves. Here we report the molecular characterization of Oo-API (sequence analysis, developmental expression and localization) and a vaccine trial in cattle with the native and recombinant baculo-expressed antigen. The full-length open reading frame of api encodes a protein of 28 kDa. The sequence showed 82% significant homology to an Aspin homologue from Trichostrongylus colubriformis (AA034715). The cDNA encoding the full-length sequence was cloned in a bacterial pET expression vector and the pVec 35 baculovirus vector. Polyclonal rabbit serum against the Escherichia coli-expressed protein was used to develop Western Blots of extracts and ES and to localize the antigen on L3, L4 and adult worm sections. The protein was expressed in all life stages, which was confirmed by real-time polymerase chain reaction (RT-PCR), and was mainly localized in the cuticle of L3, the intestinal cells of L4, and the gut and sphincter of adult worms. Polyclonal serum was also used to affinity purify the native protein. Vaccination of calves with native Oo-API and baculovirus-expressed Oo-rbAPI in combination with QuilA resulted in no protection against Ostertagia challenge infections.
