Biomedical Engineering Reference
In-Depth Information
and decreased transmission, particularly if the vaccine impairs feeding and,
therefore, prevents blood loss.
In the interest of developing a safe, stable and effective vaccine against
human hookworm, a catalytic variant of Na-APR-1 (Na-APR-1
mut
) was made
in which both active site aspartic acid residues were mutated, rendering the
recombinant molecule enzymatically inactive but conformationally unchan-
ged.
3
This mutant protease was tested as a vaccine in a canine model of het-
erologous infection with A. caninum and resulted in a 67% decrease in faecal
egg output, reduced weight loss, and a 29% decrease in adult worm burden
between vaccinated and control animals.
3
Moreover, IgG from animals vac-
cinated with catalytically inactive Na-APR-1 inhibited the ability of recombi-
nant Na-APR-1 to cleave substrate, implying that vaccination induces
protective antibodies which act to neutralize Na-APR-1, compromising the
parasite's capacity to feed.
3
Despite the ability to express Na-APR-1 in E. coli
inclusion bodies and refold the protein, this process proved dicult to scale-up
at an industrial level. We therefore generated monoclonal antibodies, some of
which neutralized the enzymatic activity of Na-APR-1, and mapped the neu-
tralizing epitope to a 13-mer peptide (A
291
Y) that was predicted to form a
solvent-exposed alpha helix proximal to the active site Asp
284
.
21
Chimeras
consisting of hookworm and schistosome vaccine antigens fused to a fragment
of Na-APR-1 containing the A
291
Y epitope were produced in soluble form and
induced antibodies that neutralized APR-1
wt
enzymatic activity, further bol-
stering the vaccine potential of this antigen.
Orthologues of Na-APR-1 have been reported from the gastrointestinal
tracts of other hematophagous helminths including the human blood flukes
Schistosoma japonicum (Sj-CatD) and S. mansoni (Sm-CatD)
14
and the liver
fluke O. viverreni (Ov-APR-1).
22
Furthermore, Sm-CatD and Sj-CatD are the
apical proteases of the Hb digestion cascades in schistosomes.
14,15
Suppression
of Sm-CatD mRNA expression in vitro and subsequent injection of these
double-stranded RNA-treated schistosomes into mice resulted in a lethal
developmental phenotype,
23
and a vaccine trial with recombinant Sj-CatD
showed significant reductions in worm burdens between vaccinated and control
animals,
24
highlighting the central role of these proteases in schistosome biol-
ogy. RNAi has not yet been successfully applied to hookworms, precluding the
silencing of these genes and subsequent assessment of their functions.
The cathepsin-D-like aspartic proteases are not the only aspartic hemoglo-
binases to be identified in hookworms. Na-APR-2 is an aspartic protease from
N. americanus which belongs to a group of proteases that resembles mamma-
lian pepsin more closely than they do cathepsin D.
25
Na-APR-2 was localized
primarily to the intestinal microvilli in adult worms and digested intact Hb but
shared only 25% of its cleavage sites with Na-APR-1. Antiserum to Na-APR-2
inhibited skin penetration of infective hookworm larvae by 50%,
25
suggesting a
role for this and/or related enzymes in skin penetration of L3,
26
as well as Hb
digestion by the adult worm. An aspartic protease from H. contortus (HcPep1),
also homologous to mammalian pepsin, has been localized to the gut of adult
worms, where it is believed be involved in digestion of the blood meal.
27
