Vaccine containing a thiol protease

Details Vaccine containing a thiol protease Vaccine containing a thiol protease

1995-05-25

2003-09-23

Duffy, Patricia A. (Department: 1645)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C424S094630, C424S094100, C424S184100, C424S185100, C424S191100, C424S265100, C424S266100, C435S183000, C435S212000, C435S219000

Reexamination Certificate

active

06623735

ABSTRACT:

This application claims priority from British Application No. 9222156.3, filed Oct. 21, 1992 and British Application No. 9307028.2, filed Apr. 2, 1993.
FIELD OF THE INVENTION
The invention relates to the use of a class of thiol proteases as protective antigens against helminth parasites, namely the Cathepsin L-like proteases frequently released as excretory/secretory products by such parasites.
DESCRIPTION OF THE RELATED ART
Each species of domestic animal can be parasitised by a number of different species of helminths, a process which usually causes disease. For example the parasitic trematode
Fasciola hepatica
is known to be the cause of the economically important disease fascioliasis in ruminants, such as cattle and sheep. The parasite enters the mammalian host by penetrating the gut wall and spends approximately seven weeks feeding on and burrowing through the liver mass before migrating into the bile duct. Following infection, development of immunity in the host is poor and resistance to reinfection in already infected hosts is only partial or non-existent. Other parasitic flukes include
Fasciola gigantica
and Dicrocoelium spp. and also Paramphistomum spp.
Problems are also caused by nematodes such as hookworms (e.g. Necator, Ancylostoma, Uncinaria and Bunostomum spp.).
Of the blood feeding nematodes the genus Haemonchus infects the lining of the abomasum of ruminants, causing anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated.
Other parasitic worms of economic importance include the various species of the following helminth genera: Trichostrongylus, Nematodirus, Dictyocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris and Strongylus. In addition to domestic livestock, pets and humans may also be infected, not infrequently with fatal results and helminth infections and infestations thus pose a problem of considerable worldwide significance.
Control of helminth parasites of grazing livestock currently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques are often unsatisfactory firstly, because anthelmintic drugs may have to be administered frequently, secondly because resistance against anthelmintic drugs is becoming increasingly widespread and thirdly because appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
Numerous attempts have been made to control helminth parasites of domestic animals by immunological means. With very few exceptions (e.g. the cattle lungworm,
Dictyocaulus viviparus
) this has not proved possible.
A vaccine against
F.hepatica
has been proposed in WO90/08819 comprising a glutathione-S-transferase from
F. hepatica
as antigenic material.
Bennett (UK Patent No. 2169606B) extracted various antigens from Fasciola organisms by a process which separates antigens specific to the juvenile stage from antigens present throughout the juvenile and adult stages.
It is known that in vitro cultured
F. hepatica
release protease enzymes which are capable of cleaving immunoglobulins with a papain or Cathepsin-B type of activity (Chapman and Mitchell, Vet. Parasitol. 11 (1982), p. 165-178). It has been suggested that these protease enzymes may assist in evading the immune response in combination with the known ability of the worms to slough off the surface glycocalyx thus shedding bound antibody (Hanna, Exp. Parasitol 50 (1980), p. 155-70). Furthermore crude in vitro excretory/secretory products can under some circumstances confer immunity on rats (Rajasekariah et al, Parasitol. 79 (1979), p. 393-400) perhaps by raising antibodies to such enzymes thus inhibiting them. However, the precise nature of the enzymes is far from clear.
A study of excretory/secretory proteases involving gelatin substrate polyacrylamide gel electrophoresis (GS-PAGE) (Dalton and Heffernan, Mol. Biochem. Parasitol. 35 (1989), p. 161-166) showed a number of cysteine proteases with a wide range of molecular weights and falling generally into two groups, namely from 27.5 KDa to 46 KDa active at pH 4.5″8.0 and from 60 KDa to 88 KDa active at pH 3.0-4.5. It was suggested that the latter group might correspond to the immunoglobulin cleaving enzymes of Chapman and Mitchell and that autolysis and/or aggregation of one or more protease enzymes might be giving the multiple band structure.
Subsequently an HPLC procedure was used and three peaks resolved. Protein from the 15 kDa peak was found to have the ability to cleave IgG at an optimum of pH 4.5 (Heffernan et al, Biochem. Soc. Trans. 19 (1991), page 275).
Another study attempting to characterise the protease enzymes of adult
F. hepatica
is that of Rege et. al. (Mol. Biochem. Parasitol. 35 (1989), p. 89-96) in which a 14,500 Da protein was purified by cation exchange chromatography and molecular sieve HPLC. Maximal hydrolysis of the substrate CBZ-Phe-Arg-AFC was found at pH 6.0. Rege et al used lyophilised whole worms as the source of their protease so that it is not clear whether their protease is excreted or not. They speculated that the protease might be involved in immune evasion or nutrition.
A protease isolated from “Fasciola spp.” has been reported by Yamasaki et al. (Japan J. Parasitol., 38 (1989), p. 373-384). The protease had a molecular weight of 27 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was capable of hydrolysing haemoglobin and this hydrolysing activity was inhibited by cysteine protease inhibitors. Monoclonal antibodies specific for the protease could also inhibit the haemoglbin hydrolysis.
Other studies relating to proteases released by helminth parasites are Fagbemi and Hillyer, Vet. Parasitol. 40 (1991), p. 217-226 relating to
Fasciola gigantica
; Knox and Kennedy, Mol. Biochem. Parasitol. 28 (1988), p. 207-216 relating to Ascaris suum; and Yamakami and Hamajima, Comp. Biochem. Physiol. 87B (1987), p. 643-648 relating to
Paragonimus westermani.
SUMMARY OF THE INVENTION
It has now been found that the group of cysteine proteases disclosed by Dalton and Heffernan as having a range of molecular weights in the range 27.5-88 KDa on gelatin substrate (GS) PAGE can in fact be resolved as two proteases of 27 KDa and 29.5 by SDS-PAGE under reducing conditions; that these proteases are two distinct Cathepsin L-like activities as determined by substrate specificity, affinity for ion exchange columns and N-terminal sequencing; that the proteases also have the ability to cleave immunoglobulins; that immunisation of rabbits with purified proteases can stimulate antibodies capable of neutralizing the enzyme activity; and that this discovery opens up the possibility of an effective vaccine against helminth parasites and in particular
F. hepatica
using well-characterised purified protective antigens and avoiding the drawbacks in terms of toxicity and side-effects such as immune suppression or dominance which are inherent in the use of unresolved crude excretory/secretory products.
Accordingly a first aspect of the present invention provides a vaccine for use in combating a parasitic infestation of helminths in a mammal wherein the antigenic material comprises a protease having enzyme activity of the Cathepsin L type, in at least partially purified form, or an antigenic fragment or epitope thereof, together with a carrier and/or adjuvant.
The invention also provides a method of combating a parasitic infestation of helminths in a mammal comprising administering to said mammal a vaccine according to the invention as hereinbefore defined in an amount effective to combat said infestation.
The mammal is preferably a ruminant, for example cattle or sheep, but the vaccine and method of the invention may also find application in humans.
DETAILED DESCRIPTION OF THE INVENTION
Preferably the Cathepsin L-like protease is derived from flukes such as Fasciola or Dicrocoelium, in particular from the liver fluke
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