VACCINES PROM TAXONOMICAL Y SIMILAR ORGANISMS

Technical Field

This invention is directed to vaccines and their preparation from organisms taxonomically similar to the infective organism.

Background Art

Immunotherapy with respect to infectious organisms has generally followed the theory of using either the organism itself or some intermediate state in its deve¬ lopment as the basis for production of an immunizing vaccine. In this regard, the art is replete with exam¬ ples of the effectiveness of this approach. For in¬ stance, immunization of chickens against cecal cocci- diosis by establishing a controlled subclinical infection (U.S. Patent Number 3,147,185); non-living vaccine pro¬ duced by incubation of third-stage ne atode larvae into the histotrophic stages (U.S. Patent Number 3,395,218 and German Auslegesschrif 1,160,139); canine hookworm vac- cine comprising a physiologically acceptable aqueous vehicle containing attenuated premigratory live hookworm larvae (U.S. Patent Number 3,657,415); an Ascaris suum vaccine comprising sonicated third-stage larvae, soni¬ cated second stage larvae, larvae hatching fluid and second-stage larval culture fluid (U.S. Patent Number

3,676,547); an antigen preparation for immunoprecipitin diagnostic testing for Chagas' disease, caused by Trypanosoma cruzi, comprising purified, water-soluble antigen obtained from tissue culture of Trypanosoma cruzi

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to essentially only the trypomastigote and amastigote growth stages(U.S. Patent Number 3,911,097); vaccine com¬ prising a live, but attenuated, metazoan endoparasite which is pathogenic to domestic animals and is a nema- tode, trematode or cestode in the form of eggs or a pre- migratory or a migratory immature form (British Patent Number 819,830 and Canadian Patent Number 602,465); a vaccine comprising a metazoan or protozoan endoparasite, which elicits an immune response in the host, attenuated with a sub-lethal dose of ultraviolet radiation (British Patent Number 902,760) and a vaccine comprising an anti¬ gen of Schistosoma mansoni separated from a development stage of the parasite (German Offenlegungsschrift 2,742,835). Additionally, a vaccine for canine hookworm has been produced which comprises a physiologically acceptable aqueous vehicle containing premigratory live hookworm larvae of a hookworm species which is specific to cats (British Patent Number 1,277,134).

However, a need continues to exist for the prepara- tion of vaccines against infectious organisms, which are either difficult or impossible to culture, in vitro, on a commercially practical scale. Exemplary of infectious organisms which cannot be readily cultured, in vitro, on a commercial scale are Ichthyophthirius multifiliis and Cryptocaryon irritans.

Ichthyophthirius multifilius and Cryptocaryon ir¬ ritans are some of the most damaging parasites of warm water and salt water fish, respectively, and some of the most difficult to control. Experimental immunization of catfish with Ichthyophthirius multifiliis has been repor¬ ted (Becker et al,"Some Host Response of White Catfish to Ichthyophthirius multifiliis Fouquet", Proc. 18th Ann. Conf. S.E. Assoc. Game and Fish Comm. , 1964). Additional studies have shown that channel catfish injected intra- peritoneally with vaccine prepared from the ground tro-

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phozoites, with and without Freund's adjuvant, survived challenge whereas controls suffered 100% mortality after seven days (Areerat, S., "The Immume Response of Channel Catfish, Ictalurus punctatus (Rafinesque) to Ichthyoph- thirius multifiliis". Masters Thesis, Auburn University, Auburn, Alabama (1974)).

No known method exists for growing the protozoans Ichthyophthirius multifiliis and Cryptocaryon irritans, in vitro. Both Ichthyophthirius multifiliis and Cryp- tocaryon irritans are obligate parasites, the infective tomites must penetrate a host within 24-48 hours or perish. For this reason, maintenance of the parasites requires continual passage to susceptible hosts. Therefor, the accumulation of sufficient antigen to im- munize large numbers of fish is very time-consuming and impractical.

Ichthyophthirius multifiliis and Cryptocaryon irritans are devastating parasites because they have a high morbidity and mortality. The diseases affect not only food fish, including trout, salmon, catfish, carp, eel, tuna, and bonita, but also ornamental fish. At present, chemical treatments are the only practical way of controlling the disease. These treatments including malachite green, formalin, methylene blue, potassium permanganate and others are not approved for use on food fish by the USDA.

Furthermore, malachite green, the most effective treatment, may soon be banned from use on any fish. Because of the limitations of chemotherapy, immunotherapy may be the only successful and practical approach to controlling these diseases.

Disclosure of the Invention

Accordingly, one object of the invention is to provide an effective vaccine for immunization against infectious organisms which are difficult or impossible to culture, in vitro A further object of the invention is to provide a vaccine for immunization against a pathogen derived from a taxonomically similar organism.

A further object of the invention is to provide a commercially feasible method of producing a vaccine for immunization against infectious organisms on a scale suitable for commercial operations.

A further object of the invention is to provide an effective vaccine for the immunization of fish against Ichthyophthirius multifiliis and Cryptocaryon irritans. A further object of the invention is to provide a commercially feasible method of producing a vaccine for fish on a scale suitable for aquacultural operations.

Briefly, these objects and other objects of the invention as hereinafter will become more readily ap- parent can be obtained by providing a vaccine for pro¬ tection against infection by an organism which cannot be readily cultured in vitro which comprises an antigen derived from a taxonomicaly similar organism which may be readily cultured, in vitro.

Best Mode for Carrying Out the Invention

Taxonomy is the orderly classification of organisms into appropriate categories on the basis of relationships among them, e.g. species, genus, family, order or class. Applicants have discovered that by careful analysis of these relationships, it is possible to select suitable pairs of organisms wherein antigenic cross- reactivity may be used to develop a vaccine for one of

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the organisms based on the other of the organisms.

Taxonomic similarity is now found to be an ideal basis for selection of organisms suitable for use in the preparation of a vaccine. Taxonomic similarity is deter- mined through fluorescent staining techniques. In parti¬ cular, an animal, such as a rabbit, is innoculated with the pathogen; blood serum is collected from the rabbit, i.e. antibodies are collected; the pathogen is then contacted with the blood serum, allowing interaction of the antigen and antibody; the so-contacted pathogen is then stained with a fluorescent stain which is selective for the site of antibody-antigen interactin, e.g. stain¬ ing with FITC labelled anti-rabbit IgG; the gross mor¬ phology of the so-stained pathogen is then examined to determine the sites of greatest intensity of staining, e.g. cilia, flagella, mouth and the like; based on the localization of high intensity staining, an organism, suitable for use as a source of vaccine, is selected which has identical physical features to those producing the highest intensity of staining in the pathogen.

Desirability, additional factors such as body shape, pathogenicity for the host and ease of culturation may be used in selection of the organism suitable for prepara¬ tion of a vaccine. For example, the minimum taxonomic relationship appropriate as an indicia of antigenic cross-reactivity for the parasites Ichthyophthirius multifiliis and Cryptocaryon irritans would be that of a ciliate. In particular, for the protozoans Ichthyoph- tirius multifiliis and Cryptocaryon irritans, a protozoan having the characteristics of being easily cultivable, having cilia and being pathogenic for fish would be highly suitable. Such ciliate organisms would induce immunity, to Ichthyophthirius multifiliis and Crypto¬ caryon irritans, when used in the preparation of vaccine. This approach allows the selection of a particular

organism, to be cultured for the production of antigen, wherein the organism selected may be selected on the basis of commercial viability - i.e. ease of production and cost, rather than solely on the criteria that it is the disease source. More importantly, this approach allows the use of large-scale commercial culturing tech¬ niques for the production of antigens for infectious organisms which organisms are not generally amenable to such culturing techniques. Typically, the organisms which are not susceptible to culturation, in vitro, are those which cannot be sustained in presently available culture media, i.e. organisms which only propagate in a living host. Exemplary of such organisms are Ichthyoph- tirius multifiliis and Cryptocaryon irritans. Ichthyophthiriasis, and its saltwater counterpart, produced by Cryptocaryon irritans, are epizootic diseases which claim high mortalities in aqua-culture. Ichthy¬ ophthirius multifiliis and Cryptocaryon irritans are holotrichous ciliates which are ectoparasitic on the gill and hypodermis of fish. The infective stage or to ite penetrates the epithelium where it encysts and develops into the parasitic stage. Macroscopically, these encys¬ ted forms produce the condition popularly known as "white spot disease" or "ick". These parasites are known to cause severe epizootics in the culture of food fish, as well as ornamental fish. They are a major problem in high density ponds because the common chemical treatments are ineffective in removing the encysted stage. The encysted stage, after several maturation processes, divides into thousands of infective tomites, perpetuating the infection. Addi¬ tionally, the common chemical treatments are also toxic to certain species and may be particularly dangerous in the treatment of young fish. The trend towards high density food fish production has increased the importance

of this disease.

The infective tomites of Ichthyophthirius multi¬ filiis and Cryptocaryon irritans morphologically resemble the holotrichous ciliate, Tetrahymena pyriformis. Tetrahymena pyriformis, a carnivorous protozoan, is con¬ sidered an opportunistic parasite of fish, rarely produc¬ ing a primary disease. The close taxonomic association between these ciliates allows the use of antigenic cross- reactivity similar to the antigenic relatedness of canine distemper and measles virus.

The adult form of Tetrahymena pyriformis and the tomities of Ichthyophthirius multifiliis and Cryptocaryon irritans are similar in that they are all covered by cilia and that they are all pear (pyriform) shaped. The differences are that tomities of Ichthyophthirius multi¬ filiis and Cryptocaryon irritans actively penetrate the skin of fish to form adult forms called trophozoites. Tetrahymena pyriformis does not induce classical "Ick" disease. Taxonomically, therefore, the three organisms are ciliates but belong to different genera.

The development of Tetrahymena pyriformis as an "heterotropic vaccine" for Ichthyophthirius multifiliis or Cryptocaryon irritans is efficacious for several reasons: 1) Tetrahymena pyriformis, a free living protozoan, is easily maintained in vitro using simple medium. Tetrahymena pyriformis is easily cultivated in the la¬ boratory using a medium of water and almost any nitro¬ genous compound. Suitably, the medium is as simple as water with protein source and a carbohydrate source. A medium of serile water plus almost any other source of organic material found in a wide diversity ordinary bacteriological culture mediums is suitable, such as, sterile water plus 2-5% of sterile calf serum. A

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particularly preferred medium is Elliot Medium #2 (Biology of Tetrahymena; Elliot, A.M.; Bowden, Hutchinson and Ross, Inc.; Stroudsburg, Pa. (1973)).

FORMULA FOR ELLIOT MEDIUM #2

Sodium Acetate 3H ₂ 0 l.Og 5 KH ₂ P0 ₄ l.Og

Proteose peptone 20.Og Liver Extract "L" l.Og Glucose* 5.0g

Water q.s. to 1000 ml

10 * glucose - filter, sterilize and add to medium after autoclaving

Cultivation takes place at a temperature in the range of about 0 to 35°C, preferably at about 26°C.

15 2) Tetrahymena pyriformis is naturally found in the same environment as Ichthyophthirius multifiliis.

3) Tetrahymena pyriformis, a relatively benign organism, can be used to protect against the pathogens Ichthyophthirius multifiliis and Cryptocaryon irritans 20. based on the fact that selected antigens, i.e. cilia and mouth parts, are identical between these organisms.

A vaccine may be prepared by using the whole pro¬ tozoan to produce a vaccine, i.e. disintegrating the whole protozoan to produce a vaccine. However, the cilia

25 are readily removable and such use of the cilia is effi¬ cacious. The disintegration of the whole protozoan may be accomplished by techniques well-known in the art, e.g. grinding with a mortar and pestle, sonication, chemical treatment and enzyme treatment. The important point is

30 that ciliary antigens protect the fish by producing an

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immunity .

Preferentially, the vaccine comprise cilia stripped from Tetrahymena pyriformis according to the method of Rosenbaum (Rosenbaum, J.L. "Cilia Regeneration in Tetra- hymena and Its Inhibition by Colchicine", J Cell Bio 40 , p. 415 (1969)).

The antigenic material, i.e. the ciliary protein, may be separated from the whole cilia to more effectively concentrate the antigen. Concentration may be achieved by methods known in the art, e.g., pervaporation, centrifugation and concen¬ tration by utlization of carbowax. Preferentially, the protein is concentrated with methyl cellulose and dialy- zed against phosphate buffered saline (PBS) at 4°C. The protein concentration can then be assayed by the method of Lowry (Lowry et al, "Protein Measurement with the Follin Phenol Reagent", J Biol Chem. , 193, p. 265 (1951)).

The vaccine additionally may comprise acceptable diluents, excipients or medicinal agents. Any substance which would not destroy or interfere with the antigenic material, i.e. ciliary protein, may be incorporated in the vaccine. Such substances include buffers, stabili¬ zers, antibiotics, bacterial vaccines and nutritional supplements. Any acceptable diluent may be used, prefer¬ ably, the vaccine is prepared in aqueous form. The vac¬ cine may also include one or more adjuvants.

The vaccine may be administered by intraperitoneal injection, in capsule form, incorporated into food, and in the case of fish, by immersion of fish, or by spraying fish suspended in a net.

For mass aquacultural operations, immersion or spraying are preferred techniques. Such techniques are

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well known for the administration of bacterial vac¬ cines. Preferably, the vaccine is mixed with one or more bacterial vaccines when administered by immersion or spraying. Additionally, when administration is by the spray technique, it is preferable to include Bentonite in the spray formulation so as to enhance penetration and uptake of the vaccine.

The dose of antigen may be determined by the method of Hall (Hall et al, "Characterization Of A Teleost Immunogloublin: The Immune Macroglobulin From Channel Catfish", Comm. Bioche . Physiol. 46 , pp. 187-197 (1973)).

In the case of a vaccine for Ichthyophthirius multi¬ filiis or Cryptocaryon irritans derived from Tetrahymena pyriformis, the dosage rate is generally at least 2.5 micrograms of ciliary protein per 20 grams of fish. Preferably, the dose rate is about 5 micrograms of cili¬ ary protein per 20 grams of fish.

The vaccine may be prepared as a concentrate and then diluted for use as needed. Lyophilization is a suitable method for the preparation of a concentrate. Alternatively, the vaccine may be stored at room tem¬ perature for short periods of time or may be kept frozen when long storage times are contemplated. Vaccines prepared by the techniques set forth herein may suitably be applied to both lower and higher forms of life, e.g., fish, domestic animals and man. Typical ex¬ amples include trout, salmon, catfish, carp, eel, tuna, bonito, dogs, cats, sheep cattle, horse and human. Having essentially described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting upon the scope of the invention.

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EXAMPLE 1

Cultivation of T. pyriformis:

An axenic culture of T. pyriformis was obtained (Midwest Culture Service, Terre Haute, Ind.). Cultures were maintained in an enriched medium at pH 7.2 to 7.4 (Elliot Medium #2). All cultures were incubated aerobi- cally at 26°C. At approximately three-week intervals fresh media was inoculated with 10% (v/v) of the old culture.

EXAMPLE 2

Cultivation of I. multifiliis:

I. multifiliis was maintained by serial passage in channel catfish (Ictalurus punctatus), which were kept in 38 liter (10 gallon) glass aquarium equipped with an undergravel filter. I. multifiliis was maintained in this manner for eight months.

EXAMPLE 3

Maintenance of Channel Catfish:

"Young of the Year" channel catfish, Ictalurus punctatus Rafinesque, were obtained from a commercial farm (J. Sims, Winder, Ga). Fish were kept in a 950 liter (250 gallon) flow-through stainless steel tank and were fed flake food (Kordon Corp., Hayward, California) supplemented with ground chicken liver on alternating days. For experimentation, fish were removed from the holding tank and allotted into 38 liter (10 gallon) glass aquaria equipped with undergravel filters. Aeration was provided by a central air supply attached to the air lift system of the gravel filter. Fish were sustained on the same diet schedule. Ammonia measured as NH3-N and ni-

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trate measure as NO2- were monitored throughout the experiments by standard test kits (LaMotte, Chestertown, Md.). Water was changed as necessary to avoid the ac¬ cumulation of toxic products.

EXAMPLE 4

Preparation of T. pyriformis for Antigen:

Ten- to fourteen-day old cultures were used for antigen preparation. Organisms from 20 ml of culture were concentrated by centrifugation at 500 xG for 10 minutes (IEC Universal Model UV, International Equipment Co., Needham Hts., Mass.). The sediment was washed twice with phosphate buffered saline (PBS), resuspended in 2.5 ml of PBS and immediately used in the deciliation proce¬ dure. Deciliation was effected by a cold osmotic shock process followed by shearing through an 18 g needle (Rosenbaum et al). Deciliated cells were non obile. Ten ml of PBS was added to the tubes containing the decilia¬ ted cells, and the cells were concentrated by centrifu¬ gation for 10 minutes at 500 xG. The supernatant con¬ taining free ailia was decanted, placed in dialysis tubing, and concentrated with carboxymethyl cellulose. The concentrated cilia then were dialyzed overnight in PBS (pH 7.2) at 4°C. The deciliated cells were resus¬ pended in PBS and stored at 4°C for later use as whole cell antigen. The protein concentrations of whole cell and ciliary antigen were determined (Lowry et al).

EXAMPLE 5

Preparation of I. multifiliis for Antigen:

Encysted trophozoites were removed from infected channel catfish by gentle scraping. The cysts were incubated at room temperature (20°C) in sterile distilled water containing 10 ml/1 chloromycetin sodium succinate

(Parke-Davis, Detroit, Mich.) and 0.025 mg/1 fungizone (GIBCO, Grand Island, N.Y.). After 18-24 hours, free swimming tomites were decanted, concentrated and decilia¬ ted in the same manner as described for T. pyriformis in Example 4.

EXAMPLE 6

In Vitro Testing:

Sera from fish and rabbits immunized with cilia and whole T. pyriformis cross-reacted wtih tomites of I. multifiliis. The antigenic relationship was demonstrated using three in vitro tests:

A. Immobilization Test -

In immobilization studies, live T. pyriformis expo¬ sed to rabbit antitetrahymena serum agglutinated at low dilutions and were immobilized at high dilutions. Tomites of I. multifiliis exposed to the same serum were immobilized within two hours. Titers in this hetero- logous test were 1:32 and 1:128. In contrast, immobili¬ zation titers in the homologous system were 1:1024.

B. Indirect Flourescent Antibody Test -

The treatment of both T. pyriformis and I. multi¬ filiis with either rabbit anti-tetrahymena or rabbit anti-Ichthyophthirius sera lead to similar patterns of fluorescence when the organisms were stained with FITC labelled anti-rabbit IgG. With both organisms, the high- test staining was seen in the surface structures.

C. Passive Hemagglutination Test -

The passive hemagglutination test was used to pro¬ vide a more sensitive indication of the cross-reactivity between I. multifiliis and T. pyriformis. In this test heterologous titers of 1:1024 were obtained (sheep erythrocytes tanned with Tetrahymena and rabbit anti-

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Ichthyophthirius). These tests also showed cross- reactivity and indicate that an antigenic relationship exists between these two organisms, localized on ciliary antigens.

EXAMPLE 7

In Vivo Testing:

Twenty-five catfish, each weighing approximately 20 g, were allotted into 38 liter glass aquaria. Antigen was prepared as described in Example 4 and Example 5. The dosage of antigen per body weight of fish was deter¬ mined according to Hall et al (Com. Biochem. Physiol. 46:187-197 (1973)). Fish were immunized by intraperi- tional injection of antigen diluted to working concen¬ tration with phosphate buffered saline (PBS). Each antigen dose was run in duplicate. Twenty-five non- immunized fish served as controls. The experimental groups were: (1) Tetrahymena cilia 5 μg; (2) deciliated Tetrahymena 10 μg; (3) Ichthyophthirius cilia 2.5 μg; and (4) deciliated Ichthyophthirius 2.5 μg. Higher concentra¬ tion of tetrahymenid antigen were used because of the cross-reacting relation. Fifteen days after the initial immunization, booster injections were administered. Fish immunized with the tetrahymenid antigens received the same dose, while fish immunized with Ichthyophthirius cilia received 3.5 μg and deciliated Ichthyophthirius 4.5 μg, respectively. Fifteen days following the booster injections, an "Ich" infected contact fish was placed into each tank. The fish were observed for clinical signs of infection and mortality. The results are set forth in the following table:

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TABLE I

Mortality of Channel Catfish Immunized with Ciliary and Whole Antigens of T. pyriformis, and I. multifiliis, and Challenged With I. multifiliis,* Two Weeks Following Secondary Immunization

Primary Secondary Total % Antigen Dose Dose # Fish Mortality

T. pyriformis cilia 5.0 μg 5.0 g 137 11.6

Whole (deciliated) 10.0 μg 10.0 g 89 100.0

I. multifiliis cilia 3.5 μg 3.5 g 120 42.5

Whole (deciliated) 2.5 μg 4.5 g 76 77.6

Noni munized 25 100.0

*Fish with clinical Ichthyophthiriasis were placed in each tank to expose experimental fish to disease.

Primary immunization of catfish with 5 μg of T. pyriformis ciliary antigen, followed by a secondary immunization with ^* 5 μg of antigen two weeks later, conferred an excellent degree of protection against challenge with I. multifiliis when the fish were chal¬ lenged two weeks after booster. Only 11.6% of the fish in these groups developed clinical disease. Fish immu¬ nized with 10 μg of deciliated whole cells, followed two weeks later with the same dose, were not protected against infection and died when exposed to disease. Homologous immunization with deciliated whole I. multifiliis cells (2.5 μg, 4.5 μg) resulted in 77.6% mortality following challenge with I. multifiliis. Mortality was 42.5% in fish immunized with homologous

ciliary antigen (3.5 μg, 3.5 μg) .Mortality was 100% in the control groups.

Having now fully described this invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention set forth herein.