DD289201A5 - METHOD FOR PRODUCING AN IMMUNOMODULATING VACCINE AGAINST VIRUSES - Google Patents

Abstract

The invention relates to a method for producing an immunomodulating vaccine against viruses. It relates to the production of a combination vaccine with intensified effect and high efficacy against viruses in the human and veterinary medical field. Essentially, the production of the combination vaccine is based on physiological, easily isolatable natural products in conjunction with viruses or viral components or even attentuierten viruses or genetically engineered bioproducts in the form of genetically engineered microorganisms. According to the invention, an extract is obtained from raw potatoes or wheat seedlings or other natural products, from which lectin is obtained as an immunomodulator by uncomplicated centrifugation and concentration steps. In lyophilized form, the glycoproteins with the antigen components mentioned are converted into enteric-coated oral vaccines or parenteral vaccines. In contrast to the production of vaccines without lectin, these preparations produce a highly effective preparation. {Bacteria; Gamma rays; glycoproteids; Immunomodulators; immunostimulants; inactivation; interferon inducers; potatoes; lectins; lyophilisates; Vaccine; viruses; Wheat}

Description

Field of application of the invention

The application relates to the production of an immunomodulatory vaccine with intensified action against viral infections in the human and veterinary field.

The application is possible for facilities / establishments producing vaccines, antibodies and biostimulators for human or veterinary medicine.

Characteristic of the known state of the art

Methods for the production of vaccines against various viral infections are widely used internationally. This includes both the production of parenteral and nonparenteral vaccines. Likewise, possibilities of using immunomodulators are known, but not in the form of lectins. However, methods for isolating lectins are set forth in many ways.

The specific direction of producing a combination vaccine involving lectin isolations, however, has not previously been described. There is also a lack of references to the lectins as immunomodulators in the literature. This applies equally to parenteral and nonparenteral vaccines.

Object of the invention

The aim of the invention are those measures from which a method results, which leads under consideration of application criteria for the production of a combination vaccine with intensified and at the same time broad action against viral infections.

The special production measures result in a vaccine which, in addition to the formation of specific antibodies, also simultaneously triggers an interferon induction and a strong activation of the phagocytosis killing system or an activation of intracellular and extracellular enzymes of alveolar macrophages. Essentially, the production of the vaccine will be based on a physiological, easily isolatable natural product in combination with antigens which are also inexpensive to obtain as inactivated viruses or modified virus components or as attenuated viruses or as genetically engineered bioproducts, in the form of an air, water and acid renatural end-preparation form suitable for oral administration.

Explanation of the essence of the invention

The object to be solved by the invention is to provide such measures that a natural immunomodulator is prepared in such a specific form, in connection with inactivated or disrupted or partially degraded virus or purified virus or virus or virus attenuated genetically engineered bioproducts into a qualitatively novel vaccine with broad specificity against viral infections or a clear activation of intra- and extracellular enzymes of alveolar macrophages or strong activation of the phagocytosis killing system and at the same time interferon induction

 These measures are aimed at the

simple production of the immunomodulator from natural products (eg potatoes, wheat seedlings) in the form of lectins, which have specific binding sites for carbohydrate structures and react differently with cells, but do not trigger any pathological reactions

easy to combine with inactivated or partially split / purified virus / viral components or attenuated viruses or genetically modified bioproducts (microorganisms)

- ease of use of the vaccine without side effects

- intensified w ‡ kung in this combination

- good storability of the vaccine

The object is achieved in that from the natural products mentioned a pressed juice / preparation obtained and optionally mixed with an antioxidant and a simple lyophilized immunomodulator is obtained by uncomplicated Zentrifugations- and concentration steps, in low concentrations after combination with said ingredients and after production an enteric final preparation gives an optimally effective oral vaccine.

It has surprisingly been found that it is precisely these lectin-antigen combinations (for example 2-20 μg of immunomodulator in the form of lectin and, for example, 20 μg of hemagglutinin from influenza viruses) that can bring these high effects against viral infections.

The process according to the invention is suitable for the production of optimally active oral vaccines for use in the human and veterinary field and, after obtaining correspondingly purified components, can also be transferred to parenteral vaccines.

embodiments

example 1

For the preparation of a combination vaccine against infection with influenza virus strain A / Leningrad / 360/86, the following preparation steps are necessary:

a) Preparation of the immunomodulator (Rohlectin) from potatoes

- Mechanical comminution of raw potatoes and extraction of the pressed juice. Optionally, add 0.2mg of L-ascorbic acid to 10ml of pressed juice and Zenirifugation for 20min at 6000g.

- Testing of the supernatant (lectinhaltig) on its Lectingehalt by agglutination titers against untreated red blood cells (preferably human, blood group B).

- Depending on the live germ count (> 10Vml) cold sterilization with cobalt 60 gamma rays (<1OkGy).

- Lyophilization (previous deep freezing to -40 ° C) and re-determination of the Lectingohaltes: Determination of Lectingehaltes in pg (using a reference lectin).

- Adjusting for a lectin content, the untreated human B erythrocytes 1: 4 still agglutinated (4HAU).

b) Preparation of the antigen

With a sterile needle, 10-day embryonated chicken eggs are opened above the air-bag boundary and inoculated with a virus dilution (strain A / Leningrad / 360/86). It follows a Nachbrütungsperiode (50h, 36 ⁰ C). Subsequently, the embryos are killed: (20 ± 4) h at (4 ± 2) ^C C, and from the disinfected eggs, the allantoic fluid is sucked by means of a pump via cannula into an infusion bottle. The virus-containing allantoic fluid is centrifuged to max. 5000g freed from cell debris and Eibsstandteilen and stored at temperatures of +4 ⁰ C. After 24 hours of exposure to HCHO 1: (9,9kGy cobalt-60) at 22 ⁰ C. After the addition of max 12500 inactivation of influenza virus by gamma-rays is carried out. 5% by volume of milk (containing about 1.5% total fat) as a stabilizer, the allantoic fluid is lyophilized in a commercial facility

 combination vaccine

The lyophilizates are mixed in such a way that 10 μg of hemagglutinin from influenza virus strain A / Leningrad / 360/86 (= combination vaccine) are obtained on 10 μg lectin from Weizon seedlings. The lyophilisates are added in admixture of physiologically indifferent excipients in a known manner to compacts, z. As tablets, dragee cores, processed, including below

According to the invention, granules, pellets or tablets, dragee cores, granules or pellets or also powder-filled gelatin capsules are to be understood. The mixing ratio between the combination vaccines according to the invention and the tablet excipients is between 1: 100 and 100: 1, preferably between 1:20 and 20: 1.

The granules, pellets or compacts according to the invention or else the gelatin capsules mentioned above are protected

provided with a coating in a known manner against environmental influences and digestive juices of the stomach

Protection of active ingredients in the range of pH 1 to pH 6 guaranteed. According to the invention for this purpose are organic solutions of Polyacrylates and Polymethacrylatan, vinyl polymers or cellulose derivatives using plasticizers such as Polyethylene glycol, dibutyl phthalate, triacetin, castor oil, citric acid ester used for drug protection, with a Intestinal juice solubility is guaranteed from pH 6 within 10 to 60 min. Applikatipn To avoid / prevent / prevent viral infections and activation or reactivation of the defense performance

1 to 3 gastro-resistant tablets administered per os over a period of 1 to 3 months. Another application form, eg. In

Form of enteric-coated gelatin capsules is also possible. Example 2 For the preparation of a combination vaccine against infection with influenza virus strain B / Yamagata / 6/88 are the following Preparation steps necessary:

a) Preparation of an immunomodulator (Rohlectin) from wheat seedlings

- 50g wheat germ are extracted with 250ml extractant. Extractant: 2% formic acid.

- The supernatant is centrifuged for 20 min at 5000g and then filtered again = Rohlectin.

- Testing of the lectin content by agglutination of washed and otherwise untreated B-erythrocytes (human).

- In the case of use as an immunomodulator lyophilization of Rohlectins (previous deep freezing to about -40 ° C) and re-determination of the biological activity of the lectin (agglutination titer).

- Setting the lectin content to a titer of 1: 4 (4 HAU) against untreated B-erythrocytes (human) (with physiological saline).

b) Preparation of the antigen

Until the isolation of the allantoic fluid with influenza virus strain B / Yamagata / 6/88, the procedure of Example 1. Then, after exposure to HCH01: 12000, the virus-containing allantoic fluid is concentrated by means of an ultrafiltration membrane. The final inactivation of influenza viruses is by gamma rays (cobalt 60, 8, OkGy). After addition of stabilizers (5% by volume milk with 2% fat and 3.3% by volume gelatin solution 5%), the concentrate is lyophilized in a commercial plant. The further procedure is as indicated under Example 1.

Example 3 The procedure is as set out in Examples 1 and 2. Instead of inactivated influenza viruses, however, come

attenuated or recombined influenza viruses of types A, B or C.

Example 4

Approximately 61 cell culture fluids, the human Fl cells of which were used to propagate adenovirus type 2 by a conventional method, are centrifuged for 15 minutes at 2000 rpm in a refrigerated centrifuge. The sediment is taken up in 150 ml of phosphate buffered saline, added with twice the volume of chloroform for analysis and mechanically shaken for 30 minutes at room temperature. After renewed centrifugation, the upper aqueous phase can be removed aseptically. At least 2 mg of lectin from potatoes are added to this virus concentrate, mixed and portioned to obtain a high titer antiserum to adenovirus type 2. For this purpose, the combination vaccine domestic rabbits of different breeds is administered intravenously to 2 ml repeated according to a standard immunization scheme. In comparison to an antigen without lectin, the combination vaccine induces multiply higher serum antibody titers.

Example 5

Poliovirus of a commercial poliomyelitis vaccine is inactivated by gamma radiation in a conventional procedure. After addition of lectin, prepared according to the procedure described in Example 1, in the ratio of 1 μg of lectin MOOpg protein, these components are mixed. This mixture is lyophilized in a plant on an industrial scale. The lyophilisate is mixed in a ratio of 1: 1 with lactose and pressed into tablets of about 100 g.

Claims (6)

1. A process for the preparation of an immunomodulating vaccine against viruses, characterized in that from raw peeled or unpeeled potatoes by mechanical comminution and by obtaining a pressed juice from these and optionally by addition of antioxidants, preferably from 0.5 to 20 mg! - ascorbic acid per 11 pressed juice, followed by centrifugation to max. 10000g for 20min in the form ofÜuberstandes a raw or pure preparation (crude or pure lectin), which can be optionally still pasteurized / sterilized is obtained and lyophilized for prolonged storage and further processing / use as a vaccine component after prior deep freezing to about -4O ⁰ C. or that from wheat seedlings dried at 35-4O ⁰ C or in the native state, with organic acids, preferably formic acid or acetic acid, or other extractants such as hydrochloric acid or phosphate-buffered saline, a crude extract is prepared and the decanted supernatant to max. 10000g over a period of max. Centrifuged for 30 min and then filtered and after determination of the immunomodulator contained in the filtrate, in the form of lectin by agglutination of washed and otherwise untreated B-erythrocytes (human), optionally still pasteurized / sterilized and lyophilized or that from the seeds peanut, pea, pea, broom, sapling, soybean, laburnum, lentil, lima bean, common bean, twine tree, gorse, vetch, horse, broad bean, pokeweed, pea, robinia or other natural products. Lectins are obtained in a modified manner and lyophilised the resulting lectins having a biological activity which may correspond to between 2-50 μg of pure lectin / dose, lyophilized inactivated viruses or viral components or attenuated viruses or genetically engineered bioproducts in the form of genetically manipulated microorganisms or preparations, optionally before Lyophilization are still mixed with stabilizers, and these combinations eventually in the form of tablets, dragees, granules or capsules in an air-, water- and acid-resistant enclosure of, for example, cellulose acetate phthalate, with a plasticizer and organic solvents are included. 2. The method according to claim 1, characterized in that in the combinations still bacteria or modified bacterial components are mitverarbeitet. 3. Process according to claims 1 and 2, characterized in that vaccines for parenteral administration can be prepared with highly purified preparations. 4. The method according to claims 1 and 3, characterized in that in the preparation of a mixture of lectin and antigen, the specific binding between these components is exploited in such a way by the precipitation of lectins, a precipitation and thus purification and concentration of the viruses. 5. The method according to claims 1, 3 and 4, characterized in that targeted coupling to / into the lymphocytes is achieved by a coupling between lectins and viral antibodies or antivirals and thus a general immunosuppression is counteracted. 6. Process according to claims 1 to 5, characterized in that highly-specific antibody preparations are prepared from the raised antibodies.