Necator americanus: characterisation of secreted proteinases and vaccine development

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Brown, A.P., 2000. Necator americanus: characterisation of secreted proteinases and vaccine development. PhD, Nottingham Trent University.

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Abstract

The proteinases present in Necator americanus larval excretory/secertory (ES) products were characterised using FITC labelled casein and shown to have a pH optimum of pH 6.5 with lesser peak of activity at pH 8. At pH 6.5 the presence of an aspartyl proteinase was demonstrated for the first time. At pH 8 a serine proteinase activity was shown to predominate. Metallo-proteinase activity was present at both pH points while cysteinyl proteinase activity was observed at pH 6.5. The mixture of proteinases secreted by both the adult and larval stages of N. americanus was further characterised by the use of a number of fluorogenic substrates. Both life cycle stages were shown to possess 'trypsin- like' serine proteinase activity together with cathepsin B and L-'like' cysteinyl proteinase activities.

Larval ES products were shown to degrade the human skin macromolecules collagen (type I, III, IV and V), fibronectin, laminin and elastin. The aspartyl proteinase activity was capable of degrading all the skin macromolecules except type V collagen. The metallo- proteinase degraded collagen and elastin while the serine proteinase degraded elastin alone.

A number of larval and adult proteinases were purified from ES products by affinity chromatography. Cathepsin B and L 'like' cysteinyl proteinases with an approximate molecular mass of 55 kDa were purified using activated thiol Sepharose 4B, an aspaityl proteinase with a mass of 66 kDa was purified using pepstatin A agarose. A 42 kDa metallo-proteinase was purified from larval ES using Z-Gly-D-Leu-AH-Sepharose.

A mixture of larval aspartyl and cathepsin B and L 'like' enzymes were used to vaccinate BALB/c mice against challenge infection with live N. americanus larvae, resulting in an apparent reduction in worm burden of 37.7 % compared with non-vaccinated control animals. Immunity to N. americanus challenge infection was induced for the first time by vaccinating BALB/c mice with y-irradiated larvae. ELISA analysis demonstrated that irradiated larvae induce a Th2 response characterised by IgGl and IL5 production typical of N. americanus infection, but with no significant difference to that induced by the same vaccination protocol using normal larvae. Further study is required to discover the mechanism or molecule(s) responsible for this immunity.

Item Type:

Thesis

Creators:

Brown, A.P.

Date:

2000

ISBN:

9781369325614

Identifiers: