Process for the isolation of membrane proteins from Neisseria meningitidis and vaccines containing same

Membrane proteins are isolated from Neisseria meningitidis by direct treatment of the cells with a detergent. The membrane proteins can be worked up into a vaccine.

This invention relates to an improved process for the isolation of membrane 
proteins from Neisseria meningitidis and to vaccines containing the said 
membrane proteins. 
The causative organism of Meningitis cerebrospinalis epidemica is Neisseria 
meningitidis, also called meningococcus. Meningitis is spread by droplet 
infection and occurs mostly in children and people in mass quarters. It 
is, therefore, desirable to immunize the endangered groups. 
According to the present knowledge, the major outer membrane protein of 
Neisseria meningitidis is in a position to induce bactericidal antibodies. 
These antibodies offer a type-specific protection against the disease. 
Other components of Neisseria, especially the capsular polysaccharides of 
Neisseria groups A and C, may ensure a group specific protection, but they 
do not induce an effective protection against Neisseria group B. Moreover, 
it has not yet been ascertained whether the capsular polysaccharide of 
group B is in a position to induce such protection. Therefore, the 
membrane proteins of Neisseria meningitidis group B are of special 
interest. 
For the manufacture of a vaccine, the major membrane protein from Neisseria 
meningitidis group B must be isolated in a sufficient amount. The process 
described by Frasch in J. Bact. 127, 973-981 (1976) yields a useful 
preparation. The yield obtained in this process is, however, at the lower, 
economically acceptable limit. The essential features of the Frasch 
process are washing the cells of Neisseria meningitidis with salt 
solutions (CaCl.sub.2 or LiCl solutions) and then treating the extract 
with deoxycholate. As mentioned above, rather poor yields are obtained. 
It has now been found, surprisingly, that the yield can be considerably 
improved by subjecting the bacterial cell mass to a direct treatment with 
detergents. The material obtained by the process according to the 
invention does not differ noticeably from the membrane protein 
preparations according to Frasch. It has the same electrophoretic 
properties in SDS-containing buffer solutions. Its endotoxin content can 
be compared with that of the Frasch material as well as its ability to 
induce protective antibodies in animals. 
It is, therefore, the object of the present invention to provide a process 
for the isolation of membrane protein from Neisseria meningitidis, 
especially membrane proteins of group B, which comprises contacting the 
organisms with the aqueous solution of a detergent in a concentration of 
from 0.1 to 2% and allowing the mixture to stand for 15 minutes to 24 
hours, optionally with agitation, separating the extract from the bacteria 
residue and further purifying the extract, if desired. 
The preferred detergent is deoxycholate, especially its readily 
water-soluble sodium salt. The concentration of the detergent of 0.1 to 2% 
is related to the weight of the detergent in grams per volume in 
milliliters. The proportion of moist bacteria mass to detergent solution 
is preferably in the range of from 1:3 to 1:20, more preferably 1:5 (w/v). 
When sodium deoxycholate is used as the detergent, its concentration is in 
the range of from 0.3 to 1% in an aqueous solution. Suitable aqueous 
solutions are mainly the buffer solutions generally used in biochemistry 
and microbiology. 
As the starting material, Neisseria meningitidis is grown in usual manner 
and the cell mass is separated from the supernatant of the culture medium, 
preferably by centrifugation. The residue of centrifugation is suspended 
in the detergent-containing solution and allowed to stand for some time. 
Next, the extract is separated from the cell residue. This is preferably 
also done by centrifugation. 
The temperature at which the extraction is carried out is limited, the 
lower limit is the freezing point of the solutions used and the upper 
limit is determined by the loss of immunogeneity of the membrane proteins 
by heat denaturation. It is, therefore, advisable to operate at a 
temperature between room temperature and approximately 60.degree. C. 
If desired, the extract can be further purified. To this end, the residue 
obtained by ultracentrifugation is subjected again to an extraction with 
the detergent-containing solution and, if necessary, this extraction is 
repeated once more. In each operation, the supernatant of the 
ultracentrifugation is rejected and the residue is suspended in the 
detergent-containing aqueous solution. 
An enrichment or purification is likewise possible using precipitating 
agents, as is usual in biochemistry for proteins and corresponding 
antigens. The Neisseria meningitidis antigen can be precipitated, for 
example, by adding 4 parts by volume of ethanol and the precipitate can be 
taken up again in aqueous solution. 
The Neisseria meningitidis antigen filtered under sterile conditions and 
optionally lyophilized is capable of inducing protective antibodies 
against the causative organism. 
The antigen can be admixed with adjuvants, stabilizers, fillers and similar 
substances as used in the preparation of vaccines. 
The membrane proteins display an immunogenic effect. It is, therefore, 
another object of the present invention to provide a vaccine containing 
the membrane proteins prepared in accordance with the process of the 
invention in an amount sufficient to induce an immunizing effect. The 
vaccine can be administered in a dosage of from about 10 to 200 .mu.g per 
dose.

The following examples illustrate the invention. 
EXAMPLE 1 
300 g (wet weight) of sedimented Neisseria meningitidis group B organisms, 
(strain 986), were resuspended in 1,500 ml of sodium deoxycholate solution 
(0.5% of sodium deoxycholate in 0.01 M tris-HCl, pH 8.5, 0.01 M EDTA) 
which had previously been heated to 60.degree. C. and the suspension was 
kept for 15 minutes in a water bath (56.degree. C.). The bacterial cell 
mass was then separated by centrifugation (Sorvall GSA Rotor, 10,000 rpm). 
The residue was again treated as described above for 15 minutes with 
sodium deoxycholate solution. The suspension was centrifuged and the 
second supernatant was combined with the first one. The combined 
supernatants were then centrifuged in a Beckmann L 75 centrifuge at 40,000 
rpm for 60 minutes in a 45 Ti Rotor. The sediment was suspended in 150 ml 
of sodium deoxycholate solution and stirred overnight at 4.degree. C. 
For further purification, the suspension was heated on the following day in 
a water bath (56.degree. C., 15 minutes), centrifuged again in the 
ultracentrifuge as used above (Rotor 45 Ti, 40,000 rpm, 60 minutes) and 
the sediment was resuspended in 150 ml of sodium deoxycholate solution. 
For resuspension, an Ultrasonic Cleaner of Laboratory Supplies Co., Inc., 
Hickville N.Y., USA was used. Aliquots of 50 ml each were treated for 3 
minutes with ultrasonic waves and then combined. Particulate material was 
separated by another centrifugation (Sorvall, SS-34 Rotor, 20,000 rpm, 20 
minutes) and the supernatant was filtered. The membrane protein was 
precipitated under sterile conditions by the addition of 600 ml of 96% 
ethanol and allowed to stand for 60 minutes. The precipitate was separated 
by centrifugation (Sorvall, GSA Rotor), washed once with 100 ml of 
ethanol, taken up under sterile conditions in 150 ml of 5% raffinose and 
stirred overnight at 4.degree. C. 
The optical density (O.D.) of the material (280 nm) was determined and 
adjusted to O.D..sub.280 =1.2 by adding 0.5% raffinose. The concentrate 
obtained in this manner was worked up to a vaccine in a usual manner. 
For the determination of the optical density an aliquot portion was taken 
under sterile conditions, 1 part by volume of trichloroacetic acid (TCA) 
of 10% strength was added to bring about precipitation, the precipitate 
was washed in 5% TCA and taken up in 1 M KOH. 
EXAMPLE 2 
50 g of sedimented organisms were worked up as described in Example 1 with 
the exception that a solution of 0.1% of sodium deoxycholate was used 
instead of a 0.5% sodium deoxycholate solution. 0.8 mg/g of membrane 
proteins of cell mass was obtained, while the yield in Example 1 was 2.3 
mg/g. 
EXAMPLE 3 
50 g of cells were worked up as described in Example 1, but a solution with 
2.0% of sodium deoxycholate was used instead of the 0.5% sodium 
deoxycholate solution. 2.2 mg of membrane proteins were obtained per gram 
of germs, while the yield in Example 1 was 2.3 mg/g. 
EXAMPLE 4 
50 g of cells were worked up as described in Example 1, but instead of 0.5% 
sodium deoxycholate in 0.01 M tris-HCl-0.01 M EDTA, 0.5% sodium 
deoxycholate was used in (a) 0.1 M phosphate buffer, pH 7.8, (b) in Hepes 
buffer (0.1 M, pH 7.8) and (c) in 0.1 M sodium carbonate buffer, pH 8.5. 
The yields obtained varied between 1.8 and 2.5 mg of membrane proteins per 
gram. 
EXAMPLE 5 
The extraction described in Example 1 was carried out with other 
detergents. The yields are listed in the following table. 
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yield 
Detergent concentration 
(mg/g wet weight) 
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urea 4 M 1.2 
Emulphogen.sup.(R) 1 % 
1 % 1.3 
Tween 20.sup.(R) 
1 % 2.6 
Triton .times. 100.sup.(R) 
1 % 2.7 
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