DE2026045A1 - Process for the desensitization of cell lysates with halogenated hydrocarbons - Google Patents

Description

Certain organic solvents and halogenated hydrocarbons have been used as agents for Purification of suspensions of viruses or virus antigens described on animals, on pieces of tissue or parts of organs or have been cultivated on isolated cells, in particular for the purpose of reduce the protein content.

For example, Xther, acetone, Chloroform, carbon tetrachloride, hexachlorobutadiene, Tetrachloroethane, perchlorethylene, trichlorethylene and the freon species received attention. Among the related work of various authors can .man name the following *

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Armbruster (1957) - Brown-Cartwright (I960) -Bachrach (1053-1960-1964)

Klemina et al. (1965) - Klimov and Mltarb. (1961) Mussgay (1959) - Prostlakov (1963) - PyI (1956) Schijfer-Armbruster (1954), regarding foot and mouth disease,

Epstein (195Q) - Gessler (1956) - Kaplan-Vaientine (1959) Marquardt (1963-1966) - Lanterose (1962), re Vaccina,

Norcross (1963) on the herpes virus, Halonen (1963) regarding the ECHO viruses,

Huxnmeler-Hamparian (1957) , Manson and Hitarb. (1957) regarding the polio virus,

Werner (1957) regarding adenoviruses, Van-Aert (1966) regarding swine fever,

Franklin (1959) regarding Bncephalomyelltia der

Mouse and various other rabies viruses.

Hamparian (1958), Halonen (1962), Isupov-Tagirova (1964) Gesseler (1956) - Shemanova-Ageeva (1964).

Other authors have commented on the value of the same solvent and halogenated carbon dioxide in the isolation or purification of tumor antigens reported, for example: Beaty-Hodes (1966) - Radzikhovskaya (1565) Stones-Moore (1959) - Taylor (1959), also with the aim of reducing the protein content.

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It has now surprisingly been found that. the halved hydrocarbons also have a desensitivity Chattering effect on lysates of cells that have mechanical or are physically preserved and antigenic to the recipients for whom they are intended are heterologous (or homologous). ■ "

In addition, these are halogenated hydrocarbons

enables the sensitizing antigenic cells to: - functions or constituents contained in the cell sucker rates are, for example, the multiplication of viruses in vitro have guaranteed, in part or in full, depending on to ellminate according to the methods used.

Know these nensitizing antigone substances

are available, so they call to the Frapfungern severe intolerance disorders that generally among the allergic manifestations, be enqueued until the death of the recipient can lead, and the easier the higher the higher the doses administered are and depending on the 19er these: ,,.

administered or the more sensitive the'Era- * * '*' is pfanger.

Are these sensitizing substances, whose chemical components are complexes of proteins, lipids and polysaccharides, partially or completely removed, it is found that when it comes to vaccine preparations acts whose protective effect is not only at least as good as that of the raw material> but that these are also .completely compatible and not disturbing Cause side effects in recipients for whom these substrates are initially antigenically heterologous or were simply homologous.

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The method of the present invention includes "<Sas cell lysate to be desensitized with a halide Treat hydrocarbon.

In particular, there is the method according to the invention therein, a suspension of the cell lysate to be desensitized and a halogenated hydrocarbon to form, to keep this suspension at low temperature, to homogenize it in a colloid mill, centrifuge the homogenate obtained and repeat this cycle several times.

According to a particular implementation of the invention Process is a suspension of cell lysate and halogenated hydrocarbon using a content of about 30 percent by volume of halogenated Hydrocarbon brought to a temperature of about 0 to 4 degrees C, then in a Colloid mill homogenized and then centrifuged for 30 minutes at at least 2500 g, and this cycle is repeated about 2 to 7 times.

The halogenated hydrocarbon is preferably trichlorethylene or a freon, but can be also all other halogenated hydrocarbons - especially chloroform, carbon tetrachloride, Hexachlorobutadiene, tetrachloroethane and perchlorethylene suitable.

The cell lysate to be desensitized is preferential a lysate of line 21 of kidney cells from newborn HaT.ster (Babv Hamster Kidney BHX. 21), which mechanically or is obtained physically or by exposure to various viruses. It should be noted, however, that the

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The invention generally relates to various cell lysates, virus suspensions or vaccines which are / can be used according to the techniques with alcohols according to Bachrach <5 or according to the two-phase techniques with aqueous media according to Albertson.

The invention is further illustrated by the following examples explained.

example 1

This example serves to demonstrate the desensitizing effects of halogenated hydrocarbons to show mechanically or physically obtained cell lysates, the different cells being homologous or heterologous to the species for which the vaccines which they ultimately produce are intended should.

Line 21 of the kidney cells from newborn hamsters (baby hamsters Kidney BHK 21).

Preparation of the raw cell lysate

The culture media for the various cells used are those described for the cells themselves Media or any other classic media or for the cultivation of tissues and cells of the type in question adapted media or media produced by derived from the aforementioned media by changing the recipe.

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The cells are grown in suspension or in various devices that allow the extraction of unicellular Allow layers to be formed by sedimentation in their culture medium has been obtained. The cells are initially abundant and at least four times with isotonic Washed phosphate buffer from pH 7/2 to 7.4, to remove all traces of the tested cell itself from freezing antigens, if it is sera, of vitamins, antibiotics, wetting agents, thickeners or to free analogous materials. The washed cells are centrifuged for one hour at 500 to 1000 rpm, concentrated.

The cells are physically and mechanically by successive freezing and thawing in hypotonic or isotonic medium or by crushing with the Ultra-Turax or by exposure to it lysed by ultrasound or by a combination of these various means.

The lysate or minced product of cells is dosed at high speed for one hour with 25,000 g centrifuged and, for example, over asbestos plates or a plate made of cellulose acetate, e.g. fine-pored Membranes of suitable gradation filtered. Finally, the protein content and the nitrogen content are determined.

Preparation of the desensitized cell lysate

Such a cell lysate is then desensitized in the following manner: Take 30 volumes halogenated hydrocarbon and 70 volwiiaa lysate, which are brought to a low temperature (0 to ψ 4 degrees C), and homogenized by means of an Ultra-Turase with twice 30 seconds each with a time interval

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of 30 seconds (you can

also every other proportion and every other Apply duration). The emulsion obtained is centrifuged at at least 25OO g for 30 minutes. Man leads 2 to 7 consecutive work steps through and determined after each treatment the content of proteins and lipids.

Testing of the SenBlhill3ier \ m "in guinea pigs with deaensilized Eelly sat

All tested with this desensitized cell lysate Individuals, for example pigs, cattle and the like, reacted in the same way,

but since "guinea pigs as experimental animals "

proved of choice, since it is particularly well sensitized by all foreign antigens introduced into its organism.

In the case of the guinea pig, in particular, the Prove sensitivity to foreign substances by the guinea pig is exposed to these substances for the first time and then after a period of time of 15 or 30 days by administration by venous route or by administration by intracutaneous route Weg subjects a second contact with a test lysate, that is genetically homologous to the substances used for the first contact,

Animal that has a sufficient amount of sensitizing harmful substance will receive

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called "sensitized" if it is at the time of intravenous administration of the antigenic fraction homologous to that used for the first contact has served, dies. In this case death is due to a typical anaphylactic peak.

Knowing which honologous antigenic fraction is administered into the animal's skin shows up the presence of a possible £ onfs.tbil.lsicrun <T by the appearance of an erythematous point within 24 hours of the intracutaneous reaction, which is an allergy that is delayed Body sensitivity is called, which also occurs in pigs and cattle occurs.

The sensitization of the guinea pig occurs by that administered by the parenteral route

raw cell lysate alone at a dose of 10 mg equals,

When the lysate is combined with an adjuvant, the dose required is lower. It is, for example, 1 mg with the incomplete adjuvant according to Freund.

A, Testing of the sensation of guinea pigs with raw lyeates or lyeates treated according to the invention

The sensitivity of guinea pigs inoculated subcutaneously with 13 mg proteins from crude lysate of cells BHK is determined here for comparison

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are, with the administration three times consecutively takes place at time intervals of 8 days, and from! 11 mg proteins from Lvsat from cells BHK, which twice treated with 30% trichlorethylene, are vaccinated, the administration twice in succession takes place at 8-day intervals.

The detection of the sensitization is done by venous route with lysates with a content of 1/1000 of crude proteins or 1/1000 of treated proteins.

The results are compiled in Table I below.

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Tabel

Guinea pigs vaccinated with raw lysate C 13th

vaccinated with treated lysate Guinea pigs (total dose:.

11 mg)

raw test lysate i.ü.

Concentration 1 t

.. all dead .. all dead

administered doses 5 ng

β ng

0 shock 0 shock

treated test lysate i.o., concentration 1 t

Cans?

0 shock 0 shock

administered doses s 2 rg 5.5 ng mg

0 shock 0 shock 0 shock

fs »O

CD

- Il -

From the table above it can be seen that

- those vaccinated with the treated Lyaat and with the raw lysate or the treated Lyiat-tested guinea pigs survived: no case of anaphylactic shock;

- those inoculated with the raw lysate and with the treated one Lysate-tested guinea pigs survived: none

Case of anaphylactic shock;

- those inoculated with the raw lysate and with the raw lysate tested guinea pigs died: anaphylactic shock.

The treatment of the lysate · with Trlehlorlthylen has at Use the same for the person he is contacting or 41 · Test reaction thus eliminating the need to flex the needle ends

or drawing items. '',

^D * ^Verguc ^ ^ * ^g Zanzibillization of guinea pigs with raw or treated lysates », lusamites ralt an Adjuvana , ·

One leads an awareness series

with a raw cell lysate BHK, mixed with 50% incomplete adjuvant according to Freund. '

The administration takes place in such a way that group of ' Guinea pigs intramuscularly in the Thigh 0.45, 0.90 and 1.8 ng of lysate, respectively

as protein weight.

You conduct another series of experiments with one Carry out BHK cell lysate, but before using the cell lysate, two treatments with 30% trichlorethylene according to the working method described above «, Sen was. ₀₀₉ e _{t8 / 1 | 87}

The packaged lysate is incomplete with 50 Freund's adjuvant mixed. One also administers 0.45, 0.90 and 1.8 mg treated lysate, respectively, expressed as weight protein.

One month later, all groups are tested venously with 1 ml of hollow cell lysate containing 15 mg of proteins.

The guinea pigs vaccinated with 0.45, 0.90 and 1.8 mg of crude lysate are found to have anaphylaxis Die in shock.

Those treated with amounts of 0.45, 0.9 and 1.8 mg Lysate-vaccinated guinea pigs survive.

It can be seen that treatment with trichlorethylene eliminates the sensitizing substances.

Example 2

This example is intended to demonstrate the de-sensitizing effect of halogenated hydrocarbons on preparations intended for the production of vaccines. The cell lysates treated in this way are cell lysates which are obtained spontaneously by the action of various viruses on cells BHK 21, in particular among others with rabies viruses, Newcattle dieease viruses, viruses of the plague, viruses of foot and mouth disease and the like. In the present case, viruses of three European OAC species of foot and mouth disease were cultivated on BHK 21 cells under certain conditions with regard to Dcu «* r, pH value and temperature.

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The desensitization took place in virulent

or attenuated suspensions with deactivation agents

materials, such as, in particular, formaldehyde; Glycidaldehyde, acetylethyleneimine, ß-propiolactone and like that.

The general technique of desensitization and that of testing the state of sensitization are the same as given in Example 1 above.

virulent suspensions are never desensitized, which are concentrated or not concentrated, centrifuged and filtered, according to the common techniques for making vaccines that are intended for species susceptible to viral disease.

A. Testing of desensitized lysate for suppression of anaphylactic shock in guinea pigs vaccinated with raw vaccines

Guinea pigs are treated three times at intervals of 15 days with a formaldehyde, .-> '' · - ' alumina and 'saponin treated'. Vaccine against foot-and-mouth disease, type C, obtained on cells BHK 21, vaccinated '.

These guinea pigs will then go through venous administration of 5 mg of lysate obtained by a physical process from cells BHK is tested, they die of anaphylactic shock.

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Cordon the guinea pigs with the same amount (5 ng) lysate treated according to the invention tested, this is how they survive with no case of anaphyxactic shock.

The same observation is made with guinea pigs that have received four vaccinations.

The purification of the test fly according to the invention thus switches off the sensitization factor.

B. Testing of vaccines desensitized to suppress sensitization; Evidence by a Testing with a raw lysate

This experiment was carried out with guinea pigs. ,

One puts a suspension of virus of the mouth "and Mouth disease, type C, cultivated on cells BHK 21, produced by mild exposure to glycidaldehyde is inactivated.

The inactivated virus is divided into three batches:

normal virus called rqhvirus; ... virus treated with trichlorethylene ? Virus treated with Freon 113.

Each batch is mixed with 50 Freund's adjuvant to give three finished vaecins;

. Raw vacuums

Treated with trichlorethylene © Vaccine Vaccine treated with Freon 113.

These 3 batches serve to unite three groups of iron guinea pigs, each being

intramuscularly into the thigh in quantities of 1 ml, 0.5 ml b2w. 0.05 nl can be administered.

21 days after vaccination, all animals become

by venous administration of 30 mg of crude lysate of cells

BITK checked.

The results are compiled in Table II below »

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Table II

Dose raw vaccine with trichlorethylene with Preon 113

treated vaccines' traded vaccines

ml «· O O

+ O O

+ 0 0

+ 0 O

+ O O

0.5 Bl + 0 0

+ 0 0

+ O O

■ ♦ ■ 0 0

+ 0. 0

0.05 al 0. 0 0

0 0 0

0 0 0

0 0 '0

OO ¹ O

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A dose of 0.05 ml raw vaccine is insufficient to allow the awareness to occur, as well also to achieve protection, as will be shown below. · ·

Sensitization occurs with 0.5 and 1 ml raw vaccine on, and the animals die of an anaphylactic Shock.

The same doses of desensitized according to the invention Vaccines do not bring about any sensitization.

The treatments with trichlorethylene and with Preon have thus switched off the elements that lead to sensitization of the vaccinated animals.

In the following it is shown that the halogenated hydrocarbons the protective value of terminal vaccines, taking into account the infectious titers in the various vaccinated species, e.g. in cattle, pigs, guinea pigs and the like, Don `t change.

C, Vaccination comparison tests in guinea pigs with raw vaccines and vaccines desensitized with trichlorethylene or freon

The guinea pigs of Experiment B were on intracutaneous plantar Ways tested with 10,000 infectious doses.

The disease is manifested by foot blisters (A) (others than the primary focus on the vaccinated

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straighten hind paw occurred) on the other three limbs C days after the virulence test.

The results are shown in Table III below compiled;

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Raw vaccine Table III • dose 0 with trichlorethylene
deeensitized
Vaccine old freon deeen-
go through
Vaooine 1 ml 0 0 0 0 0 0 • 0 0 0 0 0 •
0 A. 0 0 0.5 ml 0 0 0 - 0 0 0 0 0 0 0 0 0 A. 0 0 0.05 nl A, A. A. A. A. A. A. A. A. .A 0 A. 0 A.

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-JO-

The vaccines desensitized according to the invention are therefore just as good as the raw vaccine.

Example 3

This example is intended to help the sensitizers To show the effect of halogenated hydrocarbons on concentrated virus suspensions.

The vaccine to be treated is a vaccine against haul, and type 0, di® mouth disease on cells. BHK 21 is received.

The blood suspension is filtered on a clarifying filter and then inactivated with Glycidald & hyd and with Aluminum hydroxide and saponin added as adjuvant. A raw vaccine is made from this Vimssuspenslon. Part of this pension will be with trichlorethylene according to that described in Example I. Procedures dealt with. The viruses desensitized in this way are dried by lyophilization. The lyophilisate is then placed in distilled water in an amount sufficient to provide the following protein levels to achieve, added: *

The protein contents are expressed as:

Protein precipitable by trichloroacetic acid 1 "Protein determinable by the Folin reaction."

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Vaccines desensitized with trichlorethylene

0.05 g per liter of rait TriehloressigsHure precipitable protein

0.70 g per liter of what can be determined with the Folin reagent protein

Desensitized with trichlorethylene and focused

Vaccine

0.49 g per liter of protein which can be precipitated with trichloroacetic acid

2.4 g per liter of what can be determined by the Folin reagent Protein "

Raw vaccine

0.52 g per liter of trichloroacetic acid precipitable Protein "

0.88 g per liter of protein determinable with the Folin reagent.

These vaccines were administered to groups of 7 guinea pigs with two vaccinations at intervals of 3 weeks with variable amounts of vaccines from 0.25 to 2 ml.

15 days after the second vaccination all received Animals an intravenous test application with 0.2 ml

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_ ₂₂ _ 2028045

raw BHK cell lysate.

The results obtained are shown in the table below. With the Rohvaeeine, indifferent what the dose used is, nan notes that more than 50% of the animals are fatal anaphylactic Suffer shock. With those who are tired Vaccines, even if the vaccines are of a test content concentrated above or equal to that of the raw vein is} one does not have anaphylactic vision Shock solid.

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dose

Table IV

Vaccine

Results

2 ml

1 ml

B V ' 5 + / 7 tW ² * 0/1 TCE Κοηε. ^ 0/7 B. 4 + / 7 TOE 0/1 TCE Cons. 0/7

0.5 ml

TCE

TCE Cons.

4 + / 7

0.25 ml

TCB Cons.

TCE

0/7

(1) Raw vaccine

(2) Trichlorethylene desensitized vaccines

(3) Desensitivity with trichlorofithylene and concentrated vaccines

(4) 5 deaths in 7 animals tested.

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Example 4.

In this example an experiment on the sensihili description of young cattle.

The vaccine to be treated is a trivalent vaccine, (OAC), which is produced from virus suspensions, which are suspended from cells by BHK cells

come. ·

The virus suspensions are infected with glycidaldehyde

■ ·

^ fourth and with aluminum hydroxide and saponin as ™ adjuvant added.

A Rohvaccine is prepared from dieter virus suspension, and a desensibllieierte Vaccine is prepared from the same Virueeuspeneion, but aim by the method of Example 1 was treated with trichlorethylene.

Each vaccine will be groups of IO calves of 6 months administered with 3 consecutive vaccinations. Between the first and second Vaccination is an interval of 1 month and between fe the second and third vaccination a distance of 6 months.

Testing of anti-Hanseatic antibodies

The anti-haroster erythrocyte hemolysins and HSmagglutinins are checked 15 days after each vaccination. The table below shows the average content of the antibodies.

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Table V

Raw vaccine

With trichlorethylene desensitized Vaccine

1. Vaccination:

- hemolysins

- hemagglutinins

1/32 1/32

1/16 1/8

2. Vaccination:

- hemolysins

- hemagglutinins

1/256 1/128

1/16 1/52

3. Vaccination:

- hemolyin

- hemagglutinins

1/16

1/128

* The serum diluted to 1/32 still has a haemolytic effect

and hemagglutinizing effect on the red blood cells of the hamster.

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Table V shows that treatment of the vaccines with trichlorethylene reduces the amount of heterologous antigen *! *, As shown by the reduction in the antibody response .

Examination of Immediate type sensitivity

15 days after the third vaccination, the animals received an intravenous sample of 100 ml raw DH ^ -Scllysat not a content by trichloroacetic acid precipitable protein of 5 g per liter. The results obtained are in the table below compiled.

The desensitization "of the vaccines with trichlorethylene considerably reduces the anaphylactic reactions of the immediate type and even switches them off.

Table VI

Dose 1.17.

Raw vaccine

Results

100 ml fatal anaphylactic shocks :. 1 death every 10

fatal anaphylactic shock that developed. 9 survivors award 10 each

with trichlorethylene desen eibilisierte Vaccine

100 ml

lack of anaphylactic shock

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Claims (7)

l. Procedure for desensitization of felly «» ten, mechanically or physically or by action are obtained from different viruses, characterized in that these fellysate with a halogener th hydrocarbon treated. 2. The method according to claim 1, characterized in that that nan produces a suspension of crude cell lysate and halogenated hydrocarbon, this suspension contributes Keep them low in a colloidal chair homogenized, centrifuged the Romofenlsate obtained and repeated this cycle of operations several times. ·. · 3. The method according to claim 2, characterized in that a suspension of cell lysate and halogenated hydrocarbon with a content of about 30 percent by volume halogenated hydrocarbon at a temperature * from et ν? a 0 to 4 degrees C is brought, dl suspension is then homogenized in a colloid mill and the * Homogen! sat for 30 minutes at least. 2500 g is centrifuged and this work cycle is repeated 2 to 7 times. 4. The method according to any one of claims 1 to 3, characterized in that the halogenated hydrocarbon is trichlorethylene or a freon. 009849/1897 5. The method according to one of claims 1 to 4, characterized in that the raw Zellyaafc a lysate of line 21 of neonatal Mieren cells Hamsters are, æaa, mechanically or physically obsolete is. 6. The method according to any one of claims 1 to 4, characterized in that the crude cell lysate is a lysate of cells BHK 21, which by action cir.es virus, such as, for example, from foot and mouth disease viruses, is preserved. 7. fiechanisch or physically or by action Bellysates obtained from various viruses which r.nch the ^ experience according to one of claims 1 to are desensitized. 009849/1897