CS258922B1 - Vaccine against infection caused by tapewormTaania crassiceps and method of production - Google Patents

Abstract

The solution relates to a vaccine against infection caused by the parasitic helminth of the species Taenia crassiceps and a method of its production. This vaccine contains a suspension of live cells of the larval stages of this tapeworm in physiological solution in an amount of 10^ to 10® in 1 ml. The production method consists in washing the larvae of Taenia crassiceps with at least 1:: physiological solution, homogenizing them so that the larvae burst and the vesicular fluid is released, then the larvae are further homogenized and or pressed through a metal sieve so that individual cells are released, the cell suspension is washed at least 1x with physiological solution and the concentration of live cells is adjusted to 10^ to 10®/ml.

Description

The invention relates to a vaccine against infection caused by the tapeworm Taenia crassiceps and a method for its preparation.

Research into the treatment of infections caused by parasitic helminths is ongoing in several directions.

Over the past decade, numerous attempts have been made to develop effective and safe vaccines, but the focus of current therapy remains on chemotherapy. Intensive research into synthetically prepared substances is leading to the gradual elimination of side effects on the host and an ever-increasing degree of efficacy.

One of the most modern means for the treatment of parasitic infections is paraziquantel (2-cyclohexylcarbonyl)-1,2,3,5,7,11b-hexahydro-2-pyrazino(2,1a)isoquinoline-4, with minor side effects (Nash T. E. et al., Am. J. Trop. Med. Hyg., 1982, 31, 977-982). In a short time after administration, the substance accumulates in the parasitic organism (Xiao, S. H. et al., Acta Pharmacol Sinica, 1981, 2, 204-211) and its effectiveness is almost 100% in practically all species of parasitic helminths.

A number of other chemotherapeutic agents vary in their effectiveness against individual helminth species and in their toxicity to the host organism.

Despite the high efficacy of chemotherapy, there are many reasons for research and development of vaccines. The disadvantage of chemotherapeutics is that they can only be treated when the infection becomes clinically apparent. However, a certain period of time elapses from the moment of invasion to the manifestation of clinical symptoms of the disease, during which the parasites damage the host organism, reduce the productivity of livestock, etc. (Děla Cruz B. et al., Proc. of Symp. Vienna, 29 June - 3 July 1981 (AIEA, FAO, UNEP), International Atomic Agency, Vienna, 1982, 127-133).

Cases of inoperable hydatidosis treated with chemotherapy have been described, where the cyst ruptured and an allergic reaction occurred. Surgical procedures for hydatidosis also often end fatally. Patients recover slowly and in some cases are unable to perform their original work. Neurocysticercosis and intraocular cysticercosis are very difficult to treat, often accompanied by complications - epilepsy, high blood pressure, etc.

Another problem associated with chemotherapy is the recurrence of infection. For example, reinfection of grazing calves with helminths of the genera Cooperia, Oesophagostoma, Haemonchus, Bunostomum, and Trichostrongylus was monitored after treatment with tetramisole hydrochloride and febendazole and it was found that after approximately 6 weeks the level of infection in treated calves was the same as in untreated calves (Lima W. et al., Arquivo Brasileiro de Medicina Veterinaria e Zootecnica, 1983, 35, 101-112). Another serious problem with the use of chemotherapy is the ability of helminths to become resistant to anthelmintics (Dias L. C. et al., Transaction of the Royal Society of Tropical Medicine and Hygiene, 1982, 76, 652-659).

Further development of vaccines capable of preventing the development of parasites at an early stage is therefore justified. However, the preparation of vaccines for helminths is complicated by the complexity of the parasitic organism, antigenic changes during ontogenesis and also by the lack of starting material. The vaccines used so far can be divided into two groups, depending on the immunogen used:

1) Vaccination with the whole parasitic organism and 2) vaccination with antigens. In the first case, vaccines prepared from live or radiation-inhibited worms were tested. The use of live, uninhibited worms is advantageous in terms of unchanged surface antigens and is very effective. It was found, for example, that mice that were subcutaneously implanted with 3 live larvae of the tapeworm Taenia crassiceps were resistant to secondary infection in up to 99% of cases (Siebert A. E. Jr et al., Int. J. Parasitol., 1978, 8, 39-43).

Live eggs of the tapeworm Taenia pisiformis have also been implanted subcutaneously and it has been found that secondary infection was eradicated in almost 100% of cases. The disadvantage of these vaccination methods is that the whole larvae or eggs of the helminths used as an immunizing agent develop and can infect the entire host organism. For these reasons, the parasites have been previously irradiated with either x-rays or ultraviolet radiation. The efficacy of these vaccines is on average 80% (Urban J. F. Jr., Tromba F. G., Vet. Immunol. Immunopathol., 1982, 3, 399-409).

Similar results have been achieved using antigens either in the form of a crude homogenate of the parasitic organism or purified antigens. A relatively high efficiency, around 80%, has been achieved by applying antigens released into the culture medium and by in vitro cultivation of T. ovi-s oncospheres and T. saginata larvae (Rickard M. D., Fourth. Inter. Congr. Parasit., Section C 1978, 134-135).

The disadvantages of the currently used methods of therapy can be summarized as follows:

A. Chemotherapy.

1) Side effects of chemotherapeutic drugs on the host.

2) Damage to the host caused by developing parasites.

3) Recurrent infections.

B. Vaccination.

1) When using live uninhibited eggs or larvae, their development leads to de facto infection.

2) When irradiated eggs or larvae are used for immunization, the vaccine is less effective.

3) When using antigens, the effectiveness is also lower.

These disadvantages are eliminated by the vaccine against infections caused by the tapeworm Taenia crassiceps, which contains a suspension of live cells of the larval stages of the tapeworm in physiological solution in an amount of 10 to 10 cells in 1 ml. The suspension of cells in physiological solution is injected into the peritoneal cavity or intramuscularly or subcutaneously.

The essence of the preparation method is that the larvae or adults of Taenia crassiceps are washed at least once with physiological solution, pressed through a metal sieve to release individual cells, the cell suspension is washed at least once with physiological solution and the concentration

6 live cells are adjusted to 10 to 10 cells per milliliter.

The advantages of the proposed procedure are mainly the following:

1) Antigens on the cell surface are in an unchanged form.

2) Infection cannot occur because parasites cannot develop from these cells.

3) No need for irradiation equipment.

4) No need for antigen purification equipment.

5) Cells can also be used for diagnostics.

An example of a method of producing and using the composition according to the invention is given below.

Example L

The larvae of the tapeworm Taenia crassiceps were washed out of the peritoneal cavity with phosphate-buffered saline (PBS) and homogenized in a glass homogenizer so that the larvae ruptured and the vesicle fluid was released. The suspension was washed 5 times with PBS, the larval remains were pressed through a steel sieve (100 mesh). The released cells were washed 3 times with PBS and their concentration was adjusted to 10 live cells/ml.

This sample was used in 6-10 week old NIH strain mice (females) that were immunized intraperitoneally. On day zero, control mice were injected with PBS only.

„ 4

Two other groups of mice were immunized with live cells at a concentration of 10/mouse.

After 14 days, control mice were injected with PBS and then one of the experimental groups

- 4 5 was immunized with live cells at a concentration of 10 and the second 10 cells per mouse. On day 28, all mice in all three groups were infected with 3 larvae of the tapeworm Taenia crassiceps. Larvae numbers

were determined 2 months after the invasion. Experimental results are listed in the following table: 1st group 2. group 3rd group mouse no PBS mouse no. 10 ⁴ mouse no. 10 ⁵ 1 339 6 202 11 0 2 110 7 0 12 0 3 494 8 147 13 0 4 257 9 0 14 27 5 322 10 0 15 0

a - numbers of larvae

PBS - controls injected with PBS only and then infected with 3 larvae

4

- mice immunized with 10 cells/mouse.

5

- mice immunized with 10 cells/mouse.

The table shows that an increased dose of live cells increases the effectiveness of the vaccine.

Table 2 summarizes the results of experiments in which mice were immunized against Taenia crassiceps infection. Due to the strain of mice used (outbred ICR mice, female, 6 to 10 weeks old) and their higher resistance to infection, larval numbers were monitored up to 3 months after infection. Mice in groups 3 and 4 were immunized as in the first experiment (see text for the first table), mice in group 2 were immunized with T. crassiceps larval antigen (crude homogenate, lyophilized) and mice in groups 5 to 7 were injected with an increased dose of 5x 10^ and 10 cells/mouse. In addition, mice in group 6 were immunized subcutaneously and mice in group 7 intramuscularly.

Table 2

Numbers of larvae found in outbred ICR mice 3 months after infection.. Group Number of mice Immunization dose Number of inf. mouse Number of larvae 1 13 2xPBS 5 30+18 2 10 0.2 and 1 mg 2 1,430 3 13 2xl0 ⁴ 3 13+10 4 24 10 ⁴ , 10 ⁵ 3 20+10 5 18 10 ⁴ , 10 ⁶ 0 0 6 11 10 ⁴ , 5xl0 ⁵ 0 0 7 5 10 ⁴ , 5xl0 ⁵ 0 0

The results show that the immunization procedure used in groups 5 to 7 is optimal, as 100% of the mice were completely resistant to infection.

Claims (7)

What is claimed is: 1. A vaccine against infection caused by parasitic helminth of the species Taenia crassiceps, characterized in that it contains a suspension of live tapeworm cells in saline in an amount of 10 ⁴ to 10 ⁶ per ml. 2. The vaccine of claim 1, wherein live cells of the larval stage or adult tapeworm are used. 3. A method according to claim 1, wherein the larvae or adult Taenia crassiceps are washed at least 1X with saline, homogenized so that the larvae crack and release the bladder fluid, then the larvae are further homogenized or passed through a metal sieve. so as to release single cells, the cell suspension is washed at least 1X with saline, and the viable cell concentration is adjusted to 10 to 10 ^ / ml. 4. A method according to claim 3, characterized in that the larvae are passed through a 100x100 [mu] m steel mesh. 5. A method according to claim 3, wherein the larvae are passed through a bronze sieve with a mesh size of 100 * 100 yum. 6. A process according to claim 3, wherein a phosphate solution is used for washing. 7. A method according to any one of claims 3 to 6, wherein any larval tissue residue is removed by filtration or centrifugation.