Note: Descriptions are shown in the official language in which they were submitted.
Process for the production of capsular
polyosides, capsular polyosides obtained
thereby, and their application to the
preparation of vaccines
This invention relates to a ~rocess for the produc
tion of capsular polyosides, to the polyosides obtained
by said process, and the application of said polyosides
to the preparation of vaccines.
It is known that it is possible to combat infectious
diseases at two different levels: either by direct
action against the pathogenic germ (antibiotics, anti-
septics)~ or by an indirect action via the reinforcement
of the systemic defences (vaccination, serotherapy, im-
munomodulation).
The use of agents likely to exert a direct action
frequently comes up against certain difficulties which
may limit theix action (particularly: toxicity of the
molecule and bacterial resistance).
In contrast, the reinforcement of the systemic
defences via the acquisition of specific antibodies has
definite advantages, particularly from the standpoint
of the duration of the protection and of the specificity.
However, vaccination with whole microbial bodies is not
always free from drawbacks: hypersensitization phenomena,
poor tolerance or pyrogenicity are among the most fre-
~uent. They are corelated to the presence of multiple
antigens of the bacteria.
It is therefore ~uite naturally that searchers
have specifically directed their efforts to the isola-
tion of the antigen responsible for the protectiveantibodies.
In the case of capsulated bacteriae, the capsular
polyosides are found to be immunogenic in humans, with
little side-reactions.
3~$
~20~9~5
A first application in humans of such capsular
polyosides is the development of antimeningococcic vac-
cine A and C and, quite recently, of a polyvalent vacci-
ne containing the purified polyosides of fourteen sero-
logic types of pneumococcus.
Several other capsulated bacteriae are involved ina variety of pathological processes, such as:
- Hemophilus influenzae, type b
- Klebsiella pneumoniae,
- Escherichia c _ , or
- Streptococci, group B,
and corresponding vaccines may be prepared from their
capsular polyosides.
In European patent application 0 002 404 is descri-
bed a process for the production of capsular polyosidesfrom Streptococcus pneumoniae.
According to this process, the microorganism is cul-
tivated with complex culture media. Then, after inacti-
vation with phenol, and without separating the microor-
ganisms from the culture medium, a first addition of analcohol such as ethanol is effected, the impurities
which have precipitated out are separated, and a second
addition o~ alcohol is then effected to precipitate out
the polyoside. The contaminants (proteins, nucleic acids)
are then removed from the re-suspended polyoside, by an
enzyme treatment or by treatment with a cationic surfac-
tant tcitrimonium bromide), which treatments may be com-
pleted by a diailtration.
In fact, this process is not generally applicable,
because the specific conditions concerning each step
vary with the polyoside to be separated. Thus, it is
nesessary to effect pre-tests to determine the conditions
of the various precipitations. In addition, due to the
use of certain media, this process may cause a degrada-
tion of the polyoside chains.
69~
The object of this invention is to pro~ide a moregenerally applicable, simpler and less degradint process for
the production of polyosides of very high molecular weight.
Therefore, this invention relates to process for the
production of capsular polyosides from capsulated bacteriae
containing same,..comprising cultivating the bacteriae in a
synthetic or semi-synthetic medium; at the end of the growth
stage of said culture separating the microbial bodies from a
supernatant phase; submitting said supernatant phase to a
filtration through a membrane which retains the molecules of
a molecular weight of 100,000 daltons or more, to give a capsu-
lar polyoside-rich retained material, submitting said retained
material to an enzyme hydrolysis, adding a butanol-chloroform
mixture thereto, separating the aqueous phase, submitting
said aqueous phase to a dialysis and then to a filtration
through a membrane which retains the molecules having a mole-
cular weight in excess of 100~000 daltons and recovering the
capsular polyosides from the retained material.
In particular, the present invention provides a
process for the production of capsular polyosides from capsul-
ated bacteriae containing same, comprising cultivating the
bacteriae in a synthetic medium containing up to 0.5 wt~ of
protein hydrolysates; at the end of the growth stage of said
culture separating the microbial bodies from a supernatant
phase; submitting said supernatant phase to a filtration
~hrough a membrane which retains the molecules of a molecular
weight of 100,000 da~tons or more, to give a capsular poly-
oside-rich retained material, submitting said retained material
to an enzyme hydrolysis, adding a butanol-chloroform mixture
thereto, separating the aqueous phase, submitting said aqueous
phase to a dialysis and then to a filtration through a mem-
brane which retains the molecules having a molecular weight in
excess o 100,000 daltons and recovering the capsular polyosides
from the retained material.
. 1;~069(~5
The present in~ention also provides capsular
polyosides produced by a process co~prising culti~ating
bacteriae in a synthetic or semi-synthetic mediumi at the
end of the growth stage of said culture separating the micro-
bial bodies from a supernatant phase, submitting said super-
natant phase to a filtration through a membrane which retains
the molecules of a molecular weight of 100,000 daltons or more,
to give a capsular polyoside-rich retained material, submit-
ting said retained materialt~ an e~ZYmehydrolysis, adding a
lG butanol~orofOrm mixture thereto, separating the aqueous
phase, submitting said aqueous phase to a dialysis and then to
a filtration through a membrane which retains the molecules
having a molecular weight in excess of 100,000 daltons and
recovering the capsular polyosides from the retained material.
The present invention further provides a vaccine
comprising at least one capsular polyoside, produced by a
process comprising cultivating bacteriae in a synthetic or
semi-synthetic medium; at the end of the growth stage of
said culture separating the microbial bodies from a super-
natant phase; sumbitting said supernatant phase to a filtra-
tion through a membra~e which retains the molecules of a mole-
cular weight of 100,000 daltons or more, to give a capsular
polyoside-rich retained material, submitting said retained
nla~rial to anenzyme hydrolysis, adding a butanol-chloroform
mixture thereto, separating the aqueous phase, submitting
said aqueous phase to a dialysis and then to a filtration
through a membrane which retains the molecules having a mole-
cular weight in excess of 100~000 daltons and recovering
the capsular polyosides from the retained material.
Thus, according to an essential feature of the
present invention, the bacterial culture is effected in
synthetic or semi-synthetic medium. By synthetic medium
are meant aqueous solutions of known chemicals, of low mole-
cular weight, which are present in definite amounts.
- 3a -
,~
~69~5
By asemi-synthetic medium is meant a medium
analogous to the former, but which may contain additionally
a small amount (generally up to 0.5 wt%) of naturally occur-
ring materials such as protein hydrolysates.
Moreover, according to the present invention,
the extraction of the polyosides is effected on the super-
natant of the culture after removai of the microbial bodies,
and not on the o~erall culture medium.
Filtration through a membrane whose porosity is
capable of retaining molecules of a molecular weight of
100,000 dal~ons or more, makes it possible to obtain rapidly
a retained material comprising predominantly the polyosides,
together with small amounts of proteins, nucleic acids and
lipids.
B
;9~D5
Removal of the contaminants is advantageously effec-
ted in the following manner: the retained material is
submitted to an enzyme hydrolysis, typically with a
mixture of Pronase, RNase and DNase, a butanol-chloro-
form mixture (preferably a 1:1 mixture, by volume) isadded thereto, the aqueous phase is separated, the
a~ueous phase is submitted to a dialysis after which
the resulting material is filtered through a membrane
which retains the molecules of a molecular weight of
100,000 daltons or more.
The purified polyoside may be separated, in known
manner, from the retained material, by precipitation
with an alcohol such as ethanol.
The following non-limiting Examples illustrate the
present invention.
EXAMPLE 1
Pneumococcic polyoside, type 3
A freeze-dried strain of Streptococcus pneumoniae,
type 3, is used as starting material.
a) Preparation of seeding materials
The freeze-dried strain is added to T-broth.
Cultivation is effected for 16 hours at 37C.
5 ml of the resulting culture are mixed with 15
ml sterilized skim milk. The material is filled into
ampoules in an amount of 0.5 ml/ampoule and is then
freeze-dried. The freeze-dried materials are stored at
+4C.
b) Pre-culture
A seeding material (freeze-dried material stored
in an ampoule) is added to a flask containing 200 ml of
the semi-synthetic medium described below, and cultiva-
tion is effected at 37C for 6 hours.
Culture medium:
Pancreatic peptone from casein ~IBF) 1500 mg
Q-cystine 150 mg
~06g~
dl-Tryptophane 20 mg
Q-Tyrosine 200 m~
Dipotassium phosphate 4960 mg
d-G]ucose, anhydrous 1250G mg
Magnesium sulfate. 7H20 500 mg
Ferrous sulfate. 7H20 5 mg
Zinc sulfate . 7H20 0.8 mg
Manganese sulfate . 7H2O 0.3 mg
Fuming hydrochloric acid RP17.8 mg
d-Biotine 15 mg
Nicotinic acid 1 mg
Pyridoxine HCl 1 mg
Riboflavine 1 mg
Thiamine 1 mg
Calcium pentothenate 5 mg
Adenine HCl 10 mg
Uracil 10 mg
Choline chloride 10 mg
Asparagine 100 mg
Distilled water, sufficient to make 1000 ml
c) Culture
The culture itself is effected in a fermenter con-
tainin~ 18 litres of the culture medium defined in b).
Seeding is effected in an amount of 1~ with the pre-
culture.
The culture is effected at 37C, at pH 7.4, with
stirring for a period of time of generally about 16 hrs,
to the end of the exponential growth phase.
d) Separation of the supernatant
At the end of the exponen~ial phase, the superna-
tant is immediately separated by continuous centrifuga-
tion at 5000 rpm. The supernatant is submitted to a
sterilizing filtration through Millipore membrane 0.22
and is used immediately or is stored at -25C.
~z06~05
e) Extraction
A volume of supernatant of about 20 litres is con-
centrated to a volume of 2 1itres by filtration through
a Millipore 10 membrane capable of retaining the mole-
cules of a molecular weight of 100,000 daltons or mGre.
The xesulting retained material is filtered through
a Diaflo 105 membrane after addition of 5 litres apyroge-
nic steril~ distilled water to complete the separation
according to the molecular weight.
Several successive diafiltrations permit a concen-
tration to about 350 ml which are then lyophilized in 1
litre flasks.
All the above operations are effected at a tempera-
ture of up to 8C maximum.
f) Purification
The lyophilized intermediate product is dissolved
in 900 ml phosphate buffer 0.01 M - pH 7.
20 mg ~Nase type 1 , 20 mg pancreatic DNase and
180 ~1 (20 units/ml) RNase Tl are added thereto. The
material is incubated for 4 hrs at 37C, after which
170 mg Pronase type 9, 800 ~1 pure phenol and 2 drops
toluene are added thereto.
The mixture is incubated overnight at 37C. The
reaction is terminated and an equal volume (900 ml) of
butanol-chloroform (1:1) is added thereto. The resulting
material is stirred for 1 hour. The two phases are
decanted by centrifugation at 10,000 r.p.m. for 30 mn.
The aqueous phase which contains the polyoside is
dialyzed against 20 litres apyrogenic sterile distilled
water for a period of time of about 60 hrs.
The dialyzate is submitted to a diafiltration
through an Amicon 105 membrane. The volume of the retai-
ned material is 1140 ml. 160 ml 40% sodium acetate
(final concentration 5%)and 3,9 litres absolute ethanol
(3 volumes) are then added.
~ZQ6!~0~
The precipitate is collected by centrifugation at
12,000 r.p.m. for 30 mn. Said precipitate is dissolved
in apyrogenic distilled water. The resulting solution
is filled in vials containing ~ ml solution each. The
material is freeze-dried _ vacuo, to give 9 g of the
desired product. Said product is the purified polysaccharide
which contains:
- sugars 64
- proteins 0.8%
- nucleic acid <0.8%
All the above steps (apart from the incubations)
are conducted between 0 and +4C.
~XAMPLE 2
Pneumococcic polyoside type 23
lS A freeze-dried strain of _treptococcus ~neumoniae
type 23 is used as starting material.
The culture and the separation of the supernatant
are effected under the conditions defined in Example 1
(steps a - d).
Extraction and purification of the polyoside are
effected in the following manner:
e) Extraction
A 15 litre volume of supernatant is concentrated to
a volume of 1.5 litre under the conditions defined in
Example l-e.
Three successive diafiltrations make it possible
to concentrate, and then to freeze-dry, to give 1.8 g
of a material of the following composition:
- sugars 60~
- proteins 19%
- nucleic acid 3.6%
f) Purification
The freeze-dried intermediate product is taken up
into 200 ml O.OlM phosphate buffer (pH 7). 1 mg RNase,
1 mg DNase and 20 units RNase Tl/ml are then added. The
-
~2~;~69(i'5
resulting material is incubated at 37C for 3 hrs, after
which Pronase type 8 (180 ~g/ml), phenol(l ~l/ml) and
tGluene (1 drop) are added thereto. The whole is incu-
bated overnight at 37C.
Three successive extractions are effected with an
equal volume (200 ml)of butanol-chloroform 1:1. The resul-
ting mixture is then stirred mechanically.
The final aqueous phase is dialyzed for about 60
hrs against 4 litres apyrogenic sterile distilled water.
The dialyzate is filtered through Amicon 105 mem-
brane. The material is concentrated to 50 ml. Absolute
ethanol (3 volumes) and sodium acetate (final concentra-
tion 5%) are added thereto and the mixture is left aside
overnight at -20C.
The resulting precipitate is collected by centrifu-
gation. Said precipitate is dissolved in apyrogenic ste-
rile distilled water, and is then freeze-dried, to
give 1050 g of a product which contains:
- sugars 75%
- proteins 1.7%
- nucleic acid 0.7~
All the above steps (except otherwise indicated)
are effected between 0 and +6~C.
E~AM~E 3
Pneumococcic polyoside type 19
A freeze-dried strain of Streptococcus pneumoniae
type 19 is used as starting material.
The culture and the separation of the supernatant
are effected under the conditions defined in Example 1
(steps a - d).
Extraction and purification of the polyoside are
effected in the following manner:
e) Extraction
Using the procedure of Example l-e, a 75 litre vo-
lume of supernatant is concentrated to 2 litres by three
~206~(~5
diafiltrations, to give 2.3 g having the following com-
position:
- sugars 30
- proteins 15%
- nucleic acid 3.4%
f) Purification
The freeze-dried intermediate product is dissolved
with 150 ml O.OlM phosphate buffer (pH 7). DNase (3 mg),
RNase ~3 mg) and RNase Tl (30 ,ul) are added thereto, and -
the resulting material is then incubated at 37C for 4hrs, after which Pronase (62 mg), phenol (150 ,ul) and
toluene (1 drop) are added. The whole is allowed to
react overnight at 37C.
Deproteination is effected with an equal volume (3
successive extractions) of chl.oroform-butanol 1:1.
The aqueous phase is dialyzed for 60 hrs against 4
litres ap~Troyenic sterile distilled water. The dialyzate
is submitted to a diafiltration through Amicon XM 100
membrane.
The retained material is precipitated with 3 volu-
mes ethanol containing 5% sodium acetate.
After overnight storage in a freezer, the precipi-
tate is dissolved in .apyrogenic steril.edistilled water,
and freeze-dried, to give 730 mg of product which con-
tains
- sugars 73%
- proteins 0.2%
- nucleic acid 0.5%.
Here, again, all the above steps (aside from the
incubations) are effected.between 0 and +4C.
EXAMPLE 4
Pneumococcic polyoside type 1
A freeze-dried strain of Streptococcus pneumoniae
type 1 is used as starting material. The culture and the
separation of the supernatant are effected under the
~lZ0~9Q~
conditions defined in Example 1~
Extraction and purification of the polyoside are
effected in the following manner:
e) ~xtraction
100 Litres of the culture supernatant are submitted
to a diafiltration through Pellicon* cassettes (Milli-
pore 105 membrane~. The volume of the final retained
material is 3 litres which are freeze-dried in flasks,
to give 6.1 g of product which contains:
- galacturonic acid 46.5%
- proteins 7.7%
f) Purification
The resulting product is dissolved in 800 ml apyro-
genic sterile distilled water. To the solution are added
- 16 mg RNase
- 16 mg DNase
- 160 ~1 RNase Tl.
The material is incubated at 37C for 4 hrs, after
which phenol (300 ,ul), toluene (2 drops) and Pronase
type 8 (150 mg) are added thereto and the whole is then
incubated overnight at 37C. The proteins are extrac-
ted with an equal volume of chloroform-butanol (1:1)~
The aqueous phase is dialyzed for 60 hrs. Diafiltration
is effected through Amicon~XM 100 membrane The volume
of the final retained material is 150 ml. The solution
is precipitated with 3 volumes cold ethanol containing
5~ sodium acetate.
The plug is taken up into 250 ml apyrogenic sterile
distilled water and freeze-dried in flasks in vacuo, to
give 3.7 g purified polyoside which contains:
- galacturonic acid 55.5% (which corresponds
to 85% sugars)
- proteins 1%
The diffusion coefficient in the KD gel of the
polyoside is 0.05. Therefore, the mclecular weight is
~'~ * Trade mark
~2(g~g~
at least 2 x 10 .
EXAMPLE 5
Polyoside from K. neumoniae,type 2
A strain of Klebsiella pneumoniae type 2 is used
as starting material.
The culture and the separation of the supernatant
are effected under the conditions defined in Example 1
(steps a - _), but using as culture medium the following
synthetic culture medium:
Trisodium citrate 0.85 g
Ammonium sulfate 0.17 g
Magnesium sulfate 0.17 g
Glutamic acid 0.17 g
d-Glucose 16.70 g
lS Dipotassium phosphate 10 g
Monopotassium phosphate 6.66 g
Distilled water, sufficient, to make 1000 ml
final pH : 6.8
Extraction and purification of the polyoside are
effected in the following manner:
e) Extraction
90 litres of the culture supernatant are used. Dia-
filtration is effected through Pellicon cassettes (2
cassettes, 105 membrane). After concentration to 1.5
litre, the material is washed 3 times with 10 litres water. The
final retained material is 2.6 litres. The material is
freeze-dried in flas]cs ,to give 6.1 g of product which
contains:
- sugars 50%
- proteins 15%
f) Purification
The resulting product is dissolved in 350 ml water.
There are added thereto:
- 7 ml 0.5M phosphate buffer, pH 7.0
- 7 mg RNase
~20691~5
- 7 m~ DNase
- 70 ,ul RNase Tl
(prior filtration of the enzymes through 0.22 ~).
The whole is incubated at 37C for 4 hrs, after
which protease type 8 (70 mg), phenol (350 ~1) and to-
luene (1 drop) are added thereto, and the mixture is
incubated overnight at 37C. The reaction is terminated
and deproteination is completed by 3 extractions with
butanol-chloroform (1:1). The a~ueous phase is dialyzed
for 60 hrs against apyrogenic sterile distilled water.
Diafiltration is effected through Amicon XM 100 membra-
ne. The volume of the final retained material is 150 ml.
The solution is precipitated with 3 volumes cold absolu-
te ethanol containing 5% sodium acetate. The precipitate
is collected by centrifugation, dissolved in 250 ml
water and then freeze-dried in flasks,ln vacuo. The pure
polyoside thus obtained contains:
- sugars 76~ (Hexoses/uronic acid = 3:1)
- proteins 3.8%
The KD of the polyoside obtained is 0.1. Thus, the
molecular weiyht is in excess of 107.
EXAMPLE 6
Polyoside from H. influenza,type b
A freeze-dried strain of Hemophilus lnfluenzae type
b is used as starting material.
The culture and the separation of the supernatant
are effected under the conditions de~ined in Example 1
(steps a - d), but using as culture medium the following
semi-synthetic culture medium:
Proteose peptone 5 g
Sodium chloride 3.5 g
d-Glucose ) 4 g
Monopotassium phosphate 1.3 g
Dipotassium phosphate 3.5 g
NAD (nicotine adenosine dinucleotide) 0.001 g
, ; ,
~ 2~ 5
Globular extract 50 ml
Distilled water, sufficient, to make lOOO ml
Extraction and purification of the polyoside are
effected in the following manner:
e) Extraction
30 Litres of the culture supernatant are used. Dia-
filtration is effected through Millipore 100,000 membra-
ne (2 cassettes ). The first concentration gives a
volume of 1 litre. The material is washed with 3 x 10
litres sterile distilled water. It is concentrated to
1.3 litre and lyophilized in flasks, to give 3.9 g of
a product which contains:
- sugars 53%
- proteins 13~
The product thus obtained is dissolved in 200
ml water.
After filtration through 0.22 ~, the following
materials are added:
4.5 ml 0.5M phosphate buffer, pH 7
4 mg RNase
4 mg DNase
40 ~1 RNase T1
The whole is incubated at 37C for 4 hrs, after
which protease type 8 (65 mg), phenol (200 ~ul) and to-
luene (2 drops) are added and the resulting material is
incubated overnight at 37C. The reaction is terminated
with 3 extractions with butanol-chloroform (1:1). The
aqueous phase is collected by centrifugation at 12,000
r.p.m. at +4C for 30 minutes. It is dialyzed for 60 hrs
against apyrogenic sterile dist-lled water (frequent
changes of the dialysis medium). Concentration is effec-
ted through Amicon XM 100 membrane ( 2 washings x lO0
ml). The volume of the diafiltrate is 100 ml. The dia-
filtrate is precipitated with 3 volumes cold absolute
ethanol containing 5% sodium acetate. The precipitate,
' ,;
~z~6~
14
collected by centrifugation, is taken up into 100 ml
apyrogenic sterile distilled water. The solution is
freeze-dried in flasks, in vacuo, to give 1.3 g pure
polyoside which contains:
- sugars (PRP) 63%
- proteins 2~
he ratio of the polyoside components is the following:
Ribose: Ribitol: Phosphorus : 0.9 : 1 : 0.98 (theore-
tical ratio: 1 : 1 : 1).
The KD is close to 0.1, which corresponds to a mole-
cular weight of about 107.
The purified immunogenic capsular polyosides obtai-
ned according to the present invention may be used for
the preparation of vaccines intended to be administered
to humans and to animals for preventive (or curative)
purposes. The vaccines may be prepared by incorporation
of the purified capsular polyosides obtained according
to the present invention in a physiologically acceptable
liquid carrier such a physiologic solution or water for
injection.
For this purpose, one or - preferably - more puri-
fied capsular polyosides obtained according to the pre-
sent invention and from differen~ serologic types may
be incorporated. In addition, polyvalent vaccines may be
prepared by incorporation of two or more purified capsu-
lar polyosides obtained from different germs.
The results of tests which demonstrate the immuno-
genic activity of the polyosides produced according to
the present invention are given hereinafter.
a) Titration of the serum antibodies in mice
~) Male Swis5 mice of 20 g body weight were used
in groups of 10 animals each:
1 reference group was not given any treatment.
1 group was treated by the I.P. route at the rate
of several doses of the polyoside of Example 1 per animal.
.
~2~)69C 5
The titration ofthe hemagglutinating antibodies was
effected according to the techniques described in the
literature, using the red blood corpuscle of sheep
sensitized by the polyoside by means of chromium chlo-
ride.
The results reported in Table I demonstrate anincrease of the serum level of hemagglutinating antibo-
dies with respect to the reference animals, together
with a conventional immunitary paralysis at high do-
sages.
~ ) - Analogous tests were conducted in mice with
the polyoside of Example 5 (polyoside from Klebsiella
pneumoniae, type 2).
The results obtained are given in Table II.
b) Titration of serum antibodies in rats.
The same study was conducted with male Sprague
Dawley rats of 400-450 g body weight.
The groups of 10 animals were administered an I.P.
injection of 2 and 20 ~g polvoside from Streptococcus
~neumoniae, type 3, per animal.
Titration of thehemagglutinating antibodies was
effected on the 4th, 6th and 14th day after administra-
tion.
The results obtained are given in Table III.
Consistent with the literature, the cerum antibody
level increases gradually to reach a maximum level on
the 6th day.
c) Specific protection
~- Groups of 10 male Swi.ss strain mice were im-
munized with the polyoside from Streptococcus pneumoniae
type 1, obtained according to the procedure of Example
4, by the sub-cutaneous route (two injections at a one
week interval); dosages injected: 0.47 - 4.7 - 47 and
470 ug/kg.
On the 10th day following the 2nd immunization,
lZ069(35
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19
the animals were infected with the virulent pneumococcus(~ype 1), by the sub-cutaneous route (1250 germs/animal).
After ten days observation, the death rate was as
follows:
- untreated animals 100%
- treated animals (0.47 ~g/kg) 30%
- treated animals (4.7 ~g/kg) 10%
- treated animals (47 ug/kg) 30%
- treated animals (470 Jug/kg) 40%
10 ~- Groups of 10 male Swiss mice were immunized with
the KP2 polyoside by the sub-cutaneous route (2 injec-
tions at a weekly interval; dosages injected: 53 - 533 -
5332 ~g/kg).
On the 8th day after the last treatment, Klebsiella
pneumoniae type 2 strain was injected to the animals at
a rate of 650 germs/animal. After 10 days observation,
the death rate was found to be as follows:
- untreated animals 100
- treated animals (53 ~g/kg) o%
- treated animals (533 /ug/kg) 0%
- treated animals (5333 ~g/kg) 0%
The polyosides produced according to the present
invention are typically useful for the preparation of
the following vaccines:
a) vaccines for the preventive and curative treat-
ment of acute and chronic respiratory diseases, compri-
sing one of the purified polyosides corresponding to
each of the following germs:
Streptococcus E_eumoniae
_ mophilus _nfluenzae, type b
Klebsiella pneumoniae
Escherichia coli
For example:
lZ069V5
1) mixture of polyosides from S. pneumoniae
types 1, 6, 14, 23 . . . . . . . . . . . . . . . 50 ~ug
polyoside from K. pneumoniae, type 2 . . 20 jug
polyoside from H. _nfluenzae, type b . . 20 ,ug
final volume: 0.5 ml physiological solution
2) mixture of polyosides from S. pneumoniae
types 1, 6, 14, 23 . . . . . . . . . . . . . . . 25 ,ug
polyoside from K.pneumoniae, type 2 . . 10 ~g
polyoside from H.influenzae, type b . . 10 ,ug
b) Antipneumococcic vaccine comprising the purified poly-
osides from the following types of Streptococcus pneu-
moniae:
1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18L, l9F, 23,
2, llA, 15F.
For example:
- mixture comprising 50 ~g active material in a
volume of 0.5 ml physiological solution,
- mixture comprising 25 ,ug active material in a
volume of 0.5 ml physiological solution.
