DE1792609A1 - Process for the production of a vaccine - Google Patents

Description

Prcf, i) r, Π ie tli he J η cherts Medical college institute of microbiology

i lan η over - ^ k lje ejF £} Jl el.

The representatives:

Dr, Friedrich lier. ^ I.-inii

Dr, Gerhard Irnisch

Dr. K ar 1-1 · 'rl ed rich Gross

p.> \ ur, Farbwerke Hoechst AG Vorr.aSs' leister Lucius q 3 riming - Patent Department -

24. Se ρ tent) er

Appendix I.

for patent application Fw 5874 Λ - Ma 11ο Process for the manufacture of a vaccine

At present, either so-called dead vaccines, which contain inactivated pathogens, or so-called live vaccines, which contain avirulent pathogens, are used for active innuinization. In addition, toxoids (detoxified bacterial toxins) s-owic subunits of viruses are used as vaccines. Dic'se herkön; nlichen vaccines make high demands on the care of the manufacturer;> eJ ύοη Totvaccinen nut to monitor the Inaktivierun.fi sorfjfälti ^ since insufficient inactivation; of the natural antigen can lead to undesirable side effects in injection, but on the other hand the natural antigen can be changed or damaged to a certain extent by the inactivation, also an insufficient attenuation of the viruses in vaccines

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. Viruses capable of replicating could have unmistakable effects Therefore, in the production of vaccines, complex, time-consuming and costly controls are necessary. Nevertheless, intolerance reactions have been described

According to the invention, these disadvantages can be overcome by not using the antigen itself for active immunization, but rather a so-called "regulator protein" is used. This regulator-α protein was surprisingly found in studies of antibody synthesis detected in immunologically competent cells. The '' processes involved can be briefly summarized

■ explain as follows: ■ x ^N -. '

* ■ ■>

If macrophages are stimulated with an antigen, these cells synthesize a ribonucleic acid (R? \ ^! S), which can be transferred to lynphatic cells and induces them to synthesize antibodies. The RNA contains the information for the amino-

■ acid sequence of the antibody to be formed and is therefore called "informational RN ¹ S" in the following.

The antibodies produced by them have the following properties:

a) They are antigen-specific, i.e. with regard to their immunological Specificity homologous to the antigen offered to the macrophages;

b) They have the same allotypic characteristics as the donor the macrophages,

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The informational RNA is species-specific, i.e. it can. can only be successfully transmitted within the same species, nine transmission from one species to another does not lead for antibody synthesis. . . · · - *

So if you isolate the informational RNA from customs of an individual Λ and add this RMS to lymphatic cells of another individual ß of the same species, the latter initially form antibodies with the allotypic characteristics of individual A (-indivldual-specific foreign antibodies), after some time they synthesize. However, antibodies with the allotypic characteristics of the individual B (= individual-specific ejLgene_Antikkörpers), This change in the production of individual-specific foreign antibodies to the formation of individual-specific antibodies takes place through a switching process that is controlled by the regulator protein. The regulator protein is a subunit of the antibody formed by the informational RNA in lymphatic cells, '..

The invention therefore relates to a process for the production ' a vaccine, which is characterized in that in a cell-free system by means of informational RNA antibodies and The regulator protein is synthesized, the regulator protein is isolated, it is sterilized and, if necessary, an adjuvant is added. The invention also relates to the vaccine obtained by this process. . .

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109884/1819

Bs has already been suggested using microorganisms To immunize informational RX S, D Appropriate attempts z, i3, on .houses, guinea pigs and monkeys are undertaken been. Here, however, the following stages had to be observed in the organism be run through:

1, receiving a specific individual foreign RNA 2, the synthesis of individual-specific antibodies fre mden

3, synthesis * of regulator protein

4, Synthesis of individual-specific own informational RNA 5. Synthesis of individual-specific own antibodies

By the method according to the invention, in which the regulator protein even for immun i s ie run f! is used, the fall Stages 1 - 3 in the macroorganism gone, one extremely rapid and Safe immunization is possible in a diosc manner. procedure for the production of vaccines, which are an antigen-free Regulator protein, are not yet known,

The informational R ^ S _s can obtained from immunologically competent cells with antigens by stimulating a culture of immunologically competent cells or a cell-free system. "Immunologically competent cells" are to be understood as meaning cells which are able to react to a primary antigen stinulus with the formation of informational RNA. The competent cells are in the lymph, in lymph nodes, in the spleen, in exudates - for example in the

under

Peritoncalex .'- 'udat - and especially the leukocytes

of the peripheral

Blood · Here cells can be of human or animal origin

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■■ ".. 179260S. ·.

be, depending on whether dio according to the invention erlialtc.no Vaccine at Should be used for humans or animals. To !! reap a , Vaccine for a particular species is therefore expedient of immmolof.i5J.ch competent cells of this species went out.

Antigens and toxins from bacteria, viruses or their subunits as well as snake poisons are used to stimulate the immunologically competent cell material the metabolic or breakdown products of jJO-rde Tella pertussis, Mycobarterium tuberculosis as well as 5a, lmonel len, ßrucellen Shigella and streptococcus, viruses and their subunits are e.g., H ₁ influenza viruses InfluenzahaeiMagglutinin, polio viruses or their Pxoteinhülle, _Ma3ern-> Toiiu ^f ut- ·, MkS-, smallpox-, Schv / eitiopesl; -, distemper-, ileptatitis contagiosa canis-. .as well as encephalitis viruses,.

If you go to the extraction of the informational UN'S! by oinow cellular system from., so! .anti you proceed as follows:

A cell culture iranunologi ', ch kftnpetenter lines (to .sei -, wio i human leukocytes) is nit to be sowed beforehand: i ntiiiiura optimal dose of the antigen ("iir'dov rule an antl ^ eapari i 1 i; l per loo cells) stimulated uuti S ~ 60 minutes, vorzugsuei va about 2o minutes at ca, 'M incubated ⁰ C, Then the cell culture is quickly cooled to mindestyns "ϊ ° Π, and washed. Au .; U ^ n

109884/1819

washed cells, the informational \ VA "> is isolated according to known methods, for example by means of phenol, so that it is free of proteins and nuclases»

If you go from. a duty-free system, then ν rC if \ όί ~ parthafi "as follows:"

Washed _} iinr> unolo <* i sch competent cells (?, Ui.i example human or animal "leukocytes from the peripheral" blood) are in a buffer solution of the holarity 0.0I - 0.15, preferably about 0.1 from pH-V.'ert 7.6 - 8.6, preferably about 7.8, to; -c noninen, A; a tris / maleic acid or tri.; /

"" N

HCl buffer. The cells are destroyed by thawing and thawing out five animals and the customs homogcnat is about 3o minuton at 3o, ooo % unJ U ~ 'C or below ^{0 0} C, centri τυρί ο π -, The supernatant obtained after centrifugation int ά \ ν zellfxv.u; Syr.tei.i, there: -. contains all soluble ingredients of the Zeilun, Anscli liei'onc; is it with e'jior optiv.ialan dose of a «vitspi-eclieriden antigen r> tiinulie t- and 2 · 60 minutes lan-- ;, vorr-'igsw't-i-; e about Io MiiiuLen i ·,:; ;,: ··:.; vAa; ü 3 / ^l 'C inkub j. « ⁵ r i., Die Weilert: Ve j iriici tini.", cr!' n \ ,. j i-'iv- über ei.-.

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long, incubated at 37 ° C. The entire batch is then centrifuged through a gradient of 5-25% sucrose for 15 hours at too ooo g. The term "gradient" means that the concentration of the .Sucrose from the 3-ode of the centrifuge beaker continuously decreases upwards, from 25 to 5%. This gradient is overlaid with the protcinol solution. The proteins sediment during centrifugation, depending on their sedimentation coefficient, differently - far towards the bottom of the centrifugal cup. After the centrifugation, the beaker is drilled at the bottom and the contents collected in fractions. The concentration gradient of the sucrose prevents premixing of the protein fractions. The individual fractions are then tested for their ability to initiate the synthesis of antibodies. For this purpose, nan uses a test system that is produced in the following manner:

A cell suspension of immunologically competent cells, e.g. 3, leukocytes, spleen cells or periΐoneal exudate cells, is frozen, the bisblock is crushed and thawed. The resulting suspension is centrifuged for 30 "to 60 minutes at 30,000 g, the sediment containing the cell trimmer is discarded and the supernatant is centrifuged for 3 to A hours at 50,000 g. ο set, whereby a precipitate separates out, which is centrifuged and dissolved in Tris buffer with a pH of 7.8. The solution, which is referred to as "nil 5 fraction", contains the enzymes and building blocks that are necessary for the construction of the Antibodies are needed.

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The test system consists of a pH 5 fraction and DNA, which according to Kirby (Isolation and fractionation of nucleic acids _r ' in Davidson and Cohn, "Progress in nucleic research and molecular biology", VoI 3, Academy Press 1964, New York-London) can be obtained.

Those fractions that are able to stimulate the synthesis of antibodies in this test system contain the regulator protein, They are sterilized and, if necessary, with a Adiuvans (for example aluminum hydroxide) are added. The product represents the finished vaccine. ·

According to the “method according to the invention, vaccines against all pathogens or poisons mentioned in the description can be obtained. Your form of an injection, feet _(r satisfy a safe vaccination löv ^ per kg of animal; the

The quantities required are therefore several powers of ten below those required for conventional vaccination.

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B is ρ ie 1 e. ^; ", '_'

1) By stimulating a cell-free system of immunologically competent cells from mice by means of Influcnzavirus-Haämaggiutinin, an informational RNA is formed and allowed to act on a cell-free system of immunologically competent cells from mice. The regulator protein formed in this way is isolated in the manner described and injected intraperitoneally into mice treated at the birth. Blood is taken from the mice at intervals of 2 days and the antibody titer is determined. Antibodies can be detected in the blood of the mice as early as 2 days after application of the regulator protein. The determination with the blood of control animals was negative,. '""-' ·. '

2) In an analogous way, a regulator protein for the synthesis of informational KAS _t, which triggers the formation of antibodies against measles-IIemagglutinin in guinea pigs, is obtained and injected intraperitoneally into untreated guinea pigs. At intervals of 2 days the guinea pig blood is taken and the antibody titer bestinmt, already 2 days after application of the regulatory protein, antibodies were detected, however, not in the blood of control animals ·, in the blood of guinea _pigs.

3) In an analogous way, a regulator protein for the synthesis of informational R \ ^T S, which triggers the formation of antibodies against inf luenza-ilanagglutinin in monkeys, is obtained and

injected intraperitoneally into untreated monkeys. In Λ 2 days the monkey blood is taken and the

109884/1819

. Antibody titer determined. Just 2 days after application of the regulator protein, antibodies could be detected in the blood of the monkeys. No antibodies were found in the control animals to be established.

4). The «einäß Example 1 obtained regulator protein for Synthesis of informational RNA that leads to the formation of Antibodies to influenza virus hemagglutinin in mice is started intraperitoncal in untreated mice. - injected, 8 days after the application of the regulator protein a stress test is carried out with these mice. the Mice turn out to be virulent after exposure Influenza virus as immune. The control animals, however, succumbed 'of stress infection. -

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Claims (2)

; i792609 P a'ten t address: 1) Process for the production of a vaccine, characterized in that one synthesizes _f from it in a cell-free system by means of info motor RKS antibodies ■ Regulator protein isolated, this protein sterilized and, if necessary, admixed with an Adyavans. 2) Vacciiie, produced according to claim 1 *. . . '*. New documents (Art. 1% 1 Para. 2 No., t 10988471819 BATH