A. viteae jird infection model
by Richard Lucius
Rodent infections with the filaria Acanthocheilonema viteae have
been used to study protective immunity induced by immunization with irradiation-attenuated
infective larvae (L3) and with various antigen preparations. As in other
models of filariasis, vaccination with attenuated L3 induces a stable, though
partial immunity (around 90% protection), and immunization with exported
antigens of L3 results in about 50% protection (see Lucius et al., Exp.
Parasitol. 73, 1991).
Experiments of Storey and Al Mukhtar (Tropenmed. Parasitol. 33, 1982) have
shown a considerable degree of cross protection between two filarial species
(Brugia malayi and Litomosoides carinii ). Therefore, it
is conceivable that important protective antigens are common to several
species and that Onchocerca volvulus and A. viteae share
protective components. Consequently, the A. viteae model can possibly
be used to evaluate the protective potential of recombinant O. volvulus
and A. viteae antigens in our animal model and which can now serve
to screen recombinant polypeptides submitted through the Clark Foundation
network. Some factors like the route of immunization, the use of adjuvants
and the timing is currently under investigation to provide an optimal screening
protocol.
The screening procedure consists of 3 (subcutaneous) immunizations of jirds
with doses of 25 ug protein each. The doses are given together with a mild
adjuvant (mixture of squalene, Tween and Pleuronic) in bi-weekly intervals.
Two weeks after the last immunization a challenge infection of 80 L3 of
A. viteae is inoculated. Blood from the animals is taken before
immunization, before the challenge infection, and at 4, 8 and 12 weeks of
infection both to determine the antibody responses against the immunizing
antigen (by ELISA and immunoblotting) and to determine microfilarial densities.
12 weeks p.i. the animals are sacrificed and the adult filariae are counted
and measured. In parallel with the experimental group (7 animals) a negative
control group (7 animals) is immunized with an irrelevant antigen (e.g.
the fusion partner of a fusion protein) together with the adjuvant. A challenge
control group (12 animals) and a sham treated group (3 animals) are used
to monitor the worm development and the antibody titres in non-immunized
animals.
With this set up, a quantity of 750-1,000 ug of the recombinant protein
and of the irrelevant control protein is needed to perform immunizations
and antibody detection assays. The purity should be >90% to avoid artefacts
due to contaminants. The protein concentration should be determined by two
independent protein tests and not by OD reading. The quality of the product
should be illustrated by a photo of an SDS PAGE gel of the antigen batch
to be tested. The minimal time from the first immunization until the read
out will be 18 weeks.
If interesting results are obtained, the experiment will be repeated. We
have the ability to clone homologous A. viteae antigens from an
A. viteae-cDNA library and to use these in immunization experiments.
This library can also be made available to interested groups.