CA1048931A

CA1048931A - Bacterial antigen composition for treatment of oto-rhinolaringological infections

Info

Publication number: CA1048931A
Application number: CA75234882A
Authority: CA
Inventors: Jacques Debat
Original assignee: Institut de Recherches Chimiques et Biologiques Appliquees SARL IRCEBA
Current assignee: Institut de Recherches Chimiques et Biologiques Appliquees SARL IRCEBA
Priority date: 1974-09-06
Filing date: 1975-09-05
Publication date: 1979-02-20
Also published as: FR2283697B1; NL7510575A; ES440773A1; SE7509913L; JPS5161624A; FR2283697A1; AR205836A1; BE832959A; IL48066A0; DE2539622A1; CH606437A5; IL48066A; GB1518277A; DK394075A

Abstract

ANTI-GENIC COMPOSITION

ABSTRACT

The present, invention relates to a new product for the treatment of infections in the oto-rhino-laryngo-logical field constituted by purified antigenic fractions of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209 and Diplococcus Pneumoniae 210 and Neisseria catarrhalis 987, of the catalogue of the collection of the Centre International de Distribution de Souches and d'Informations sur les Types Microbiens of Lausanne.
The antigenic fractions of the nine strains are obtained by culture in a nutrient medium, enzymatic lysis with papain and then trypsin, purification and lyophilisation.

Description

lOg8931 The present invention relates to a new industrial product constituted by a therapeutic composition for nasal use containing antigens of bacterial origin. It also relates to the process for preparing the said composition, as well as to the application of the said composition for the treatment of infectious manifestations in the oto-rhino-laryngological field and in particular rhinitis and rhino-pharyngitis, otitis and sinusitis.
The new nasal composition according to the invention is characterised in that it contains 3 parts by weight of purified antigenic fraction of Staphylococcus aureus 634, 3 parts by weight of purified antigenic fraction of Staphylococcus aureus 636, 3 parts by weight of purified antigenic fraction of Staphylococcus aureus 659, 3 parts by weight of purified antigenic fraction of Streptococcus 147, 3 parts by weight of purified antigenic fraction of Streptococcus pyogenes 155, 3 parts by weight of purified antigenic fraction of Streptococcus_pyogenes 1178, 3 parts by weight of purified antigenic fraction of Diplococcus pneumoniae 209, 3 parts by weight of purified antigenic fraction of Diplococcus pneumoniae 210 and 1 part by weight of purified antigenic fraction of Neisseria catarrhalis 987, and is prepared by the process described below or by an obvious chemical equivalent thereof.
The numbers of the strains which are given above are those of the catalogue of the collection of the Centre International de Distribution de Souches et d'Informations sur les Types Microbiens of Lausanne.
The nine strains from which are derived the puri-fied antigenic fractions which enter into the compositionaccording to the invention and which form part of the ~' iC)48931 pathogenic strains frequently encountered clinically and towards which it is of particular interest to produce immunity have been selected for their ability to produce antigens.
Of course, without departing from the scope of the invention, the purified antigenic fractions extracted from these nine strains may be associated with other ingredients, as will be seen hereinafter in Example B.
In accordance with the present invention, there is provided a process for the preparation of a therapeutically-active product, which comprises separately cultivating in a liquid nutrient medium containing a source of carbon, a source of nitrogen and mineral salts the strains of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209, Diplococcus pneumoniae 210 and Neisseria catarrhalis 987 of the catalogue of the collection of the Centre International de Distribution de Souches et d'Informa-tions sur les Types Microbiens of Lausanne; for each of the strains, effecting enzymatic lysis of the strain in the culture medium at 60 to 65C using papain and then at 37C
using trypsin, destroying each enzyme after use by heating at lO0~ to 120C for 30 minutes, eliminating solid substances of bacterial origin which result from the lysi by centrifuging, adsorbing the antigenic fraction contained in the liquid medium after centrifuging on a solid support, separating the solid support from the liquid phase by centrifuging, eluting the antigenic fraction from the support using an aqueous solution of ammonia, evaporating ammonia from the eluate, concentrating the eluate, and lyophilising the concentrate ~ _ 3 ~, eluate to obtain the antigenic fraction in the form of powder; and mixing the separately-obtained lyophilised antigenic Eractions in the proportions of 3 parts by weight of each of the lyophilised antigenic fraction~ of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209 and Diplococcus pneumoniae 210 and 1 part by weight of the lyophilised antigenic fraction of Neisseria catarrhalis 987.
The extraction of the purified antigenic fractions generally necessitates two different culture media, each containing a source of carbon, a source of nitrogen and mineral salts:

A11, 1048~31 a) one med~um for the Stap~Llococcus aureus, the Streptococcus and the Diplococcus pneumoniae strains;
this medium is prepared in a vessel with a volume of 300 litres, and b) a special medium for the Neisseria catarrhalis;
this medium is prepared in a 5-litre flask.
Although it is possible to cultivate the eight strains of Staphylococcus, Streptococcus and DiPlococcus simultaneously 2nd extract their antigenic fractions sim-ultaneously, it is preferred to operate strain by strain so as to avoid any possible additional risk of contamination and especially so as proportion each of the antigenic fractions better at the time of the final mixing.
Other advantages and characteristics of the invention will be better understood on reading the following non-limitative Examples.
Example A
I - PreParation of each of the purified anti~enic fractions of Staphylococcus aureus, Streptococcus and Diplococcus pne _ niae 1) Culture medium NaCl 1,500 g Dipotassium phosphate 1,500 g Monopotassium phosphate 600 g Monoammonium phosphate 600 g Glucose 1,500 g Magnesium sulphate 60 g Glycerin 600 g Yeast autolyzate 600 g Manganese sulphate 6 g Thiamine hydrochloride 1-,50 g Pyridoxine hydrochloride 1-50 g 1~)4893~
Calcium pantothenate 1-50 g Nicotinic acid 1-50 g Riboflavin 075 g Water Quantity sufficient for 300 litres The constituents of the culture medium are dissolved in 50 litres of water while stirring. After making up the volume to 300 litres with water, the pH is adjusted to 7-8 by adding soda. After fresh stirring, the medium is sterilized at 100-120C, preferably at 110C for 30 minutes.

2) Supp~:~ of tbe str~
m e strains intended for extracting the antigenic fractions are supported by passing them on to Swiss Albino mice so as to preserve their pathogenic character and their biochemical properties. Inoculation of the animal is effected intravenously.
Immediately on the death of the animals by septicaemia, blood is removed sterilely by cardiac puncture, and a liquid enrichment medium is inoculated therewith and then, from the liquid medium, a solid isolation medium.
With the pure culture obtained in this way there is inoculated a suitable quantity of liquid medium, intended to serve as inoc~lum ~or the commercial culture, in accordance with the method described in 3 hereunder .
After each new isolation, the conformity of the biochemical characteristics is checked by con~entional identification techm ques.

3) Pre~aration of the commercial inoculum The inoculation with the pure culture obtained previously is effected on 10 ml of liquid medium (described in 1). After remaining for 24 hours in an incubator at 37C, this volume is introduced sterilely into 5 litres of medium of the same composition, previously sterilized. After 11~48931 remaining for 24 hours in an incubator at 37C, this volume is introduced sterilely into 300 litres of the same medium which has been previously sterilized.

4) Culture The temperature o~ the medium being maintained at 37C, the culture is carried out over 24 hours, aeration of the medium being ensured by the admission of filtered air (obtained by means of a Millipore diaphragm for e~ample). After 24 hours, the culture is stopped by sterilization of the medium for 1 hour at 100-120C.
At this stage, 10 ml of medium are removed under aseptic conditions for a sterility check~

5) Lysis of the bacteria This operation is effected by enzymatic hydrolysis and has the aim of liberating the antigenic fractions in the medium. After checking the sterility of the medi~um, enzymatic hydrolsis is performed in two stages:
firstly, at 60-65C by means o~ papain at pH 6-5, then, at 37C by means of trypsin at pH 7.
The object of the enzymatic lysis is to degrade the above bacterial cell so that the culture medium of point 4) above (in which the enzymatic lysis is effected and which is brought to pH 6-5 for the treatment with papain and then to pH 7 for the treatment with trypsin) is enriched with anti-gens.
The amounts of papain and trypsin are not critical.
In fact, the simplified mechanism is as follows.
The enzymes (even when present in small amount) destroy the cell wall and lay bare a portion of the cytoplasmic membrane;
the said membrane is then destroyed by reason of (i) the 1~48931 - -difference is osmotic pressure existing between the cyto-plasmic mass and the enzymatic lysis medium, and/or (ii) the enzymatic degradation; the result ~s that the antigens (located in the cell wall and located especially inside the cytoplasmic membrane), or at least a part thereo~, pass into the enzymatic lysis medium. Generally speaking, it can be considered that the enzymes enable the antigens to be separated from the substances covering or enveloping .them or from any possible substrates to which they are bound, and a bacterial extract (antigenic fraction) to be recovered which is obtained from each complete antigen and has preserved in its structure the antigenic sites of the complete antigen.
Moreover, the durations of treatment, which depend on the amounts of enzyme, are not critical either.
To obtain good results as regards the amount and the quality of the antigens extracted, it is recommended that: .
- 25 to 35 g of papain tContainirlg 660 units per milligram of enzyme, see the French Pharmacopoeia of 1949 for the definition of this content) be employed for 300 litres of medium, the treatment being effected at pH 6-5 and at 60-65C for 3 hours;
- heating be carried out at 100-120C for 30 minutes to destroy the papain;
- 12 to 18 g of trypsin (containing at least 30 International Units per milligram of enzyme, see the French Pharmacopoeia of 1965 for the definition of this content) be then employed for 300 l~tres of ~. medium, the treatment being effected at pH 7 and at 1~48931 37C for 3 hours~ and - heating be carried Ollt at 100-120C for 30 minutes to destroy the trypsin.
In the present Exarnple, the following conditions were applied, namely: treatment with 30 g of papain (con-taining 660 U/mg) for 300 litres of medium at pH 6.5 and at 60C for 3 hours; heating at 100C for 30 minutes; treat-ment with 15 g of trypsin (containing at least 30 I.U./mg for 300 litres of medium at pH 7 and at 37C for 3 hours, and then heating at 100C for 30 minutes.

6) Extraction of the anti~enic fractions -First of all, the elimination of the bacterial substances is carried out by centrifuging. The 300 litres of culture medium containing the antigenic fractions are then recovered. These antigenic fractions are then adsorbed on activated carbon ("OENO" (Trademark) carbon, for example).
A fresh passage through a centrifuge is carried out in order to recover the antigenic fractions adsorbed on the carbon, which will then be eluted with the aid of an aqueous solution of ammonia.

The antigenic fractions are then contained in the ammonia water.
The solution undergoes treatment in an evaporator, the object of this operation being to eliminate the ammonia and concentrate the aqueous solution. The concentration of the said aqueous solution is effected under conventional conditions; the aim to be attained is an antigenic fraction content which can be lyophilised. It is thus possible to concentrate from 2 volumes to 1 volume or, better still, from 5 volumes or more to 1 volume.

~,~,' ~48931 The lyophilisation of this aqueous solution constitutes the final stage of the extraction.
The purified antigenic fractions are then in the form of powder.
II - PreParation of the anti~enic fraction extracted from Neisseria Catarrhalis In this instance there is no change of principle, but the culture medium is different (this is due to the difficulty of cultivating this germ); moreover, the culture is performed in a 5-litre flask and lasts for 48 hours.
Only the differences in comparison with the preparation of the antigenic ~ractions extracted from Staphylococcus aureus, Streptococcus and Diplococcus Pneumoniae are given here.
Culture medium Horse serum 250 ml T broth quantitr æufficient for5 litres Composition of the T broth Peptone 200 g Glucose 10 g NcCl 25 g Distilled water quantity sufficient for 5 litres For the preparation of this medium, the horse serum is introduced sterilely into the sterile T broth and the pH is adjusted to 7-2.
The medium is then sterilized at 100C for 45 minutes.
The lysis of the bacteria, the extraction of the antigenic fractions and then the lyophilisation are then effected by the method hereinbefore described for the -1~)4893~
Staph~lococcus, Streptocooous and Diplococcus.
The molecular weight of each of the 9 antigenic fractions obtained according to ~ and II is about 12,000.
III - Mixin~ of the various anti~enio ~ractions 1 part by weight of the purified and lyophilised antigenic fraction of Neisseria catarrhalis is mixed with 3 parts by weight of each of the purified and lyophilised antigenic fractions of ~ aureus 634, 636 and 659, Streptococcus 147, 155 and 1178 and Diplococcus Pneumoniae 209 and 210.
Example B
The preferred composition according to the invention was prepared by reproducing Example A and mixing the antigenic fractions of the nine straîns in Stage III with sodium mercuro-thiolate, sodium chloride and water so as to obtain the following formulation:
Composition I:
Purified antigenic fraction extracted from ~ 634 3 mg Purified antigenic fraction extracted from Staphylococcus aureus 636 3 mg Purified antigenic fraction extracted from 5taphylococcus aureus 659 3 mg Purified antigenic fraction extracted from Streptococcus 147 3 mg Purified antigenic fraction extracted from Streptococcus p,~og~nes 155 3 mg Purified antigenic fraction extracted from Streptococcus E~ 1178 3 mg Purified antigenic fraction extracted from Diplococcus pneumonia 209 3 mg Purifled antigenic fraction extracted from DiPloc_ccus ~a~ 210 3 mg Purified antigenic fraction extracted from Neisseria catarrhalis 1 mg Sodium mercurothiolate 0-25 mg Sodium chloride 90 mg Distilled water quantity sufficient for 10 ml ~ y proceeding as above, a composition II was prepared observing the proportion of composition I.
Composition II
Purified antigenic fraction extracted from Staph,ylococcus aureus 634 4-5 mg Purified antigenic fraction extracted from StaphYlococcus aureus 636 4-5 mg Purified antigenic fraction extracted from Staph~lococcus aureus 659 4 ~ 5 mg Purified antigenic fraction extracted from Streptococcus 147 4a5 mg Purified antigenic fraction extracted from Streptococcus P~o~e-n-es 155 4 - 5 mg Purified antigenic fraction extracted from Strept coccus pyo~enes 1178 4~5 mg Purified antigenic fraction extracted from Diplococcus pneumoniae 209 4~ 5 mg Purified antigenic fraction extracted from Diploc_ccus pn,eumon,iae 210 4 ~ 5 mg Purified antigenic fraction extracted from Neisseria catarrhalis 987 1 5 mg ~ ; ~
Sodium mercurothiolate o~ 375 mg Sodium chloride 135 mg Distilled water quantity sufficient for 15 ml 16~48931 Composition II packaged in 15 ml bottles for the purpose of being administered by spraying into the nasal fossae has been called provisionally by the names "Rhinapten"
and "Rhinapten Nebuliseur". The results of the pharmacological ~5 and clinical tests which have been undertaken are summarized hereinafter.
Toxicity Study of the acute toxicity in mice, per os, has enabled it to be shown that the LD~0 (minimum mortal dose) of each of the nine antigenic fractions entering into compositions I and II is higher than 4 g/kg. It has also been shown that the LD-0 of the mixture of the nine anti-genic fractions is distinctly higher than 108 g/kg and that the LD-0 o~ compositions I and II is e~ual to 40 ml/kg (which corresponds to a dose of the mixture of the nine antigenic fractions of 100 mg/kg).
Study of the actue toxicity in mice and rats, ~er os, of the mixture of the nine antigenic fractions entering into compositions I and II at doses of 1 mf/kg and 10 mg/kg for 13 weeks, 5 days a week, has enabled the absence of mortality to be established.
Pharmacolo~ical tests Pharmacological experiments with animals comes up chiefly against two practical difficulties: on the one hand, it is difficult to create locally, at the level of the nasal fossae, an experimental infection which does not get better spontaneously in a space of time too short for the test to be conclusive; on the other hand, nebulization or spraying, which is a form of administration well adapted ~0 to man, is scarcely so adapted to animals, which accept 1~8~31 instillation or exposure to an aerosol mist better.
To solve these difficulties, four series of tests have been used:
a) Immunolo~ical ~roPerties in vitro of the ~ anti~enic fractions and of a mixture thereof in accordance with Com~osition I and II.
It is demonstrated that the extracts of the nine strains (Staphvlococcus aureus 634, 636 and 659, StrePtococcus 147, Streptococcus P~o~enes 115 and 1178, DiPlococcus neumoniae 209 and 210 and Neisseria catarrhalis 987) possess an anti-genic structure by employing Kolmer's method; in fact, each fraction obtained in accordance with Example A and the mixture of the said fractions give a positive reaction with the immunoserums produced in animals (rabbits) by injection of suspensions of each of the nine strains in formaldehyde (the said suspension is obtained by adding formaldehyde to the nutrient culture medium of Example A containing the strain and by the centrifuging).
b) Immunisation by the parenteral method with the anti~enic fractions and a mixture thereof in accordance with Compositions I and II
Three times a week, for 4 weeks, there is administered to rabbits by the injection method 2 mg per day of each of the antigenic fractions entering into Com-positions I and II (the antigenic fraction extracted from Neisseria catarrhalis being excluded, since this would in~olve a risk of causing shock by the parenteral method).
The serum of the animals is then taken, which is tested with the 9 antigenic fractions and the mixture thereof by Kolmer's method and that of Ouchterlony. The results of 1~4~931 the two methodsenable it to be affirmed that the antl-genic fractionq and the mixture thereof produce a good immunisation of the animals.
c) Sho inR_antibodies~at_the level of the nasal mucous membrane in treated animals.
The experiments were made by instillation (rats) and aerosol (guinea pigs); the results being concordant, - only the tests relating to the rats have been summarised hereinafter.
1~ Record The experiments were conducted on 40 rats distributed as follows:
Control batch:
Male SPF rats (average weight 250 g) Nos. 1 to 10 1~ ~emale SPF rats (average weight 250 g) Nos. 11 to 20 Treated batch:
Male SPF rats (average weight 250 g) Nos. 21 to 30 Female SPF rats (average weight 250 g) Nos. 31 to 40 The animals were treated nasally at the rate of 11 drops per nostril, twice a day, for 8 weeks, with a solution of the mixture of the nine antigenic fractions entering into the Composition I of Example B. This solution contained 5 g of mixture of purified antigenic fractions for 100 ml of distilled water.
The control batch received only distilled water under the same conditions.
At the end of eight weeks of treatment, the animals were sacrificed and the nasal mucous membranes were removed.
The nasal mucous membrances were then washed with p~ysiological serum and milled in the presence of 0 5 ml of isotonic solution of sodium chloride. Once the milled products had been centrifuged, the supernatant material was used for carrying out the Kolmer reaction against the anti-staphylococcus, antistreptococcus, antipneumococcus and antineisseria immunoserums and proving the presence of antibodies.
Results The results, animal by animal, for each batch, are given in Tables I and II; these results have moreover been regrouped in Tables III and IV, respectively, to be expressed in % according to the indications of the Kolmer reaction.
d) Showin~ the Protective ProPerties in vivo of the anti-~eni~ ractions in relation to exPerimental infections with Streptococci and Sta~hylococci.
As it was difficult to create a rhino-pharyngeal infection useful for experiments in animals, subcutaneous abscesses were caused in guinea pigs by injection of a microbial dose close to the minimum in~ectious dose (MID) of Streptococcus pyogenes 155 (MID = 005 ml of the 24-hour culture medium of this strain) and of Staphylococcus aureus 636 (MID = 0-5 ml of the 24-hour culture medium of this strain). The mixture of the antigenic fractions (excluding the Neisseria catarrhalis fraction)was then administered intramuscularly at the rate of 8 mg/kg of the said mixture every day for 5 weeks. In comparison with the infected control animals not receiving the said mixture, it was observed that the treated animals were better protected.

. 1~48931 ' CONTR~I BATCH
_ .. _. ..... _ Rats Antistaphylococcus Streptococcus Antipneumococcus .~ntineisseria Nos. antiserum antiserum antiserum antiserun : ~ ~ ~

J _ _ 1 __ . - KOL~ER REAC~J.ON
- Legend: - negative reaction + slightly positive reaction + positive reaction ++ very po itive reaction ~ 16 1~48931 ~ , . .
. ~AB~E II
TREA~'~D BA~CH

. ~ ....... _ . , Rats Antistaphylococcus Streptococcus Antipneumococcus Antineisseria No~. antiserum antiserum antiserum antiserum~ -~ L
-.
~ . ABLE TII
CONTROL B.~rCH
.~ . , eXpre~sed ¦AntiStta~hylococous Streptococcus antiseru~ nantsseeria :. _ _ - ne~ative 65 ~0 75 % 100 % 100 9 + slightly .
~ positive 15 ~ 10 90 .
+ positive 20 ~, 5 ~ _ positive - _ ~ _ TAB~E IV
TR~ATED BATCH
.

e~precsed antiserum 3 iseru~ Antipneumococcus antiseru~ 1 ,. . . .

- ne~ative 10 ~ . 15 % 25 ~ 25 + ~lightly .
~ positive 15 ~0 20 jo 45 ~0 50 ~0 + positive 55 ~ 55 ~ 30 ~ 25 '~
positivF 20 ~ 10 ~ _ ~ ' ., , . I

- I

48931 . I

. ~ . . .

Clinical experiments were carried out using t~e co~position oi ~xa~ple B. The effects of this composition on oto-rhino-laryn~ological infections and also the immunizing : power created in relation to the germ9 isolated both in children and in adults were studied. Parallel with this, in~estigætion of the level of i.~muno Bobulins o~ the secretory Ig~ type at the le~el of the na~al mucous ~as , undertaken before and after treatment.
These clinical experiments, concerned with 50 ~ætients .di~tributed in age groups as indic~ted i~ Table V and suffering ~rom . a) rhinitis and rhino-pharyngitis, 'b) recurrent otitis and/or . . c) sinusitis, gave the ag~re~ate results contained in Table VI.
'TAB~E V
. . ..
, Age No. of Cases Dosage ._ , . . .
0 - 5 years 3 ' Onc,e in each nostril . . . twice a day __ 5 - 10 years 22 . Once in e~ch nostril . . three times a dæy ... .. . . . . .
10 - 20 years 10 Twice in eac~ nostril twice a day 20 years and . . .
.above 15 Twice in each nostril . . three times a day .
. .
. Note: (a) duration of treatment: 2 - 3 weeks . .

.
.

.... . ~._ 1~48~31 ?AB~ VI

. .. . . . ._ ' . Overall results.
.__ _ _ . _ ..
Results No, o~ Caseæ ,percenta~e . . _ _ ..... _ .
Very good 3 6 1 Gooa 34 '' 68 ~ = 82 .
Average . .4 8 J
. . .
Negat~ve ~ _ 18 In the 82~" of positive re ~ ts in ~able VI,. effectiveness was observed ~rom the 15th day of treatment. This effectiveness was found to be lasting, since the majority of the ,~atients showed no recurrence of symptoms during a period of 2 to months after the end of the treatr~ent, ~o case of intolerance was observed in any of the 50 patients.

1~4893~
FLOW CEIART

CULTURE
~I
LYSIS OF BACTERIA
I

DESTRUCTION OF BACTERIA
1, SEPARATION (1) ANTIGENIC SOLID BACTERIAL RESIDUES -->
FRACTION +
LIQUID
\~
ABSORPTION OF
ANTIGENIC FRACTION

SEPARATION (2) ABSORBED LIQUID
SOLID FRACTION

\~1 !
ELUTION
~ ~"
ELUATE ABSORBANT -----~
~I
SEPARATION (3) CONCENTRATION ELUANT -~

LYOPHILISATION

ANTIGEN

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of a therapeutically-active product, which comprises separately cultivating in a liquid nutrient medium containing a source of carbon, a source of nitrogen and mineral salts the strains of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209, Diplococcus pneumoniae 210 and Neisseria catarrhalis 987 of the catalogue of the collec-tion of the Centre International de Distribution de Souches et d'Informations sur les Types Microbiens of Lausanne; for each of said strains, effecting enzymatic lysis of the strain in the culture medium at 60 to 65°C using papain and then at 37°C using trypsin, destroying each enzyme after use by heating at 100° to 120°C for 30 minutes, eliminating solid substances of bacterial origin which result from the lysis by centrifuging, adsorbing the antigenic fraction contained in the liquid medium after centri-fuging on a solid support, separating the solid support from the liquid phase by centrifuging, eluting the antigenic fraction from the support using an aqueous solution of ammonia, evapora-ting ammonia from the eluate, concentrating the eluate, and lyophilising the concentrate eluate to obtain the antigenic fraction in the form of powder; and mixing the separately-obtained lyophilised antigenic fractions in the proportions of 3 parts by weight of each of the lyophilised antigenic fractions of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209 and Diplococcus pneumoniae 210 and 1 part by weight of the lyophilised antigenic fraction of Neisseria catarrhalis 987.

2. The process of claim 1, wherein said nutrient medium for the cultivating of each of the strains with the exception of Neisseria catarrhalis 987 comprises: 1500 g of NaCl, 1500 g. of dipotassium phosphate, 600 g of monopotassium phosphate, 600 g of monoammonium phosphate, 1500 g of glucose, 60 g of magnesium sulphate, 600 g of glycerine, 600 g of yeast autolyzate, 6 g of manganese sulphate, 1.50 g of thiamine hydrochloride, 1.50 g of pyridoxine hydrochloride, 1.50 g of calcium pantothenate, 1.50 g of nicotinic acid, 0.75 g of riboflavin and water to a total of 300 liters.

3. The process of claim 1, wherein said nutrient medium for cultivating the strain of Neisseria catarrhalis 987 is grown in a nutrient medium comprising 250 ml of horse serum, 200 g of peptone, 10 g of glucose, 25 g of NaCl and water up to a volume of 5 litres.

4. The process of claim 1, wherein said enzymatic lysis using papain is carried out at pH 6.5 for 3 hours and using trypsin is carried out at pH 7 for 3 hours.

5. The process of claim 1, wherein said solid support is charcoal.

6. The process of claim 1, 2 or 3, wherein each of said antigenic fractions has a molecular weight of about 12,000.

7. The process of claim 1, 2 or 3, including the further step of mixing the mixture of antigenic fractions with 0.25 part by weight of sodium mercurothiolate, 90 parts by weight of NaCl and water to 10 parts by volume.

8. The process of claim 1, 2 or 3, including the further steps of mixing the mixture of antigenic fractions with 0.25 part by weight of sodium mercurothiolate, 90 parts by weight of NaCl and water to 10 parts by volume, and packaging the resulting aqueous composition in 15 ml nasal spray bottles

9. A therapeutically-active product prepared by the process of claim 1, or its obvious chemical equivalent thereof.

10. A therapeutically-active product prepared by the process of claim 8, or by an obvious chemical equivalent thereof.

11. A therapeutically-active product prepared by a process comprising: separately cultivating the strains of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209 and Diplococcus pneumoniae 210 of the catalogue of the Collection of the Centre International de Distribution de Souches et d'Informations sur les Types Microbiens of Lausanne, in a liquid nutrient medium comprising 1500 g of NaCl, 1500 g of dipotassium phosphate, 600 g of mono-potassium phosphate, 600 g of monoammonium phosphate, 1500 g of glucose, 60 g of magnesium sulphate, 600 g of glycerine, 600 g of yeast autolyzate, 6 g of manganese sulphate, 1.50 g of thiamine hydrochloride, 1.50 g of pyridoxine hydrochloride, 1.50 g of calcium panthothenate, 1.50 g of nicotinic acid, 0.75 g of riboflavin and water to a total of 300 litres, separately cultivating the strain of Neisseria catarrhalis 987 of the catalogue of the Collection of the Centre International de Distribution de Souches et d'Information sur les Types Microbiens of Lausanne, in a liquid nutrient medium comprising 250 ml of horse serum, 200 g of peptone, 10 g of glucose, 25 g of NaCl and water to a volume of 5 litres; for each of the strains, effecting enzymatic lysis of the strain at 60° to 65°C and a pH of 6.5 for 3 hours using papain and then at 37°C and a pH of 7 for 3 hours using trypsin, inactivating the enzymes after use by heating at 100° to 120°C for 30 minutes, eliminating solid substances of bacterial origin by centrifuging the medium resulting from the enzymatic lysis, absorbing the antigenic fraction contained in the liquid medium after centrifuging on charcoal, separating the charcoal from the liquid phase by centrifuging, eluting the antigenic fraction from the charcoal using an aqueous solution of ammonia, evaporating ammonia from the eluate, concentrating the eluate, and lyophilising the con-centrated eluate to obtain the antigenic fraction in the form of powder; and mixing the separately-obtained lyophilised antigenic fractions in the proportions of 3 parts by weight of each of the antigenic fractions of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209 and Diplococcus pneumoniae 210 and 1 part by weight of the antigenic fraction of Neisseria catarrhalis 987, or by an obvious chemical equiva-lent thereof.

12. The product of claim 11, wherein the lyophilised anti-genic fractions are also mixed with 0.25 parts by weight of sodium mercurothiolate, 90 parts by weight of NaCl and 10 parts by volume of distilled water.

13. The product of claim 12, packaged in 15 ml bottles in a manner to be sprayed or nebulized into nasal fossae and con-stituted by 4.5 mg each of the antigenic fraction of Staphylococcus aureus 634, Staphylococcus aureus 636, Staphylococcus aureus 659, Streptococcus 147, Streptococcus pyogenes 155, Streptococcus pyogenes 1178, Diplococcus pneumoniae 209 and Diplococcus pneumoniae 210, and 1.5 mg of the antigenic fraction of Neisseria catarrhalis 987, 0.375 mg of sodium mercurothiolate, 135 mg of NaCl and water in sufficient quantity to the volume of 15 ml.