DD144359A1 - METHOD FOR THE PRODUCTION OF IMMUNE PREVENTION AGAINST TEMPERATURE DISEASES - Google Patents

Abstract

The process for the preparation of immune preparations against Inflammatory diseases are applicable in the field of human and Veterinary medicine and is characterized in that an i-n ducks proliferating persistent retrovirus in duck embryo cell cultures, is then enriched and purified and that the effective but harmless bzvz. inactivated Virtisbesfcandteile as prophylactic, therapeutic and Diagnosticians-by retrovirus-related infections (Tumors) are mainly used in papillomatosis.

Description

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Method of making anti-tumor preparations

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Field of application of the invention

The invention "relates to a process for the preparation of immuno-preparations for the treatment and diagnosis of tumorous diseases in human and veterinary medicine caused by retroviruses, preferably papillomatoses"

Characteristic of the "known technical solutions

The Yöifalix s, which have been bred by the GDR patents WPA 61 K / 179 066 and WPA 61 K / 190 065, for the preparation of vaccines against human laryngeal papillornatosis and against papillomatoses of other localization in humans and animals are based on in vitro culture the pathogen, which is suspected to be a member of the family of the papovavirus "the virus is isolated from tumor tissue and grown in cell cultures," After enrichment and purification of the production of vaccines from the virus concentrate _e done parenteral administration of the vaccine results in the disappearance papillomatous growths »'Recurrence free after treatment',

Object of the invention

In further electron microscopic investigations on the viral etiology of human tumors, it has been found that other viruses are present in papillomas, for example of the larynx and bladder, and also in prostate carcinomas belonging to the family of retroviruses · The structures originally identified as papovavireii probably correspond to the inner bodies

It has also been found that in clinically healthy duck populations, a retrovirus is circulated that is vertically transmitted to the duck embryo, unfolds in normal duck embryo cells, and can be cultured with no discernable alterations in persistent viral infection. Persistent syndrome causes no Developmental Disorders in the Growing Duck Embryos «. It is apathogenic for duck and chick chicks as well as infant mice, guinea pigs, rabbits and calves. "Oncogenic properties could not be detected. Serologically, kinship relationships exist with the retroviruses contained in human papilloma tissue »

Goal of! The invention is the application of non-pathogenic retrovirus persisting in normal duck embryo cells for the immunotherapy of cancer patients. According to the invention, the persistent retrovirus is obtained directly from primary duck cell cultures. The Exfindungsgemäße solution has the advantage that the difficult and time-consuming isolation of the pathogen of the tissue growths from Geschwixistge \ vebe and its breeding in pre. cultured cells, which corresponds to the method protected by the GDR patents V / PA 61 K / 179 066 and WPA 61 K / 190 065, can be avoided «

¥ / esen the invention.

For infections with retroviruses, the human and animal organism is generally unable to prevent the onset of clinical manifestations, including, but not limited to, to express themselves in progressive growth, to dominate with the aid of his system of execrations. In order to enable the organism to build up specific cell-mediated immunity to destroy the retroviruses and the tumors they induce, additional information needs to be communicated to the immune system * For those protected by the GDR patents WPA E / 179 086 and WPA 61 K / 190 065 Inventions this happens in the case of human laryngeal papillomatosis

by repeated inoculations with the tumor material 6i? isolated and homozygous cell culture-bred species homologues »

In the inventive method is to Inimuntherapie papiilomatö'ser tumors a heterologous retrovirus use that persists in normal duck cells · It does not have pathogenic properties, "In contrast to the isolated from Geschwuistgewebe pathogens it may stop working oncogenic _c Through the virus recovery from normal, unseeded Entenemb .Tyozellkulturen is the two-phase process of the previous method, which on cell culture with

subsequent viral vaccination is based on one step _? cell culture production, reduced «

The cell cultures are made with large cell seed from young duck embryos. The cell density is chosen? that within τοη 24 h a confluent cell growth before ~ liegte After 2 * 2 d incubation, the cells from the Kui-turgefäßen removed _s minde in a serum-free medium ~ least 1Ofach in relation to the cell culture medium konisentriert and to release the intracellular virus disintegrated The by Centrifugation to remove τοη cell components is then treated with lipid solvents and further purified and concentrated with the ultracentrifuge or other suitable method. After inactivation with formalin or other suitable inactivating agents, the virus may be used with or without the addition of adjuvants Vaccine use * Repeated use of the vaccine produced from concentrated duck retrovirus, the Geschwststaclistum in acute and chronic-recurrent papillomatosis brought to a standstill and EezidiTfreiheit achieved The hitherto conventional surgical procedure can da s method according to the invention is supported, in many cases also replaced »

1. Entenembryozellkuliuren be prepared in a known manner from 11 (10-12) d old full embryos and incubated at 38 ⁰ C in a roller device. The cell diclite is adjusted to 3x10 cells, resulting in a confluent cell layer within 24 hours. After a further 2 incubation days, the cells are removed from the culture vessels by a Chelapex (0.1 $) - Trypsin sh (0.2 $), sedimented by centrifugation (1500 rpm for 5 min) and concentrated in at least 10 times the concentration Zeilzüchtungsmedium recorded with serum-free Hanks - solution. To release the intracellular virus, the cells are disrupted by atmospheric diffraction in CO ₂ - dry ice-alcohol mixture or by other suitable methods. The cell detritus is removed by centrifugation at 2000 rpm for 30 minutes from the virus-containing fluid separated from dezs sediments, mixed with 50 $ diethyl ether and shaken thoroughly for 5 min. After renewed centrifugation at 2000 U / min for 30 min, the Äthorphase settles on a small precipitation Schichiit from the virus-containing aqueous phase. The virus fluid is carefully aspirated under the Präaipitationsschiciit and then by ultracentrifugation in 80 ml rotor for 1 VAC 602 for 2 hours 25000 U / min at ⁰ ° C centrifuged (about 100000 g). the sediment is taken up in Tris buffer. After centrifugation at 5000 U / min for 20 rain in a% hlzentrifuge at 4 ⁰ C, the in relation to the starting material 100 times enriched "virus with formalin in a final concentration of 1: 4 000 inactivated. For use as a vaccine, the inactivated virus fluid, with or without the addition of adjuvants, is filled in 5 ml in-ampules 3, which are subsequently fused with one flame

and stored at 4 ⁰ C. As a rule, 5 injections are administered intramuscularly at intervals of 3 d, to which further injection series can be added every 4 weeks.

The viral content and purity of the vaccine assay are determined by negative contrasting with phosphotungstic acid. The serological identity is determined with specific hyperimmune sera and with patient sera using the MNCINI single-irinal diffusion (EANCINI, G., CARBONARAy AO, u, HEREMANS, J ₎ . Immunochem., 2 (1965), 235), the

145. Laurell - electrophoresis or the Überwanderungselektrophorese (methods taken from FRIEMEL, EU: Immunological working methods, VEB Gustav Fischer Verlag Jena, 1976) carried out. The harmlessness is tested in animal experiments on infant mice, guinea pigs and rabbits in various application methods "treated with antilymphozyteneren mice serve to complete an oncogenic activity"

2. For detection of serological relationship and cell-mediated immunological reactions, the duck retrovirus obtained according to the method of the invention is used in 1000-fold concentrated form. As antiserum for testing humoral immunity, patient sera are used in different dilutions in buffered solution. For control experiments, specific hyperimmune sera from rabbits are used. The determination of the immunological similarities or an interspecific relationship between the virus isolated from Enstenilen, the human retroviruses in particular. Human papillomaviruses as well as the diagnosis of an expired antigenic contact in the subjects are carried out with the simple radial imarand diffusion according to IAANCINI (1965). "The overexpoving elec- trophoresis, Laurell electrophoresis and immunoelectrophoresis are carried out on the basis of FRBEL (1976).

Claims (2)

Erfindungsanspruoh 1, Process for the preparation of immuno-preparations against tumors in humans and animals, which are caused by retroviruses mainly by Papillomat @ sen _? characterized by the isolation, concentration and purification of persistent retroviruses present in the cells of normal duck embryos and in the cell cultures produced therefrom and by their inactivation for use as a vaccine for parenteral administration with or without the addition of adjuvants. Method according to item 1, characterized in that persisting retroviruses are used in normal tissues or fresh cell cultures of other vertebrates. 3. Method according to items 1 and 2, characterized in that the persisting retroviruses are in active or inactive form for immunological diagnosis, by retroviruses caused infections * fo