DE2616406B2 - Means for prophylaxis and therapy of gastroenteritis - Google Patents

Description

The invention relates to an immunological, orally administrable agent for the prophylaxis and therapy of Gastroenteritis, particularly infantile gastroenteritis, in primates.

Infantile gastroenteritis (diarrhea) in humans and in animals have always been a serious problem. Has been for many years so far searched in vain for an effective prophylactic and therapeutic agent.

In 1969 the agent responsible for a large part of the diarrhea in these animals, the Nebraska Calf Diarrhea Virus (NCDV), to be isolated. In 1975 it was also known that calf diarrhea with colostrum from animals originally immune to the NCDV and to antigenetic such as morphologically indistinguishable viruses could be prevented.

Since 1973, a virus (rotavirus) has been made from a virus (rotavirus) in many cases of human infantile gastroenteritis the chair of sick children isolated. Whether the isolated virus is the causative agent of infantile gastro-enteritis in Question comes, but is not known.

Surprisingly, it has now been found that immunized from the colostrum and / or the milk Orally applied antibodies against NCDV available to ruminants provide protection against rotavirus cause experimentally induced gastroenteritis in primates. The clinical appearance in primates is very similar to the gastroenteritis known in humans, in which so far none infectious agent could be found, especially those referred to as winter diarrhea will.

The invention accordingly relates to an immunological, orally administrable medicament for prophylaxis and therapy of infantile gastroenteritis in primates, characterized in that it contains antibodies against Nebraska Diarrhea Virus (NCDV) in a conventional pharmaceutical preparation.

Secretory antibodies of the IgA, IgG and IgM classes are preferred. From such antibodies it is known that, as secretory antibodies, they have a longer half-life than serum antibodies own in the intestine. Such antibodies occur in larger quantities in colostrum or milk, also in intestinal juice and saliva. You can, for example, from the milk of immunized with NCDV Can be obtained from ruminants.

According to the invention, however, antibodies can also be used can be used in the usual manner by immunizing animals and recovering them Sera are obtained.

The viruses described in the literature in connection with infantile gastroenteritis with the different Names like Rotavirus, Orbivirus, Reo-virus-like, Duovirus appear after current knowledge to be only synonymous with a pathogen. However, they may not be the same i "and only represent relatives within a group that we call rotavirus. Since this or these pathogens are not yet classified, the nomenclature came to change. Antigenically related Viruses, the relationship of which has not yet been completely clarified, can act in the same or in a very similar way serve as an antigen.

Antigen recovery:

Rotavirus is extracted from the stool of sick children in the manner described by M e 1 η i c k and W e η η e r in Diagnostic Pro- - '»cedures for Viral and Rickettsial Infections, 4th edition, Page 561, New York, 1969, described manner by low and high speed centrifugation won.

The NCDV is propagated in tissue cultures from primary, y < embryonic or postpartum bovine kidney cells or in cells of a permanent pig kidney cell line (PK 15). After the virus has replicated optimally in these cells, the infectious culture supernatant, titer approx. 10MO ⁷ ID ₅₀ / ml, is harvested and used as the starting material for the immunization.

To produce the inactivated antigen, the viruses are concentrated by means of ultracentrifugation (Pelletizing at approx. 100,000 X g) or ultrafiltration by a factor of 5-1000, preferably 10-100. Inactivation can be carried out with formaldehyde according to Lauffer Biochemistry of Viruses Pergamon Press, Oxford 1958, or after LoGruppo, Ann. N. Y. Acad. May be. 83 (1960) is carried out with beta-propiolactone the. Other inactivation methods, e.g. B. after Wiktor et al Appln. Microbiol. 23 (1972) are possible.

Immunization procedure:

Female ruminants are infected orally with reproductive NCDV about 8 weeks before the birth of the fruit with an effective dose of 10 ⁴ to 10 '", preferably 10 ⁷ to 10 ^s , infectious doses of _5U (ID _{511) during their dry period.} 4 weeks and 1 week before birth, NCDV capable of replication and / or inactivated as described above are administered with or without adjuvant, for example in the following manner at the same time:

In each case about 5-10 ml, preferably about 10 ml, Vi reproducible (titer 10 ⁴ -10 '"ID ₅₀ / ml) or inactivated antigen is injected subcutaneously into the lymphatic drainage system of the entire udder.

In each case about 50 to 200 ml, preferably about 100 ml, of the same preparations are in each «Ι Quarter of the udder or each half of the udder through the teat canal instilled.

In each case about 2 to H) ml, preferably about 5 ml of the same antigens are injected intramuscularly.

During the lactation period,

I ₁ , stood for several, preferably about 4 weeks, in each udder quarter or half of the udder about 50 to 200 ml, preferably about 100 ml, NCDV of the preparation described above through the teat

channel instilled. In addition, at intervals of several, preferably about 12 weeks, about 5-50 ml, preferably about 10 ml, of the NCDV of the same preparation are injected intramuscularly.

Other methods of immunization are possible, e.g. B. after Pasieka et al. Can. J. Microbiol. 21: 655 (1974) or according to Watson et al. Res. Vet. Sei. 18: 182 (1975).

Mixtures of antigens can also be used.

Obtaining the antibody-containing Garr.ma globulin fraction from colostrum and milk of immunized ruminants:

The milk (colostrum) obtained from the day of birth is defatted and decaseined in the by Knop et a '.. Aust. J. Exp. Biol. Med. Sci. 49: 405 (1971).

The gamma globulins are z. 3. in that of Pasieka et al. Can. J. Microbiol. 21: 655 (1974) described manner by ammonium sulfate precipitation from the Whey isolated. They are further processed by chromatographic methods, e.g. B. according to Knop et al. Aust. J. Exp. Biol. Med. Sci. 49: 405 (1971) and Knop et al. J. Infect. Dis. 130: 368 (1974). Alternatively can e.g. B. also that of Hammer et al. Eur. J. Biochem. 6: 443 (1968) and Pasieka et al. Can. J. Microbiol. 21: 655 (1974) can be used. Then it is sterile filtered and checked for the absence of reproductive pathogens.

Such gamma globulin fractions containing antibodies against NCDV are essential Part of the agents according to the invention for the prophylaxis and therapy of infantile gastro-enteritis.

It is particularly advantageous to apply the agent according to the invention orally and in the gastrointestinal tract to take effect. The dosage should depend on the level of virus neutralizing Titers are made. With mean virus-neutralizing titers of 1: 500, approx. 0.1-10 g, preferably 0.5-5 g, antibody-containing protein preparations administered per dose. In the Usually this corresponds to 1-10OmI or 5-50 ml of an antibody solution.

The gamma globulin preparation can neutralize gastric juice Substances such as sodium bicarbonate, magnesium carbonate or carrier substances such as aluminum hydroxide and protein stabilizers such as Amino acids or peptides, e.g. B. Glycine can be added in a conventional manner. The galenic Preparation takes place in the drug known for orally administrable in the gastrointestinal tract Way.

The preparation can be used monospecifically, or together in a mixture with antibodies against other microbial such as bacterial or other viral gastroenteritis pathogens.

Determination of the activity of the antibody preparation

'■ · tion:

The antiviral activity is neutralized in the virus ^; onstest determined. It has proven to be beneficial to prepare the preparation to a virus-neutralizing titer of about 1: 100 to 1: 1000, for example 1: 320 ml,

"> set.

example 1

Five motherless monkeys of the genus Macaca obtained by caesarean section are given 1000

ι '■ ID _j0 Rotavirus infected. 1 hour later, 10 ml of the above-described antibody preparation with an antiviral activity 1: 320 / ml was administered orally using a syringe without a cannula. A control group of 5 monkeys received an equally

: ii Unimmunized immunoglobulin preparation recovered Ruminants (control globulin).

The monkeys treated with control globulin all developed diarrhea, while those treated with specific Animals treated with immunoglobulin did not

y> got sick.

Example 2

A held in the same way group of 5 Af- _{J (|.} Fen received 2 hours prior to infection with 1000 ID ₅₁₁ Rotavirus each animal 10 of the antibody preparation described above ml The associated control group of 5 animals received at this time 10 ml Kontrollglobulin. While all animals which had received control globulin developed diarrhea, all animals which had been treated with specific immunoglobulin showed no symptoms of disease.

_4I1 example 3

10 animals were _{infected with 1000 ID 50} rotavirus. After the onset of the disease, 5 animals were treated orally with 10 ml of specific immunoglobulin titer 1: 320 / ml each. 5 animals received at this point

■ r> 10 ml of control globulin each.

All animals treated with control globulin became seriously ill. Of those with specific immunoglobulin treated animals, 3 animals were symptom-free within 24 hours, 1 animal showed

> <> a strongly abbreviated and mitigated course of the disease. It was possible in one animal as well as in the control group the course of the disease cannot be influenced.

Claims (2)

Patent claims: 1. Orally administrable agent for the prophylaxis and therapy of gastroenteritis in primates, characterized by having antibodies against Nebraska Calf Diarrhea Virus (NCDV) contains in a conventional pharmaceutical preparation. 2. Means according to claim I. characterized in that the antibodies from the milk of ruminants immunized with NCDV can be obtained.