Process for the preparation of detoxified polysaccharide-outer membrane protein complexes, and their use as antibacterial vaccines

Process for preparing a detoxified polysaccharide-outer membrane protein complex from bacterial envelopes; the so-obtained products which are useful as vaccines against infection by the same bacteria and method for protecting animals against the same infection by administration of a pharmaceutical composition containing the detoxified polysaccharide-outer membrane protein complexes.

BACKGROUND OF THE INVENTION 
This invention relates generally to a process for the preparation of 
immunogenic detoxified polysaccharide-outer membrane protein complexes 
from bacteria and more particularly to the use of said complexes, as 
vaccines, to protect animals against infections by the bacteria from which 
it has been derived comprising administering to an animal a pharmaceutical 
composition containing the detoxified polysaccharide-protein complexes. 
The virulence of certain gram-negative bacteria is enhanced by the presence 
of a capsule which envelopes the outer membrane and is made up of 
different components among which are polysaccharides and 
lipopolysaccharides. 
The outer membrane proteins of gram-negative bacteria such as Haemophilus 
influenza type b, Neisseria gonorrhoeae, Escherichia coli, Pseudomonas 
aeruginosa, and Neisseria meningitidis have been shown to induce 
bactericidal antibodies in man, both when encountered in the course of 
natural infections and when given as a vaccine in which the proteins are 
noncovalently complexed to the capsular polysaccharide. Vaccines of this 
type, as exemplified by Neisseria meningitidis, have been studied by 
several groups to determine their potential for providing protection 
against group B meningococcal disease. In most instances the outer 
membrane proteins from a serotype 2a strain have been used together with 
group B polysaccharide. Although vaccines of this type have shown promise 
in terms of safety and immunogenicity, there are a number of problems 
which need to be resolved. 
When presented as a complex with the outer membrane proteins, the B 
polysaccharide induces a transient IgM antibody response. These antibodies 
are bactericidal with rabbit complement, but have little if any 
bactericidal activity with human complement. This fact together with 
reports that antibodies to group B polysaccharide cross-react with human 
fetal and neonatal brain antigens suggest to applicants that these 
antibodies are probably not protective and an alternative to group B 
polysaccharide is needed in future vaccine preparations. 
A second problem is the great antigenic diversity and variability of the 
outer membrane proteins. Meningococci possess multiple outer membrane 
proteins which are known to vary antigenically from strain to strain. 
According to the classification scheme of Tsai, et al. described in the 
Journal of Bacteriology, Volume 146, pages 69 to 78, 1981, 5 major classes 
of outer membrane proteins are recognized. Of these, classes 1, 2, 3, and 
5 have been shown to vary antigenically from strain to strain. In 
addition, lipopolysaccharides are present and exhibit antigenic 
variability. From the point of view of vaccine development this creates 
problems both in terms of vaccine formulation and evaluation of the 
antibody response to vaccination. The number of different serotype 
proteins that can safely be included in a single vaccine may be limited by 
reactogenicity resulting from the residual lipopolysaccharide (LPS) 
associated with them and probably also by reactogenicity intrinsic to the 
proteins themselves. 
Applicants have evaluated the human bactericidal antibody response to 
serotype 2b and serotype 15 outer membrane proteins, prepared these 
proteins relatively free of LPS (less than 1%), and discovered the 
immunogenicity and safety of these proteins when combined in a single 
vaccine and solubilized by the tetravalent mixture of A, C, Y, and W135 
polysaccharides. 
The existence of other methods for preparing vaccines useful against 
infections caused by gram-negative bacteria are disclosed in U.S. Pat. 
Nos. 4,451,446; 3,636,192; 3,859,434; 4,356,170; 4,123,520 and 3,978,209 
which are hereby incorporated herein by reference. 
Although partially effective vaccines are available for treatment of 
bacterial infections, most vaccines presently known to be employed have 
limited effectiveness. 
SUMMARY OF THE INVENTION 
This invention relates to a novel means for affording treatment and control 
of infections in animals, including human beings and other mammalian 
species, which may be caused by gram-negative bacteria. It is based upon 
the use or administration of immunogenically effective amounts of a 
detoxified polysaccharide-outer membrane protein complex against infection 
by the same bacteria from which it has been derived. The term 
"polysaccharide" as used herein includes lipopolysaccharides and capsular 
polysaccharides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Practical utility has been established for immunogenic detoxified 
polysaccharide-outer membrane protein complexes prepared from bacteria 
wherein the complex, unlike the bacteria from which it was derived, is 
essentially free (less than 1%) of toxic polysaccharide comprising the 
steps of: 
a. suspending the outer membrane proteins in a buffer solution (TEEN) 
containing 1% of the zwitterionic detergent Empigen BB, 0.01M 
ethylenediaminetetraacetic acid (EDTA) 0.05M tris-HCl, and 0.15M NaCl, 
said solution having a pH of about 8.0; 
b. stirring the suspension formed in step a. for about 1 hour at about room 
temperature followed by mild sonication; 
c. centrifuging the suspension formed in step b. at about 20,000.times.g 
for about 15 minutes and separating the supernatant fluid from the 
insoluble residue; 
d. adding solid ammonium sulfate to the supernatant layer formed in step c. 
in an amount of 500 grams/liter of supernatant protein solution and 
stirring until the ammonium sulfate is dissolved, and then allowing to 
stand at about room temperature for about 1 to 2 hours to allow 
precipitation of the outer membrane protein; 
e. centrifuging the product of step d. at about 20,000.times.g for about 20 
minutes and collecting the precipitated outer membrane proteins which form 
a top layer; 
f. dissolving the precipitated outer membrane proteins formed in step e. in 
a buffer solution of the type described in step a. adding ammonium 
sulfate, and separating the precipitate; 
g. repeating the process of step f.; 
h. dissolving the precipitate formed in step g. in a TEEN buffer to form a 
solution, then dialyzing said solution against TEEN buffer to remove 
(NH.sub.4).sub.2 SO.sub.4 ; 
i. centrifuging the solution formed in step h. at about 35,000.times.g for 
about 20 minutes and removing any insoluble material; 
j. filtering the solution formed in step i. sequentially through 1.2.mu., 
0.45.mu., and 0.22.mu. membrane filters; 
k. sterile filtering the solution formed in step j. through a 0.22.mu. 
membrane filter; 
l. complexing the sterilized outer membrane proteins with the sterile 
filtered polysaccharide by combining the outer membrane protein, derived 
from one or more strains, and polysaccharide, derived from one or more 
strains, in a ratio within the range of 1:2 to 2:1 in TEEN buffer and 
coprecipitating by the cold, sterile absolute ethanol to 75% 
volume/volume; 
m. centrifuging under sterile conditions, the ethanolic solution formed in 
step 1. at about 20,000.times.g for about 15 minutes and separating the 
precipitate. 
n. washing the precipitate formed in step m. with sterile absolute ethanol 
to remove essentially all components of the buffer solution yielding an 
immunogenic detoxified polysaccharide-protein complex. 
The immunogenic detoxified polysaccharide-protein complexes prepared in 
accordance with applicants' novel process are preferably stored in 
distilled water or a 3% lactose solution at about -20.degree. C. for 
future use, although other physiologically-acceptable solvents can be used 
for this purpose. 
Applicants' novel process is applicable to the preparation of detoxified 
lipopolysaccharide-outer membrane protein and capsular 
polysaccharide-outer membrane protein complexes wherein the 
lipopolysaccharide is noncovalently bonded the protein whereas capsular 
polysaccharides can be either bonded noncovalently or covalently to the 
protein to form a complex. 
The process of this invention is generally applicable to the preparation of 
detoxified polysaccharide-protein complexes derived from gram-negative 
bacteria. Although bacteria such as Neisseria meningitidis group B, 
Haemophilus influenza type b, Neissera gonorrhoeae, Escherichia coli, and 
Pseudomonas aeruginosa are preferred. 
Applicants have found that the outer membrane proteins which are referred 
to in step a. of their process can be in the form of an outer membrane 
complex, outer membrane vesicles or an extract from whole cells. When in 
the form of whole cell extract(s), the outer membrane proteins are first 
extracted from whole cells by the process comprising suspending said cells 
in a 0.15M NaCl solution, separating said cells from the solution by 
centrifugation at between 10,000 to 20,000.times.g for about 20 minutes 
and suspending the resulting cells in one volume of 1M sodium acetate 
buffer, pH 5.5, adding 9 volumes of 3% Empigen BB in 0.5M CaCl.sub.2 and 
stirring for about 1 hour, adding ethanol to 20% volume/volume, 
centrifuging at about 20,000.times.g for about 10 minutes to separate the 
precipitate from the supernatant, adding ethanol to the supernatant to a 
final concentration of about 45% volume/volume, collecting the precipitate 
by centrifugation, dissolving the collected precipitate in a buffer 
solution containing 1% Empigen BB, 0.05M tris-HCl, 0.01M 
ethylenediaminetetracetic acid (EDTA), and 0.15M NaCl, said buffer 
solution having a pH of about 8.0 and separating of any insoluble material 
by centrifugation to yield an aqueous solution from which the outer 
membrane proteins are separated. 
The outer membrane proteins referred to in step a. of applicants' process 
may be derived either from several different bacterial strains or a single 
bacterial strain. Applicants have found that the detoxified complexes 
prepared according to the process of this invention are most efficacious 
when the ratio between the polysaccharide and the outer membrane protein 
components of the complex are with the range 1:2 to 2:1, most preferably 
1:1. 
The sequential steps involved in applicants' process can be illustrated in 
the following schematic diagram I: 
SCHEMATIC DIAGRAM I 
##STR1## 
WORKING EXAMPLES 
The working examples set forth below illustrate the preparation of 
representative compositions, but in no way limit the scope of the 
invention. 
EXAMPLE 1 
Noncovalent Complex of Neisseria meningitidis Group B Outer Membrane 
Proteins and Tetravalent Mixture of Capsular Polysaccharides 
A. Purfication of outer membrane protein from outer membrane complex. 
Meningococci of serotype 2b (or 15) are grown in liquid culture (15 liters) 
under conditions which result in a heavy stationary state culture. The 
culture is inactivated with phenol at 0.5% for two hours and the organisms 
collected by centrifugation. Outer membrane complex is extracted from the 
organisms by the method of Zollinger, et al. (J. Clin. Invest. 63:836) and 
stored at -20.degree. C. in distilled water. 
The outer membrane complex is precipitated by addition of 2M NaCl to a 
final concentration of 0.15M and 2.5 volumes of absolute ethanol. The 
precipitate is collected by centrifugation at 8000.times.g for 15 minutes 
and the supernatant thoroughly drained off and discarded. 
The pellets are dissolved in TEEN buffer which contains, 0.05M 
Tris-hydrochloride, 0.01M EDTA (ethylenediaminetetraacetic acid, disodium 
salt), 0.15M NaCl, and 1% of the zwitterionic detergent Empigen BB 
(Albright and Wilson, LTD., Whitehaven, England), pH 8.0. Alternatively 
the outer membrane complex may be mixed 1:1 with a 2X solution of the TEEN 
buffer. Solubilization is aided by mild sonication such as may be obtained 
by holding the flask in a bath sonicator. The solution is centrifuged at 
30,000.times.g for 15 minutes and the supernatant collected. The pellets 
are resuspended in the TEEN buffer using a syringe and needle, sonicated 
briefly, and recentrifuged as above. The supernatants are pooled. 
Solid ammonium sulfate is added to the solution in the amount of 500 
g/liter and the mixture stirred until all the ammonium sulfate is 
dissolved. The solution is allowed to stand at room temperature for 1 hour 
and then centrifuged at 20,000.times.g for 20 minutes. The precipitated 
protein collects at the top of the tube and is recovered by drawing off 
the liquid from the bottom. Lipopolysaccharide, capsular polysaccharide, 
and nucleic acid remain in solution. When all the liquid is removed, the 
protein is redissolved in the TEEN buffer. 
The precipitation with ammonium sulfate is repeated twice with the 
centrifugation being done when all the ammonium sulfate is dissolved. The 
final precipitate is dissolved in TEEN buffer at 1-2 mg/ml and dialyzed 
against 4 changes of 20 volumes of TEEN buffer to remove the ammonium 
sulfate. After dialysis the solution is centrifuged at 35,000.times.g for 
20 minutes and then filtered sequentially through membrane filters with 
pore sizes of 1.2 .mu.m, 0.45 .mu.m, and 0.22 .mu.m. The solution is then 
sterile filtered through a 0.22 .mu.m filter and stored at -30.degree. C. 
After two precipitations with ammonium sulfate the protein contains about 
1-3% residual lipopolysaccharide. After the third precipitation, residual 
LPS is at 1% or less, capsular polysaccharide is not detectable, and 
nucleic acid is less than 1%. The ammonium sulfate precipitated proteins 
are easily soluble in the TEEN buffer. 
Polyacrylamide gel electrophoresis of the proteins by the method of Laemmli 
(Nature 227:680-685) and staining with coomassie brilliant blue shows the 
presence of two or three major bands corresponding to the class 1, class 2 
(or 3), and class 5 major outer membrane proteins as classified by Tsai 
and Frasch (J. Bacteriol. 146:69-78). These proteins constitute about 
70-90% of the total protein in the sample. 
B. Preparation of the Polysaccharide-protein complex. 
Purified capsular polysaccharides are prepared by the methods approved by 
the Office of Biologics, Food and Drug Administration and the World Health 
Organization for the currently licensed meningococcal polysaccharide 
vaccines. These methods are based on those of Gotschlich (U.S. Pat. No. 
3,636,193). A mixture of equal amounts of the capsular polysaccharide from 
serogroups A, C, Y, and W-135 is dissolved in 0.15M NaCl at 1 mg/ml total 
polysaccharide. This solution is combined with an equal volume of TEEN 
buffer and sterile filtered through a 0.22 .mu.m filter. 
The sterile filtered capsular polysaccharides are combined at 4.degree. C. 
with a mixture of equal amounts of sterile filtered outer membrane 
proteins from strains of two different serotypes. The amounts of 
polysaccharide and protein are calculated based on assays performed after 
the filtration, and are measured to give approximately equal amounts of 
total polysaccharide and total portein. In the present example a ratio of 
3 parts polysaccharide to 2 parts protein was used. Three volumes of cold, 
sterile absolute ethanol are added to precipitate the mixture and remove 
the components of the TEEN buffer. The precipitate is collected by 
centrifugation at 16,000.times.g for 15 minutes and washed three times 
with cold, sterile absolute ethanol. The final precipitate is suspended in 
a solution of 3% lactose and constitutes the bulk product. It is stored at 
-30.degree. C. until sterility is assured and then dispensed into glass 
vials and lyophilized. 
The product is reconstituted with sterile saline for injection. 
C. Immunological characteristics of the polyvalent polysaccharide-protein 
complex. 
The final product (ACYW2B15-2) was evaluated for its antigenic activity by 
examining its ability to react with monoclonal antibodies against the 
serotype specific determinants of the major outer membrane proteins in a 
solid phase radioimmunoassay (Infect. Immun. 18:424-433). Antibodies to 
the serotype proteins of the serotype 2b and 15 strains were inhibited 50% 
by a concentration of 0.45 and 1.1 .mu.g protein/ml, respectively. 
Antibodies to the class 1 protein were inhibited by 7.6 and 27.3 .mu.g 
protein/ml. 
Further immunological evaluation was done by immunizing Balb/C mice with 
this vaccine and the tetravalent capsular polysaccharide vaccine (ACYW). 
The results are given in Table A. One .mu.g of protein (ACYW2b15-2) or 2 
.mu.g of polysaccharide (ACYW) was given intraperitoneally to groups of 6 
mice at day 0 and day 21. The antibody levels were measured by SPRIA using 
outer membrane complex from the vaccine strains as antigen for 
anti-protein antibodies and poly-L-lysine followed by purified capsular 
polysaccharide as antigen for anti-polysaccharide antibodies. A good 
antibody response to the protein was observed which included a strong 
booster response to the dose given at 21 days. The antibody response to 
the capsular polysaccharides was enhanced somewhat in the 
protein-containing vaccine. 
This vaccine has also been tested in rabbits with similar results for the 
protein antigens, but the rabbits did not respond to the capsular 
polysaccharides. 
Several additional characteristics of the vaccine war given in Table B. 
About 50% of the polysaccharide was actually bound hydrophobically to the 
protein as determined by gel filtration on a column of Sepharose 4B-CL and 
comparing to a similar column run with polysaccharide alone. Some of the 
polysaccharide appears to lack a hydrophobic moiety necessary for binding 
to the proteins. The product was nonpyrogenic in rabbits at a dose of 0.1 
.mu.g/kg. The complexes were of high molecular weight with 90% of the 
polysacccharide eluting with a Kd of greater than 0.5. 
EXAMPLE 2 
Safety and Antigenicity Test of Polyvalent Protein-Polysaccharide Vaccine 
in Human Volunteers 
A noncovalent complex of outer membrane proteins (serotypes 2b and 15) and 
a tetravalent mixture (groups A, C, Y, and W-135) of capsular 
polysaccharide has been tested for safety and antigenicity in 65 adult 
volunteers. Such a vaccine if fully effective might protect against 
meningococcal disease of all 5 pathogenic serogroups (A, B, C, Y, and 
W-135). The composition on a dose basis of the vaccine lot tested is given 
in Table C. The protein used in this lot was only precipitated twice with 
ammonium sulfate (see Example 1) and, therefore, contains about 2% 
residual lipopolysaccharide. A single dose was given subcutaneously in the 
upper arm. The volunteers were mostly military recruits who routinely 
receive a licensed tetravalent meningococcal capsular polysaccharide 
vaccine. This vaccine was given by needle and syringe to a control group. 
The vaccine appeared safe in that no severe or unusual reactions occured. A 
summary of reactogenicity is given in Table D. 
The antiobody response to the protein antigens was measured by bactericidal 
assays against several different group B strains including the vaccine 
strains, 8047 and 44/76. Antibodies were also measured using an enzyme 
linked immunosorbant assay (ELISA). Seroconversions with respect to the 
group B vaccine strains are summarized in Table E. The geometric mean 
increase in bactericidal antibody to the homologous group B vaccine 
strains at two weeks is given in Table F along with the geometric mean 
increases determined by ELISA. Analysis of the bactericidal antibody 
response is shown in FIGS. 1 to 7. 
Lower responses were seen when the sera were tested against heterologous 
serotypes. This suggests that much of the antibody is directed against 
serotype specific determinants and that it is important to prepare the 
vaccine outer membrane proteins from the most prevalent group B serotypes 
serotype(s). 
EXAMPLE 3 
Preparation of Outer Membrane Protein-detoxified lipopolysaccharide 
noncovalent complexes 
A. Preparation and Characterization of Detoxified Meningococcal LPS. 
Lipopolysaccharide is purified (less than 1% nucleic acid and 1% protein) 
by standard methods such as the hot phenol-water method of Westphal, et 
al. (Z. Naturforsch, Teil B 7:148-155) from a serogroup B case strain e.g. 
8047 or 44/76. 
Purified lipopolysaccharide is detoxified by dissolving at about 4 mg/ml in 
0.1N NaOH and placing the vessel in a 60.degree. C. water bath for 3 
hours. The reaction is stopped by addition of sufficient acetic acid to 
neutralize the NaOH and five volumes of cold absolute ethanol are added to 
precipitate the lipopolysaccharide. The precipitate is collected by 
centrifugation and washed twice with cold ethanol dissolved in distilled 
water and dialyzed against 100 volumes of distilled water overnight at 
4.degree. C. The solution is then lyophilized. 
The biological and chemical properties of the detoxified lipopolysaccharide 
were studied and compared to native lipopolysaccharide. Gas liquid 
chromatographic analysis of fatty acid composition showed complete loss of 
ester linked fatty acids and retention of amide linked fatty acids. The 
relative toxicity of the native and detoxified product is compared in 
Table G. Four different toxicity tests were used and in each case a 
greater than 1000-fold decrease in toxicity was observed. A four log 
reduction in activity in the Limulus ameobocyte lysate gelation assay was 
also observed. No loss in antigenic determinants could be detected by 
binding of lipopolysaccharide-specific monoclonal antibodies in a spot 
blot assay. In addition, the detoxified product was shown to retain its 
ability to bind to and solubilize outer membrane proteins. 
B. Preparation of Outer Membrane Protein-detoxified lipopolysaccharide 
complexes. 
Detoxified lipopolysaccharide is dissolved in TEEN buffer (see Example 1) 
and sterile filtered through a 0.22 .mu.m filter. This solution is 
combined with an equal amount of purified outer membrane protein from the 
group B strain 44/76 (B:15:P1.16:L3,8) also solubilized in TEEN buffer. 
Four volumes of cold, sterile absolute ethanol is added to coprecipitate 
the mixture. The precipitate is collected by centrifugation and washed 
three times as described in Example 1. The final precipitate is dissolved 
in sterile distilled water with the aid of mild sonication using a bath 
sonicator. Sonication is often essential to facilitate the 
protein-lipopolysaccharide interaction and solubilize the protein. The 
solubilized product is centrifuged at 10,000.times.g for 10 minutes to 
remove any insoluble material and the supernatant dispensed into vials and 
lyophilized. The product is reconstituted with normal saline for 
injection. 
C. Immunological properties of detoxified lipopolysaccharide-outer membrane 
protein complexes. 
Groups of 10 mice were vaccinated with a 1 .mu.g (protein) dose of vaccine 
on days 0 and 28. Separate groups of mice were vaccinated with capsular 
polysaccharide (group C)-outer membrane protein complexes prepared using 
the same protein preparation [OMP(c)] and a different protein preparation 
from the same strain [OMP(b)]. Antibody levels against the homologous 
proteins were determined by solid phase radioimmunoassay using outer 
membrane complex as antigen. The results as given in Table H show that the 
outer membrane protein has approximately equivalent antigenicity when 
solubilized with either the detoxified lipopolysaccharide or the capsular 
polysaccharide. A strong antibody response to both the initial dose and 
the booster dose was observed. 
EXAMPLE 4 
Extraction of Meningococcal Outer Membrane Protein from Whole Cells 
Meningococcal cells of strain 44/76 (B:15:P1.16:L3,8) are harvested from 
liquid culture by centrifugation at about 16,000.times.g for 20 minutes. 
The cells are suspended in about an equal volume of 1M sodium acetate 
buffer pH 5.5, and nine volumes of a solution containing 3% Empigen BB in 
0.5M calcium chloride are added. The mixture is stirred at room 
temperature for one hour after which ethanol is added to a concentration 
of 20% volume/volume. The resulting precipitate is removed by 
centrifugation at about 20,000.times.g for 10 minutes. The pellets are 
discarded and the supernatant is brought to 45% ethanol volume/volume. The 
precipitated proteins are collected by centrifugation at about 
20,000.times.g for 10 minutes and dissolved in buffer containing 1% 
Empigen BB, 0.15M NaCl, 0.01M EDTA, and 0.05M Tris-HCl at pH 8.0. Any 
insoluble material is removed by centrifugation at about 20,000.times.g 
for 10 minutes. The proteins are further purified to remove 
lipopolysaccharide, capsular polysaccharide, and nucleic acid by ammonium 
sulfate precipitation three times as described in Example 1A. This product 
gives essentially the same band pattern on SDS polyacrylamide gels as 
outer membrane protein purified from outer membrane complex or outer 
membrane vesicles. The purified outer membrane protein is noncovalently 
complexed with capsular polysaccharide or detoxified lipopolysaccharide as 
described in Examples 1 and 2. 
Mice were vaccinated with complexes prepared from protein purified by this 
method [OMP(c)]. The procedure is given in Example 3C and the results in 
Table H. The antibody response obtained in mice with vaccines containing 
(OMP(c) are equally as good as those containing OMP(b) which was derived 
from outer membrane complex rather than whole cells. 
TABLE A 
__________________________________________________________________________ 
Immunogenicity of Meningococcal Polyvalent Polysaccharide - 
Protein Vaccine in Mice 
Antigen 
Vaccine 
Day 
8047 OMC 
44/75 OMC 
Bsss 
Asss 
Csss 
Ysss 
Wsss 
__________________________________________________________________________ 
ACYW2b15-2 
0 .33* .23 .36 
.16 
.57 
.52 
.40 
5 5.0 3.54 .86 
20.84 
4.49 
2.90 
1.66 
21 9.71 5.02 .51 
.53 
1.00 
1.78 
.74 
28 79.62 62.95 .55 
3.32 
4.45 
11.04 
1.94 
ACYW 0 .30 
.50 
.39 
.32 
5 1.37 
1.96 
1.50 
.86 
21 .58 
.82 
1.24 
.57 
28 .84 
1.36 
2.14 
.76 
__________________________________________________________________________ 
*.mu.g Antibody/ml by solidphase radioimmunoassay 
Bsss = group B capsular polysaccharide, etc. 
TABLE B 
______________________________________ 
Characterization of Meningococcal Vaccine 
lot ACYW2b15-2 
Parameter Result 
______________________________________ 
Polysaccharide bound to protein (est.) 
51% 
Rabbit pyrogen test 0.12 .mu.g/kg passed 
Size of polysaccharide on Sepharose 
90% with Kd &gt; 0.5 
CL-4B.sup.+ 
______________________________________ 
.sup.+ Based on sialic acid assay of column fractions. 
TABLE C 
______________________________________ 
Composition of Vaccine lot ACYW2b15-2 
Substance .mu.g/Dose .+-. 10% 
______________________________________ 
Protein 
(serotype 2b) 60 
(serotype 15) 60 
Polysaccharide 
(group A) 45 
(group C) 45 
(group Y) 45 
(group W-135) 45 
Lipopolysaccharide* 
4.1 
Nucleic acid 1.5 
Total.sup.+ 305 
______________________________________ 
.sup.+ Based on assay of the bulk protein for KDO. 
.sup.+ In addition, the vaccine contained 4.8 mg lactose per dose and was 
reconstituted in normal saline (0.5 ml/dose). 
TABLE D 
______________________________________ 
Summary of Side Effects of Vaccine lot ACYW2b15-2 in 
Military Recruits at Ft. Benning, GA. 
ACYW2b15-5 ACYW 
Number vaccinated 
54 47 
Question or complaint 
Response-vaccine 
Response-control 
______________________________________ 
Temperature at 6-8 hr. 
Geometric mean .+-. SEM 
37.31 .+-. 0.06 
37.14 .+-. 0.07 
Mo. with temp. 37.8.degree. C. 
3 1 
Mo. with temp. 37.6.degree. C. 
9 5 
Erythema at 24 hr. 
3.76 .+-. 0.32 
1.38 .+-. 0.25 
(Geometric mean .+-. SEM 
of largest diameter in cm) 
Number positive 44/54 23/47 
Induration Number positive 
36 10 
Sore arm (Mean soreness 
1.57 .+-. 0.11 
1.28 .+-. 0.15 
index .+-. SEM scale of 0 
to 4) 
Number positive 50 37 
______________________________________ 
TABLE E 
______________________________________ 
Seroconversions in Volunteers Vaccinated 
with Meningococcal Vaccines 
Percent seroconversion 
8047(B:2b) 
44/76(B:15) 
Vaccine No. ELISA BCT ELISA BCT 
______________________________________ 
ACYW2b15-2 54 91 66 94 76 
ACYW (Control) 
41 12 10 7.3 10 
______________________________________ 
*Based on 4fold or greater increase in bactericidal antibody (BCT), or 
2fold or greater increase in antibody in the ELISA. 
TABLE F 
__________________________________________________________________________ 
Geometric Mean Antibody Response of Human Volunteers Vaccinated with 
Meningococcial 
Polyvalent Polysaccharide-Protein Vaccine ACYW2b15-2 
Geometric Mean Antibody Level at Indicated Time 
Assay Strain or Antigen 
0 wk 2 wk 14 wk 
__________________________________________________________________________ 
Bactericidal Assay 
8047(B:2b:P1.2) 
2.2 20.1 6.9 
44/76(B:15:P1.16) 
2.1 25.5 8.6 
ELISA Assay 
8047 OMC 5.6 51.4 16.5 
44/76 OMC 
4.0 59 14.6 
__________________________________________________________________________ 
Only noncarriers are included in the data (N = 24). Values for the 
bactericidal test are reciprocal titers and those for the ELISA are .mu.g 
IgG antibody/ml. 
TABLE G 
__________________________________________________________________________ 
Characterization of Alkaline Detoxified Meningococcal Lipopolysaccharide* 
Property or Assay Untreated LPS 
Alkaline Treated LPS 
__________________________________________________________________________ 
Pyrogenicity in rabbits Pyrogenic at 
Non-pyrogenic at 
(.mu.g/Kg) 0.025 .mu.g 
50 .mu.g 
Local Shwartzman reaction 
Positive at 
Negative at 
(.mu.g for preparative dose) 
0.2 .mu.g 
500 .mu.g 
Limulus lysate assay (Lowest positive conc.) 
1 ng/ml 10 .mu.g/ml 
Chick embryo lethality test 
LD.sub.50 = &lt; 1 .mu.g 
Non-toxic at &gt; 1000 .mu.g 
Antigenic activity** 0.2 .mu.g/ml 
0.2 .mu.g/ml 
(Binding of monoclonal antibodies vs L8 & L3,7) 
1.6 .mu.g/ml 
1.6 .mu.g/ml 
Capacity to bind to and solubilize OM protein 
++ + 
Galactosamine sensitized mouse toxicity Test (LD.sub.50) 
.003 .mu.g 
3.3 .mu.g 
__________________________________________________________________________ 
*Lipopolysaccharide from strain 44/76 was treated with 0.1 N NaOH for 3 h 
at 60.degree. C. 
**The lowest concentration of antigen that gave a positive reaction in a 
dotblot assay with two different monoclonal antibodies to LPS. 
TABLE H 
______________________________________ 
Antibody Response of Mice to Meningococcal Outer Membrane 
Protein Noncovalently Complexed to Meningococcal Capsular 
Polysaccharide or Detoxified Lipopolysaccharide 
Mean antibody response 
to OMP by SPRIA 
Day 0 Day 28 Day 42 
Vaccine composition* 
(.mu.g/ml) 
______________________________________ 
OMP(b):C polysaccharide (1:1) 
0.40 27.8 1250 
OMP(c):C polysaccharide (1:1) 
0.48 22.5 910 
OMP(c):detoxified LPS (1:1) 
0.46 18.4 1175 
OMP(c):detoxified LPS (2:1) 
0.49 10.4 326 
Saline ND 0.87 1.7 
______________________________________ 
*OMP(b) is outer membrane protein prepared from vesicles of outer membran 
collected from the culture supernatant. OMP(c) is outer membrane protein 
extracted from whole cells by the method in Example 4. A serotype 15 
strain 44/76(B:15:P1.16:L3,8) as used in each case for OMP and LPS. 
UTILITY 
The detoxified polysaccharide-outer membrane protein complexes prepared 
according to applicants' novel process of this invention induce immune 
response to bacterial infections. More specifically, evidence indicates 
that these complexes have activity against bacterial infections caused by 
gram-negative bacteria including Neisseria meningitidis group B, 
Haemophilus influenza type b, Neissera gonorrhoeae, Escherichia coli, and 
Pseudomonas aeruginosa. 
Several experiments have been conducted to determine the activity of the 
detoxified polysaccharide-outer membrane protein complexes prepared 
according this invention. In order to guide one of ordinary skill in the 
practice of this invention, these experiments are described below, as well 
as results obtained in each experiment with a representative sampling of 
vaccine preparations. 
PREATION AND CHARACTERIZATION OF LOW LPS VACCINES 
Applicants' novel method for separating LPS from the outer membrane 
proteins was used to prepare several lots of vaccine which differed in 
formulation. This method which made use of the zwitterionic detergent 
Empigen BB, an alkyl betaine, (Albright and Wilson LTD, Cumbria, White 
Haven, UK), was compared to the previous method, described by Zollinger, 
et al., Seminars in Infectious Disease, Volume 4, "Bacterial Vaccines", 
pages 254-262, 1982; and Zollinger, et al., Journal of Clinical 
Investigation, Volume 63, pages 836 to 848, 1979, which involved 
preferential solubilization of LPS with sodium deoxycholate (DOC). The 
starting material in each case was outer membrane complex (OMC) prepared 
by one of several procedures. The essentials of the method include 
solubilization of the OMC in a buffer (TEEN) containing 1% Empigen BB, 
0.05M tris-hydrochloride, 0.01M EDTA and 0.15M NaCl, pH 8.0 and thrice 
precipitating the protein with ammonium sulfate added as a solid to 500 g 
ammonium sulfate per liter of protein solution. The final precipitate was 
dissolved in TEEN and dialyzed against TEEN with 0.1% Empigen BB. For 
preparation of the vaccines, the protein was sterile filtered and 
complexed with polysaccharide as described by Zollinger, et al. in 
Seminars In Infectious Disease, Volume 4, "Bacterial Vaccines", pages 254 
to 262, 1982. 
Eight different lots of vaccine were prepared for use, characterized, and 
tested for safety and immunogenicity in animals; but because of the 
considerations mentioned above, only one lot, that did not contain the 
group B polysaccharide, was tested in human volunteers. 
Several of these vaccines which differed in LPS content, method of 
preparation and composition are compared in Table 1. In experiment 1, 
vaccines which differed mainly in method of preparation were compared. Lot 
BP2b-1 was prepared by our previous method using deoxycholate to 
preferentially solubilize the LPS. Lot BP2b-2 was prepared from the same 
batch of OMC with the new Empigen BB method and contained about half as 
much LPS as lot BP2b-1. The geometric mean antibody response in mice as 
measured by solid phase radioimmunoassay (SPRIA) with homologous OMC as 
antigen was not significantly different. 
In the second experiment, all three lots were prepared by the Empigen BB 
method. The protein in lot BP2b-3 was precipitated with ammonium sulfate 
three times and contained less than 1% LPS. It was compared to two other 
vaccines which contained protein that was precipitated twice with ammonium 
sulfate and contained about 3.5% residual LPS (relative to protein). Lot 
ACYW2b15-2 contained the tetravalent A, C, Y, and W135 polysaccharide 
mixture in place of the group B polysaccharide and the protein serotypes. 
Two doses of 0.5 g protein (1.0 g of lots ACYW2b15-2 and BP215-2) was 
given intraperitoneally at 0 and 21 days. Ten to twenty-fold geometric 
mean increases in antibodies were observed after both the initial dose and 
the booster dose. The results indicate that neither more complete removal 
of LPS nor replacement of the group B polysaccharide with the tetravalent 
A, C, Y, and W135 mixture adversely affected the immunogenicity of the 
serotype 2b protein. It is important to note that experiments one and two 
cannot be directly compared because the mice were about two months older 
(15 weeks versus 9 weeks) in the second experiment. 
CHARACTERIZATION OF LOT ACYW215-2 
Lot ACYW2b15-2 was chosen for human testing because it did not contain the 
group B polysaccharide, and because it had a formulation that was 
applicable to the needs of the military. Each dose of the vaccine 
contained 60 .mu.g each of the serotype 2b and serotype 15 outer membrane 
proteins, 45 .mu.g of each of the four capsular polysaccharides, about 4 
.mu.g of LPS and 1.5 .mu.g of nucleic acid as shown in Table C. The total 
dose was approximately 300 .mu.g. Further characteristics of this vaccine 
are given in Table 2. About 50% of the polysaccharide was bound to protein 
as estimated by the elution profile on Separose CL-4B compared to that of 
the purified polysaccharide mixture. 
The vaccine was used to inhibit each of four murine monoclonal antibodies 
that were specific for determinants either on the serotype protein or the 
class one protein of the vaccine strains. The 2B strain had the P1.2 
determinant on the class 1 protein and the serotype 15 strain had the 
p1.16 determinant on the class 1 protein. The results are given as the 
concentration required to inhibit 50% and were used as an identity test to 
demonstrate the presence of these specifid determinants in an 
antigenically active state. The vaccine passed the rabbit pyrogenicity 
test at a level of 0.12 .mu.g/kg. 
CLINICAL STUDIES 
Vaccine lot ACYW2b15-2 was tested in 10 laboratory volunteers to insure 
safety and full immunogenicity of the tetravalent polysaccharide mixture. 
It was then tested for safety and immunogenicity in 54 recruit volunteers 
at Ft. Benning, GA. After obtaining informed consent, a single injection 
was given subcutaneously in the upper arm by needle and syringe. An 
additional 47 volunteers were given the regular tetravalent polysaccharide 
vaccine by needle and syringe as a control. Blood samples and throat 
cultures were obtained at 0, 2, 4, 6, 9, and 15 weeks. Temperatures were 
taken at 6 hours, and at 24 hours the vaccination site was examined and 
the individuals questioned regarding adverse reactions. 
BACTERICIDAL ANTIBODY RESPONSE 
The results reported here are limited to an analysis of the bactericidal 
antibody responses to the outer membrane protein portion of the vaccine. 
Since nasopharyngeal carriage of meningococci frequently leads to an 
increase in bactericidal antibodies, the data reported here, with several 
exceptions, are limited to those individuals who were noncarriers during 
the first 6 weeks of the study. 
The bactericidal assays were performed in microtiter plates using 25% fresh 
human serum that lacked bactericidal activity against the test strain as 
an exogenous source of complement. The results were scored as a reduction 
in colony forming units after 60 minutes, and the titer was determined as 
the highest serum dilution that resulted in greater than a 50% reduction 
in colonies. 
The pre- and post-vaccination antibody titers against the serotype 2b 
vaccine strain 8047 is shown in FIG. 1. At two weeks 66% of the volunteers 
had a 4-fold or greater rise in titer, and greater than 90% had at least 
at 2-fold rise. Of 12 individuals who lacked bactericidal antibody prior 
to vaccination, all but one acquired detectable bactericidal antibody at 
two weeks. By 15 weeks titers had decreased, but 70% were still above 
prevaccination levels. 
The bactericidal antibody titers to the serotype 15 vaccine strain, 44/76, 
were similar and are shown in FIG. 2. All but three had a 4-fold or 
greater rise in titer against this strain, and all had at least a two-fold 
rise. Again, at 15 weeks a few titers had returned to pre-vaccination 
levels, but about 85% remained at least 2-fold higher. 
These same sera were also tested against two heterologous strains that 
shared neither the class 1 protein nor the serotype protein with either of 
the two vaccine strains. In FIG. 3 the geometric mean titers at 0, 2, and 
15 weeks against the vaccine strains are compared to the titers against 
the two heterologous strains M978 (B:8) and M136 (B:11). The geometric 
mean bactericidal titers of controls who received the standard tetravalent 
A, C, Y, and W-135 polysaccharide vaccine and who were non-carriers are 
also given for comparison. Little or no increase in antibody to the 
serotype 11 strain was observed, but a significant, through reduced, 
increase was observed against the serotype 8 strain. An increase in the 
geometric mean of about 8-fold was observed against the two vaccine 
strains. These results suggest that although much of the bactericidal 
antibody is type specific, some cross reactive bactericidal antibodies are 
also induced. 
To further investigate the specificity of the bactericidal antibodies, ten 
pairs of sera which showed a 4-fold or greater rise to one of the vaccine 
strains were tested against two heterologous strains, each of which shared 
ether the same serotype or the same class one protein type with the 
respective vaccine strain but not both. The geometric mean titer of these 
ten paired pre- and 4-week post-vaccination sera gainst the serotype 2b 
vaccine strain and two related strains are compared in FIG. 4. The 
serotype 2a strain, 99M, shares the class one protein with the vaccine 
strain and strain 80182 shares the serotype protein but has no detectable 
class 1 protein. The increase in titer against the two heterologous 
strains is less, but not much less, than against the homologous strain. 
Since both heterologous strains are killed about equally well, it appears 
that the bactericidal antibody is not predominantly directed against 
either the serotype protein or the class one protein. 
A similar experiment was performed with the serotype 15 vaccine strain 
related strains (FIG. 5). In this experiment the geometric mean titer 
against strain 8242 which shared the class 1 protein was a little greater 
than against strain P355 which shared the serotype protein. 
Since it was possible that the human serum that was used as a source of 
complement in the bactericidal assays might contain either blocking 
antibodies or natural antibodies that would augment the bactericidal 
activity of vaccine-induced antibodies, the bactericidal activity of fresh 
pre- and post-vaccination sera was determined without addition of 
exogenous complement. In these experiments, 50% fresh serum was combined 
with 25% bacteria and 25% buffer. The percentage decrease in colony 
forming units after 60 minutes at 37.degree. C. was determined and 
compared to the control containing heat inactivated serum in place of 
fresh serum. The sera were tested against two recent group B case isolates 
that had been passed less than 4 times. The first (FIG. 6) was a serotype 
2b strain which also shared the class 1 protein with the vaccine strain 
8047. At two weeks all the sera but one were able to kill over 90% of the 
organisms. At 15 weeks all but two were still able to kill greater than 
90%. 
The second test strain was nontypable, but had the same class 1 protein 
type (P1.16) as the serotype 15 vaccine strain (FIG. 7). This strain was 
not killed as well by the post-vaccination sera as the serotype 2b strain. 
The 2-week sera of most individuals, however, showed an increase in 
bactericidal activity. All but three were able to kill greater than 50% of 
the organisms. By 15 weeks some had returned to pre-vaccination levels, 
but in many cases the antibody persisted through this period. These 
results support the concept that the bactericidal antibodies elicited by 
this type of vaccine will be functional in vivo in human beings. 
SEROTYPING OF MENINGOCOCCI ISOLATED FROM MILITARY PERSONNEL 
We were interested to know the percentage of cases of group B meningococcal 
disease that occurred in military personnel during the past 5 years that 
might have been prevented by use of a vaccine such as ACYW2b15-2. To 
answer this question we determined the serotype and class 1 protein type 
all of available group B case strains isolated from army personnel over 
the past 5 years. Monoclonal antibodies specific for the class 1 proteins 
and the serotype proteins of the two vaccine strains were used. In 
addition, serotype 2a and 2c-specific monoclonal antibodies were used. A 
simple spot-blot procedure 15 was used for serotype determination. The 
results indicated that most strains (58%) were serotype 2b with the P1.2 
determinant on the class 1 protein. A single serotype 15 strain was 
present. Altogether, 65% of the isolates shared at least one major protein 
antigen with the vaccine strains. Of course this number would vary greatly 
with the population, time period and epidemiological setting. 
The geometric mean bactericidal titers elicited by this vaccine were lower 
than those elicited by a previously tested vaccine which was a complex of 
serotype 2a outer membrane protein and group B polysaccharide prepared by 
the deoxychloate method. Although there are several possible explanations 
for this result, one possibility is that the amount of class 5 protein in 
the present vaccine was quite low. The vaccine strains were chosen with 
this intent because, although the class 5 proteins are known to be 
immunogenic in man, they exhibit a high degree of variability in their 
occurrence and antigenic specificty. Some evidence for this interpretation 
was obtained by testing ten pairs of sera from the previous study against 
the vaccine strain (99M) before and after passage of the strain in a 
guinea pig subcutaneous chamber (Table 3). The geometric mean titer of 
this set of sera dropped from 1:328 against the unpassed strain to 1:20 in 
the guinea pig passed variant. Several differences were identified between 
the two variants. After passage in the guinea pig the colonies were more 
opaque and one of two class 5 proteins present in the unpassed variant had 
disappeared. Thus, much of the high-titered antibody may have been 
directed against the missing class 5 protein. 
TABLE 1 
__________________________________________________________________________ 
Comparison of Antibody Responses in Mice to Vaccines Prepared with 
Different Detergents and Having Different Compositions 
PB bound 
LPS Antibody response in mice* 
Vaccine Expt. 
mg/mg prot. 
mg/mg prot. 
Pre 5 d 
21 d 
28 d 
__________________________________________________________________________ 
BP2b-1 (deoxycholate) 
1 .35 .07 .29 .35 
.59 
8.25 
BP2b-2 (Empigen) 
1 .36 .03 .25 .32 
.81 
14.5 
BP2b-3 (Empigen) 
2 .6 .01 .32 3.5 
5.2 
127 
BP2b15-2 (Empigen) 
2 .53 .034 .37 2.6 
4.6 
70.3 
ACYW2b15-2 (Empigen) 
2 .7 .034 .33 5 9.7 
79.6 
__________________________________________________________________________ 
*Mice were vaccinated i.p. on day 0 and day 21 with 0.5 .mu.g protein or 
1.0 .mu.g protein for lots containing both serotypes. Values are geometri 
mean .mu.g antibody/ml vs serotype 2b OMC for groups of six Balb c/J mice 
as determined by SPRIA. 
TABLE 2 
______________________________________ 
Characterization of Meningococcal Vaccine Lot ACYW2b15-2 
Parameter Result 
______________________________________ 
Polysaccharide bound to protein (est.) 
51% 
Antigenic activity of protein epitopes 
MIC-50 (.mu.g/ml)* 
2b 0.45 
15 1.1 
P1.2 7.6 
P1.16 27 
Rabbit pyrogen test 0.12 .mu.g/kg passed 
Size of polysaccharide on Sepharose 
90% with Kd &gt; 0.5 
CL-4B.sup.+ 
______________________________________ 
*Amount of total protein required for 50% inhibition of monoclonal 
antibody binding in the solid phase radioimmunoassay. 
.sup.+ Based on sialic acid assay of column fractions. 
TABLE 3 
______________________________________ 
Effect of Passage in a Guinea Pig Subcutaneous Chamber on the 
Suseptibility of Strain 99 M to Human Complement-Mediated 
Bactericidal Activity by Paired Sera from 11 Recruits Vaccinated 
with Meningococcal Group B Vaccine 
Pre-Vaccination 
Post-Vaccination 
Positive/ 
G. M. G. M. 
Strain/Variant 
Total Titer Positive/Total 
Titer 
______________________________________ 
99 M/Unpassed 
6/11 1:4 11/11 1:328 
99 M/G. P. Passed 
1/11 1:1.1* 9/11 1:20 
______________________________________ 
*Seronegative = 1