Antibody-mediated inhibition of proteases of African trypanosomes.
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Date
2006
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Abstract
The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in
cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may
contribute to pathogenesis of the disease, and antibody-mediated inhibition of this
enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a
trypanosomal serine peptidase, is also a potential virulence factor in African
trypanosomes because it is released into the host circulation by dead or dying parasites,
where it retains catalytic activity due to the enzyme's insensitivity to serum protease
inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and
oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing
antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these
antibodies was assessed.
The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned
and expressed in Escherichia coli, from which active recombinant enzymes were
purified. These recombinant enzymes exhibited trypsin-like specificity for peptide
substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and
lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally
stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength.
The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they
were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide.
High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of
fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography.
Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of
bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against
large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M
directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes.
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The catalytic domain of congopain, C2, was used to immunise rabbits either without
adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either
bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response.
However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was
obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes.
In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in
alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits
immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels
were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced
with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies
produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase
B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly
less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed.
It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the
production of antibodies that were better able to neutralise the proteolytic activity of C2
and congopain in vitro than that with conventional adjuvants . The immunisation of C2
complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite
congopain in vivo, and may contribute to an anti-disease vaccine against African
trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since
antibodies produced against this complex are not able to inhibit the activity of
oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce
efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against
African trypanosomosis.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.
Keywords
Immunoglobulins., Trypanosomiasis in cattle., Proteolytic enzymes., Cattle--Trypanotolerance., Trypanosoma., Theses--Biochemistry.