Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1999-12-23
2003-04-22
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S184100, C424S192100, C424S265100, C424S266100, C435S069100, C435S069300, C435S069700, C435S252300, C530S300000, C530S350000, C530S387100, C530S388600
Reexamination Certificate
active
06551594
ABSTRACT:
TECHNICAL FIELD
This invention relates to biotechnology generally, and more specifically to an ex vivo animal or challenge model as a method to measure protective circuitry directed against parasites and vaccines shown to be protective in the method.
BACKGROUND
Only a few vaccines against parasites are commercially available. Most of these vaccines are based on attenuated live parasites that induce natural, protective immunity and cause less severe pathological damage. These parasite vaccines include one directed against
Dictyocaulus viviparus
(e.g., Dictol, Glaxo), undoubtedly the most successful anti-parasite vaccine, and analogous therewith a vaccine against
Dictyocaulus filaria,
the lung worm in sheep (Sharma et al. 1988). These vaccines are based on live but irradiated third-stage larvae (Peacock & Pointer 1980). Another attenuated vaccine is directed against the hookworm
Ancylostoma caninum
in dogs. However, this vaccine has been marketed only for a short time in the USA, marketing was discontinued because the American veterinary profession did not accept this live vaccine (Urquhart 1980). An attenuated vaccine against
Babesia bovis
has been in use for nearly a century in Australia (Purnell 1980) and a dead vaccine based on metabolic products named “Pirodog” is used to vaccinate dogs against
B.canis
(Moreau 1986).
Vaccination trials in sheep with a recombinant vaccine against the tape worm
Taenia ovis
(Johnson et al. 1989) and the concealed antigen H11 from
Haemonchus contortus
(Newton 1995, review) have been performed successfully. A trial with the SPf66 malaria vaccine in Africa has recently been completed. The efficiency against clinical malaria in areas of high transmission was 31% and the product appeared to be safe. However, because it is not fully understood how SPf66 mediates protection, the development of improved vaccines is hampered (Tanner et al. 1995; review).
Problems of developing anti-parasite vaccines are abundant. Parasites have complex life cycles and each stage expresses different sets of antigens. Moreover, the different stages are often associated with different sites in the body. For most parasites little is known about the immune mechanisms involved in natural immunity and about the stage of the parasite inducing this immunity.
Most often, no reproducible animal model is available to study these mechanisms, thereby blocking a new approach in vaccine development. As mentioned above, most available vaccines are based on attenuated live parasites. These vaccines can sometimes be successful because the “vaccine parasites” follow the correct route of infection and deliver a wide array of stage-specific antigens. However, such vaccines must challenge the acceptance of the public (e.g.,
Ancylostoma caninum
vaccine), especially when they are for human use (e.g.,
Schistosoma mansoni
vaccine, Taylor et al. 1986). Moreover, live vaccines, in general, have a short shelf-life and are relatively expensive. From this perspective there is an obvious need for vaccines that are based on (recombinant) proteins derived from the parasite. However, the identification of such protective proteins meets a great number of difficulties, as shown below as an example for
Fasciola hepatica.
The trematode parasite
Fasciola hepatica
mainly infects cattle and sheep. Sometimes also humans get infected. The parasite causes considerable economic losses in, for example, western Europe, Australia and South America. The metacercariae of
Fasciola hepatica
enter its host by the oral route, penetrate the gut wall within 4-7 hours (Dawes 1963, Burden et al. 1981, Burden et al. 1983, Kawano et al 1992) and migrate through the peritoneal cavity towards the target organ, the liver. Oral infection of cattle results in almost complete protection against a challenge, whereas sheep often die from an infection and do not acquire natural immunity. Both the natural host (cattle) and the animal model (rat) acquire natural immunity after infection (Doy & Hughes 1984; Hayes, Bailer & Mitrovic 1973). Therefore, rats are often used to study resistance in cattle. In the rat a large part of natural immunity is expressed in the gut mucosa, the porte d'entree of the parasite. In immune rats, about 80% of the challenge newly excysted juvenile stages (NEJs) is eliminated in the route from the gut lumen to the peritoneal cavity (Hayes & Mitrovic 1977, Rajasekariah & Howell 1977, Doy, Hughes & Harness 1978/1981, Doy & Hughes 1982, Burden et al. 1981/1983). Based on natural immunity, a vaccine based on irradiated
Fasciola gigantica
metacercariae was developed for cattle (Bitakaramire 1973). In the seventies and eighties many vaccination experiments have been performed with antigen extracts of adult and juvenile flukes (Haroun & Hillyer 1980, review). However, these studies lead to conflicting or disputable results. For example, subcutaneous or intramuscular injection of rats with adult or juvenile fluke extracts did not result in protection (Oldham & Hughes 1982, Burden et al. 1982, Oldham 1983). Adult fluke extracts given intraperitoneally in Freund complete adjuvant (FCA) or incomplete Freund adjuvant (IFA) resulted in about 50% protection (Oldham & Hughes 1982, Oldham 1983). Using very high antigen doses of
Bordetella pertussis
as additional adjuvant this protection reached 80-86% (Oldham & Hughes 1982, Oldham 1983). Extracts of 4-week-old juveniles given intraperitoneally in AIOH
3
did not induce protection in the studies of Pfister et al. (1984/85), whereas 16-day old juvenile extracts provided 86% protection in mice, without the use of adjuvant (Lang & Hall 1977). Subcutaneous sensitization of cattle with sonicated 16-day-old juveniles resulted in more than 90% protection (Hall & Lang 1978). Intramuscular injection of calves with an isolated fraction from adult
Fasciola hepatica
(Fh
SmIII
), with an immunogenic 12 kD protein as major component, resulted in 55% protection (Hillyer et al. 1987).
Since 1990, several
Fasciola hepatica
vaccine candidate antigens have been isolated and/or produced. Most of these antigens are derived from adult flukes and share homology with
Schistosoma mansoni
antigens. Glutathion S-transferases (GST) are enzymes amongst others active in the cellular detoxification system. Immunization of sheep (n=9) with GST purified from adult
Fasciola hepatica,
injected s.c. in FCA, with a boost immunization 4 weeks later in IFA, resulted in 57% protection (Sexton et al 1990). Immunization of rats with GST provided no protection (Howell et al. 1988). Vaccination trials in cattle performed by Ciba Animal Health Research (Switzerland) and The Victorian Institute of Animal Science (Australia), resulted in 49-69% protection (Morrison et al. 1996).
Intradermal/subcutaneous immunization with recombinant
S.mansoni
fatty acid-binding protein Sm14 in FCA, provided complete protection against
Fasciola hepatica
challenge in mice (Tendler et al. 1996). PCT International Patent Publication WO 94/09142 suggests the use of proteases having cathepsin L type activity, derived of
Fasciola hepatica,
in the formulation of vaccines for combatting helminth parasites; immunisation of rabbits with the purified mature enzyme resulted in rabbit antibodies capable of decreasing the activity of the enzyme in vitro.
However, levels of protection obtained with
F. hepatica
cathepsin L or haemoglobin in cattle were only 53.7% or 43.5%, respectively (Dalton et al. 1996). Cathepsin L belongs to a family of cysteine proteinases, secreted by all stages of the developing parasite. Cathepsin L from
F. hepatica
is most active at slightly acid or neutral pH (Dalton & Heffernan, 1989). The functions of this proteinase include disruption of host immune function by cleaving host immunoglobulin in a papain-like manner (Smith et al. 1993) and preventing antibody mediated attachment of immune effector cells to the parasite (Carmona et al. 1993). Moreover, cathepsin L is capable of degradation of extracellular matrix and basement membrane components (Berasain et al. 1997), and pre
Bokhout Bernard Adri
Cornelissen Johannes Bernardus Wilhelmus Joseph
van Milligen Florine Johanna
Baskar Padmavathi
Smith Lynette R. F.
Stichting Dienst Landbouwkundig Onderzoek
TraskBritt
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