WO2000052055A1

WO2000052055A1 - SPECIFIC EGG YOLK ANTIBODIES IgY, THE OBTAINMENT AND USE THEREOF

Info

Publication number: WO2000052055A1
Application number: PCT/DE2000/000547
Authority: WO
Inventors: Hartmut Kobilke
Original assignee: Hartmut Kobilke
Priority date: 1999-02-26
Filing date: 2000-02-21
Publication date: 2000-09-08
Also published as: DE10080481D2; DE19910159A1

Abstract

The invention relates to specific egg yolk antibodies IgY and the obtainment and use thereof for immunotherapy in animal breeding and animal production. Pullets are immunised and repeatedly boostered over the entire laying period of twelve to thirteen months. The obtained IgY antibodies in the form of whole egg powder, egg yolk powder or lyophilisates are given to the animal stock via ready feed or drinking water. The IgY antibodies can be used as the basis in monospecific ELISA kits for associated diagnostics and titer quality control as well as for titer development.

Description

Specific IgY egg yolk antibodies, their extraction and their use

The invention relates to specific IgY egg yolk antibodies, their extraction and their use for immunotherapy in animal breeding and animal production. They are given in the form of whole egg powder, egg yolk powder or lyophilisate to ready-made feed or drinking water to livestock. The IgY antibodies are suitable as a basis in monospecific ELISA kits for accompanying diagnostics and titer quality control as well as for titer development.

The starting point of the present invention is the fact that immunities actively acquired from the chicken (Gallus gallus) for the protection of their offspring are transmitted as specific polyclonal IgY antibodies via the egg yolk (KLEMPERER, F., 1893 [1]) and this is a real alternative for are targeted passive immunotherapy or immunodiagnostics that replace the killing or manipulation of laboratory mammals and make them unnecessary (including KOBILKE, 1994 [2a]; KOBILKE et al, 1994 [2b]; SCHADE et al, 1993 [4], [5 ]; IBURG et al, 1994 [7]; OLTMER, 1998 [8]).

Egg yolks containing immunoglobulin with specific IgY are produced by targeted immunizations using adjuvants. These egg yolks containing immunoglobulin with specific IgY (ERHARD et al, 1993 [9]) replace missing vaccines and immune sera. Overall, the chicken ensures a high yield of such specific, thermostable and acid-resistant IgY egg yolk antibodies that belong to the immunoglobulin class IgG (KOBILKE 1994 [2], KOBILKE et al. 1994 [2b], LÖSCH 1996 [10]). DE 195 04 755 A1 discloses avian, vitelline antibodies directed against HIV antigens, as well as a process for their production and their use in HIV-positive patients for producing high antibody serum titers. It is also known from EP 0 225 254 B1 the use of a special antibody which was obtained from the egg of the chicken, which has been immunized against a pathogenic antigen, and in pharmaceutical composition against infectious intestinal disease caused by an intestinal toxin-producing E. coli neonatal mammals is used. EP 0 503 293 A1 also discloses the production of IgY antibodies and their pharmaceutical use as egg yolk powder and in particular egg yolk cleaning and degreasing.

The state of the development evident from the cited patents is restricted in its use to neonatal mammals or humans, against pathogenic E. coli strains and rotavirus or the HIV-1 p24 core antigen. The production of IgY egg yolk antibodies and their preparation for general therapy or the preparation / purification of these yolks as a pharmaceutical composition are generally described. From EP 0 225 254 B1 and 0 503 293 AI no immunization schemes and adjuvants used can be seen.

The object of the invention is to provide specific IgY antibodies permanently and in such a sufficient amount that they can be used to carry out specific passive immunotherapy and to significantly reduce the infection pressure accumulated over years in existing livestock. The object was achieved in that specific egg yolk antibodies from the chicken - taking advantage of the entire laying period - by immunization according to a new vaccination scheme with poultry-pathogenic viruses and bacterial cultures - parvovirus, adenovirus types, reovirus types or E. coli strains and Pasteurella multocida ssp. - be won. To achieve the object, the invention is based on years of practice and experience based on the fact that bacteria and viruses - especially those which attack the gastrointestinal tract and multiply there mainly in the cells - are the main causes of lossy infectious diseases and reduced performance in farmed poultry, especially waterfowl and laying hens. Despite partial immunoprophylaxis using vaccines, the poultry stocks are not adequately and consistently protected against such infections.

Furthermore, it is proposed to administer globulin-containing dry whole egg powder and / or egg yolk powder or their lyophilisate orally with the feed and / or the drinking water and / or with feed supplements and / or as a natural remedy using new uses and strategies. The properties of polyclonal egg yolk antibodies and, when given orally, their favorable attack site in the intestinal tract guarantee long-term success when used as dry whole egg or egg yolk powder in the ready feed for farmed poultry or as lyophilisate via drinking trough over the risky period. So far, immunotherapy in poultry, in particular water poultry and laying chickens, which is relevant in practice and meets the demands of animal rights activists, is not known. Surprisingly, it has been found that significant progress is made in this and in the uncomplicated and inexpensive administration via ready-made feed mixtures or drinking water and that this procedure as immunotherapy leads to considerable animal losses and reduced growth in the breeding phase, as well as reduced laying performance and fertilization in the adult breeding animals, and depressed growth in the fattening phase as well as reductions in slaughter values and the infection pressure in the production plants can be reduced considerably or a field virus can be displaced. According to the invention, the IgY antibodies are obtained by a new immunization scheme for poultry, which enables the achievement of permanently high antibodies and thus maximum antibody yields for a rational and economically justifiable therapy. The immunization schedule spans the entire laying period of 12 to 13 months. To carry out the same, 14 to 15 week old pullets of the laying direction are stabled 7 to 14 days before the basic immunization.

The strategy and use in combating the lossy poultry infections mentioned above, often with an epidemic course, and the fitting of this oral therapy for poultry, in particular water poultry and laying hens, into the objectively given gaps in immunoprophylaxis enables the aforementioned solution to the problem.

From the immunization scheme for the generation of such specific IgY antibodies via egg yolk, which the poultry produces to protect its offspring against infections, specific antibody-containing eggs with the highest possible specific IgY content are produced by targeted vaccination during the entire laying period. The laid eggs are stored in a cool place and regularly beaten - not older than four weeks - and processed as whole egg homogenate or as egg yolk homogenate to whole egg or egg yolk powder or to freeze dry material (lyophilisate) under low-germ conditions. The whole egg or egg yolk homogenate is processed and packaged natively without further additives and used in poultry feeding or resuspended via drinking troughs. This end product is obtained from the whole egg and / or the egg yolk raw mass as a powder over spray towers or as a lyophilisate.

It is within the scope of the invention to extend the applications provided by IgY egg yolk antibodies for specific, passive immunotherapy and for the construction of diagnostic KITS (e.g. ELISA) also against other pathogens and for other animal species and for humans.

All previous patent descriptions have the disadvantage that they are accompanied by the diagnostic examination methods / procedures for monitoring purposes

- the initial or herd status

- The effectiveness control diagnosis for these specific antibodies used in production foci

- the disease-diagnostic follow-up examinations and - the quantitative antibody determination are missing.

For this reason, this application for industrial property rights pursues the aim of using appropriate ELISA kits to create the appropriate test method to accompany the immunization of the test subjects or antibody donors and the immune status of the herds of user companies. This makes it possible to replace common, but time-consuming and expensive examination methods (e.g. SNT / serum neutralization test). In addition to the claims, the features of the invention also emerge from the description, the individual features representing advantageous protective designs, individually or in combination in the form of combinations, for which protection is requested with this document. The combination consists of known (immunizations, boosters) and new elements (immunization scheme, route of administration) that mutually influence each other and a new benefit in their new overall effect and result in the desired success, which lies in the fact that not only can the infection pressure accumulated over the years in animal herds be reduced, but surprisingly a field virus displacement can also be achieved.

The IgY antibodies according to the invention are obtained in that immunized SPF or preferred pullets of the laying direction are immunized subcutaneously or intramuscularly and the antigen dose is 100 μg (0.1 ml) to 1,000 μg (1 ml) in 0.5 to 1 .0 ml suspension (sterile) is divided into 3 to 4 injection depots.

The invention will be explained below using several exemplary embodiments:

Example 1:

Through highly immunogenic living or killed (inactivated) pure mono- or polyvalent homologous or heterologous antigens (against lossy pathogen-related infections of the water fowl and the laying hens), SPF or preferred pullets of the laying direction (laying hybrids with a defined status) and repeated boosters in legends Hens are produced according to a new vaccination scheme described below, egg yolks containing specific IgY antibodies that can be obtained permanently and containing highly specific, polyclonal, vitelline antibodies between 20-100 mg specific IgY per egg yolk.

Long-term immunization scheme for optimal IgY production:

In order to permanently generate a maximum antibody level in the egg yolk, the immunization scheme according to the invention extends over the entire laying period from 12 to 13 months. 14 to 15 week old pullets of the laying direction are stabled 7 to 14 days before the basic immunization and immunized according to the following scheme:

Day 0: Basic immunization = 1st immunization sbk. Or i.m. with antigen and Freund's complete adjuvant (FCA) or other alternative adjuvants (aA) Day 28: 1. Booster = 2. immunization with antigen and Freund's incomplete adjuvant (IFA) or aA

Day 38 - 42: Beginning of the antibody peak reached = harvesting / harvesting and processing of the egg yolk Day 56: 2. Boosting = 3. Immunization with antigen and IFA or aA

Day 140: 3. Boosting = 4. Immunization with antigen and IFA or aA

Day 252: 4th booster (booster shot) = 5th immunization with antigen and

IFA or aA [all immunizations from day 28 subcutaneously (sbk.)]

Day 510 - 500: End use of the immunized laying hens at the end of use after 17 to 18 months of life, d. H. in the 12th to 13th laying month.

Antigen dose: depending on the degree of purity and condition (lyophilisate or culture suspension) between 100 μg (0J ml) and 1000 μg (1 ml) in 0.5 - 1.0 ml suspension (sterile). Adjuvant: between 0J - 0.5 ml per dose well mixed in sterile syringes and cannulas as Freund's adjuvant; complete and incomplete.

Depending on the antigenic molecular weight (<15000> 30000), binding of the antigen to carrier proteins (bovine serum globulin or human serum albumin) is necessary.

Example 2:

From the immunization scheme described in Example 1 and the general immunization principle for the generation of such specific IgY antibodies via egg yolk, which the poultry generates to protect its offspring against infections, targeted vaccination is carried out, for example, on laying hens from the 15th week of life according to the long-term immunization scheme according to the invention and with the aid of Freund's adjuvant produced these specific, antibodies-containing eggs with the highest possible specific IgY content during the entire laying period. The eggs laid are stored in a cool place and regularly beaten - not older than four weeks - and as whole egg homogenate or egg yolk homogenate with 1: 1 physiol. Saline or PbS buffer solution in the pH range around 6.0 to 7.5 processed to whole egg or egg yolk powder or to freeze dry material (lyophilisate) under low-germ conditions. The whole egg or egg yolk homogenate is processed and packaged without further additives and used in poultry feeding or resuspended via drinking troughs. This end product is obtained from the egg and / or egg yolk raw mass a) as a powder via spray towers according to predetermined temperature-time parameters between 68 ° and 95 ° C by heating with at least 95% dry matter = maximum 5%

Residual moisture, b) as lyophilisate (freeze dry goods) via freeze drying systems, resuspendable, with max. 3.5% residual moisture.

Example 3:

The new products produced according to Example 2 are rich in specific IgY and processed into egg yolk or whole egg powder and / or lyophilisate and have a specific immunotherapeutic effect. They are orally mixed 1% to 5% in the finished feed or 2% to 5% resuspended in the drinking water and administered for 1 to 6 (8) weeks - if necessary repeatedly - administered.

The use via feed admixture and / or as feed additive takes place, for example, in the case of farmed poultry when there is an indicated risk, mono- or polyvalent specifically in the rearing and fattening phase and / or during the laying period or in the reproduction phase of the useful poultry as well as in the laying rest of the breeding animals against the viroses (parvo, adeno- and / or reovirus) in the water poultry and / or. enterotoxic E. coli strains and Pasteurella multocida ssp. infections in laying hens taking into account the diagnosis, the infection pressure, the economic and epizootiological loss relevance as well as the stress prevention and animal welfare.

Literature:

[1] KLEMPERER, F .: About natural immunity and its use for immunization therapy. Arch. Exptl. Pathol. Pharmathol. 31 (1893) 356 - 382) [2a] KOBILKE, H .: Reasoning and advantages for the implementation of research projects on animal experiment replacement methods and the production of polyclonal antibodies from the SPF chicken for diagnostics and therapy in medicine, veterinary medicine and life sciences. Template for the BMG, the MELF and MWFK in Potsdam and conference participants of the GV SOLAS 9/94 Munich and the BIOTEC / MEDICA 1 1/94 in Düsseldorf (4 and 8/1994) - technical argument [2b] KOBILKE, H., GIPPNER- STEPPERT, C. and SCHEUBER, H.-P .: The SPF chicken - an alternative to obtaining highly specific mono- and polyclonal IgY antibodies from egg yolk for immunodiagnostics and therapy in medicine and veterinary medicine. XXXII. Wiss. Day. d. GV SOLAS, Munich, September 12-15, 1994, abstracts and posters, conference report p. 85 [3] SCHADE, R. and HLINAK, A .: The extraction of specific antibodies from the yolk of chicken eggs - an attractive and bloodless alternative to the extraction specific antibody in mammals. Mh. Vet. Med. 48 (1993), 91-98

[4] SCHADE, R. and HLINAK, A .: Egg yolk antibodies. State of the art and future prospects Altex, Supplement-Volume 13 (1996), 5-9 [5] IBURG, M and OLTMER, S .: New strategies to prevent diarrhea in young animals Lohmann Information Sept. - Dec. 94, 5- 13

[6] OLTMER, S .: News on egg immunoglobulins for young mammals. Lohmann- Information 2/98, April - June 1998, 23-28 [7] ERHARD, M. and LÖSCH, U: Use of yolk antibodies against diarrhea in: Lectures of the Wiss. Conference from October 20/21, 1993 on "Current topics in animal nutrition and processing" Lohmann - LTE GmbH, Cuxhaven [8] LÖSCH, U .: How do the antibodies get into the egg? Altex, Supplement Volume 13 (1996), 15-17.

Claims

claims

1. Specific IgY egg yolk antibodies, characterized in that egg yolks with a high content of highly specific polyclonal vitelline antibodies are permanently obtained from according to an immunization scheme, which is based on highly immunogenic living or killed pure mono- or polyvalent homologous or heterologous antigens, immunized hens, whereby Pullets are immunized over the entire laying period of 12 to 13 months and repeatedly boosted.

2. Obtaining specific IgY egg yolk antibodies, as described in claim 1, after basic immunization of hens, characterized in that to generate a permanently maximum antibody level in the egg yolk pullets according to the following immunization scheme over the entire laying period of 12 to 13 months are immunized: basic immunization = 1. Immunization 1. Booster

= 2nd immunization - start of the antibody spike reached -2. Boosting = 3.

Immunization, 3rd booster = 4th immunization, 4th booster (booster vaccination) = 5th immunization and use of the immunized laying hens up to the 17th or 18th month of life.

3. IgY antibody according to claims 1 and 2, obtainable in eggs from hens of the laying direction, the first immunization on day 0 subcutaneously with antigens and Freund's complete adjuvant (FCA), the second immunization between the 25th and 30th day, preferably on the 28th day with antigen and incomplete Freund's adjuvant (IFA) or aA (other alternative adjuvant), in the period between the 35th and 45th day the antibody obtained reaches the start and during this time the production and processing of egg yolk begins , the third immunization between the 51st and 61st day, preferably on the 56th day with antigen and IFA or aA, the fourth immunization between the 130th and 150th day, preferably on the 140th day with antigen and IFA or aA, the fifth immunization between the 240th and

265th day, preferably on the 252nd day with antigen and IFA or aA and the end use is between the 490th and 500th day.

4. Obtaining IgY Antiköφer according to claim 2, obtainable in that immunized SPF or preferred pullets of the laying direction are immunized subcutaneously or intramuscularly and the antigen dose 100 ug (0.1 ml) to 1,000 ug (1 ml) in 0.5 to 1.0 ml suspension (sterile) is divided into 3 to 4 injection depots.

5. IgY antibodies according to claims 1 to 4, contained in whole egg homogenate and whole egg powder, which are produced under low-germ conditions.

6. IgY antibodies according to claims 1 to 4, contained in egg yolk homogenate and egg yolk powder, which are produced under low-germ conditions.

7. IgY antibodies according to claims 1 to 4, contained in whole egg homogenate and egg yolk homogenate as lyophilisates, which are produced under low-germ conditions.

8. IgY antibodies according to claims 1 to 4, contained in whole egg powder or egg yolk powder or lyophilisate as a component of animal feed and / or animal trough to reduce the infection pressure in and displacement of the field virus from the animal population.

9. IgY antibodies according to claims 1 to 4 and 8, contained in finished feed or drinking water for poultry, which in particular against viroses - parvo, adeno- and / or reovirus - and against enterotoxic E. coli strains and Pasteurella multocida ssp. Infection is susceptible.

10. IgY antibodies according to claims 1 to 4, as a basis contained in monospecific ELISA kits for accompanying diagnostics, titer and quality control and titer development.

1 1. Use of specific IgY egg yolk antibodies according to claims 1 to 10, characterized in that the egg yolk powder and egg yolk powder and lyophilisates, which are rich in specific IgY and have a specific immunotherapeutic effect, are given to ready-made poultry via feed or drinking water, in particular against viroses - Parvo, adeno- and / or reovirus - and against enterotoxic E. coli strains and Pasteurella multocida ssp. Infection is susceptible.

12. Use of specific IgY egg yolk antibodies according to claims 1 to 10 as a starting point in monospecific ELISA kits for accompanying diagnosis and titer quality control and titer development.