Vaccines for gram-negative bacteria

An immunizing composition is disclosed and comprises a detoxified polysaccharide from a gram-negative bacterium covalently coupled to a detoxified protein from said gram-negative bacterium by means of a 4-12 carbon moiety. To prepare the above immunizing agent, the lipid A portion of lipopolysaccharide from a gram-negative bacterium is separated to give a detoxified polysaccharide. Reactive aldehyde groups are generated on the detoxified polysaccharide by selective oxidation. The detoxified polysaccharide is then covalently coupled to a detoxified protein from said gram-negative bacterium by means of a 4-12 carbon moiety having functionalities reactive to the aldehyde groups on the detoxified polysaccharide and to the carboxylic groups on the detoxified protein.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to and has among its objects novel non-toxic 
immunizing compositions against gram-negative bacteria and novel methods 
for their preparation. It is a particular object of the invention to 
prepare an immunizing composition effective against Pseudomonas aeruginosa 
infections. Further objects of the invention will be evident from the 
following description wherein parts and percentages are by weight unless 
otherwise specified. 
2. Description of the Prior Art 
Gram-negative bacteria have two cell envelope membranes separated by a 
single thin layer of peptidoglycan. The inner or cytoplasmic membrane 
contains all known active transport systems and many of the cell envelop 
enzymes. The outer membrane is distinguished by a unique component 
lipopolysaccharide (lipid A plus polysaccharide) and a unique set of 
proteins. The O antigen-specific polysaccharide endows the particular 
bacterium with its main serological specificity. There is also a core 
polysaccharide, common to gram-negative bacteria, which is linked to a 
lipid component (lipid A). These complexes of lipid A, polysaccharide, and 
protein are antigenic and also exert toxic reactions in humans, thus being 
considered as endotoxins. 
Vaccines containing lipopolysaccharides (LPS) from gram-negative bacteria 
have been used for immunization of humans against infection. The vaccines 
may comprise killed cells, cell lysate, or purified LPS; however, many are 
toxic. 
Although infection with Pseudomonas aeruginosa (P. aeruginosa) is not 
common among the general population, P. aeruginosa infection is 
encountered very frequently in certain susceptible groups of patients. 
Burn victims and immunosuppressed cancer patients have been identified as 
having an unusually high risk of acquiring severe, and sometimes fatal, P. 
aeruginosa infection. P. aeruginosa infections are usually acquired during 
a hospital stay, not at home. 
Antibiotics have been used to treat patients with P. aeruginosa infections. 
However, antibiotic treatment is very expensive, effectiveness is often 
uncertain, and organisms continue to develop resistance to antibiotics. 
Vaccines have been prepared for a number of pathogenic bacteria, including 
P. aeruginosa. For example, U.S. Pat. No. 4,157,389 discloses a three 
component mixed vaccine against infections caused by P. aeruginosa which 
comprises as the antigens an infection-protective common antigen, Original 
Endotoxin Protein obtained from P. aeruginosa, an elastase toxoid obtained 
from P. aeruginosa and a protease toxoid obtained from P. aeruginosa. 
Toxoids derived from protease and elastase of P. aeruginosa which are 
effective to prevent infections caused by P. aeruginosa are described in 
U.S. Pat. No. 4,160,023. 
U.S. Pat. No. 3,987,164 discloses vaccine preparations comprising the cell 
wall protein component of P. aeruginosa as an active ingredient in a 
prophylactic pharmaceutical preparation. 
Mink infection caused by P. aeruginosa can be prevented according to U.S. 
Pat. No. 4,096,245 by administering to mink a prophylactic preparation in 
the form of vaccine whose effective component mainly consists of protein 
and a small amount of lipid and sugar derived from P. aeruginosa. 
Original endotoxin protein derived from P. aeruginosa is disclosed in U.S. 
Pat. No. 4,079,126. In the patented method of preparation the original 
endotoxin protein is processed with either proteolytic enzyme or reductant 
or further processed with proteolytic enzyme after it has been treated 
with reductant. 
A bacterial endotoxin LPS of reduced toxicity covalently coupled to a 
protein antigen is described in U.S. Pat. No. 4,185,090. The coupling was 
effected by reaction with haloacylhalide. LPS acylated with an anhydride 
of a dibasic acid is detoxified; in combination with endotoxin 
polysaccharide covalently coupled to protein antigen it developed 
synergistic immunogenic effects. 
In U.S. Pat. No. 4,285,936 a method is taught for isolating a non-toxic, 
high molecular weight polysaccharide antigen from the crude slime of a P. 
aeruginosa culture, and a method for inducing immunity in a host to said 
live organisms is described. Initially, bacterial cells are separated from 
the slime, which is dissolved in a phosphate buffer solution. After 
removal of dissolved contaminating nucleic acids, a lipid A portion of the 
contaminating LPS constituent is removed and precipitated by acetic acid 
hydrolysis. The remaining lipids are extracted with chloroform. Nearly all 
of the residual nucleic acids are then removed by digestion with 
nucleases, and the remaining protein extracted with phenol. The aqueous 
and phenol layers are separated, and the aqueous layer applied to a gel 
filter to isolate the polysaccharide antigen by column chromatography. The 
polysaccharide antigen was non-toxic and highly effective in inducing an 
immune response to the organism in a host. 
SUMMARY OF THE INVENTION 
We have discovered an immunizing composition comprising a detoxified 
protein derived from a gram-negative bacterium covalently coupled by means 
of a 4-12 carbon moiety to a detoxified polysaccharide from said 
gram-negative bacterium. The novel immunizing agent of our invention is 
prepared by a method wherein the lipid A portion of a lipopolysaccharide 
derived from a gram-negative bacterium is first separated to give a lipid 
A-free, detoxified polysaccharide, which is selectively oxidized to 
produce aldehyde groups thereon. The selectively oxidized lipid A-free 
polysaccharide is covalently coupled through the aldehyde groups to a 
protein derived from said gram-negative bacterium by means of a 4-12 
carbon moiety containing functionalities reactive to the aldehyde group on 
the lipid A-free polysaccharide and carboxylic acid group on the 
detoxified protein. The compositions of the invention are useful as 
vaccines for parenteral administration for preventing bacterial infections 
and for administration to donors to raise the levels of antibody to a 
gram-negative bacterium of said donors. Blood collected from such donors 
may be pooled and fractionated to yield an immune serum globulin having a 
very high titer of said antibody. The high titer immune serum globulin may 
be administered to patients suffering from a particular gram-negative 
bacterial infection. 
It is a particular advantage of the compositions of the invention that they 
exhibit a high degree of immunogenicity free of toxicity or endotoxic 
activity. Indeed, the immunogenicity of the immunizing agents is nearly 
equivalent to that of native lipopolysaccharide. By the phrase free of 
toxicity or endotoxin activity is meant the composition causes no weight 
loss or failure to gain weight in mice and has less than 1/1000th the 
activity of lipopolysaccharide in the Limulus amebocyte lysate assay. 
It is important to note that the lipid A-free polysaccharide and the 
detoxified protein individually are not immunogenic. Furthermore, mixtures 
of the lipid A-free polysaccharide and the detoxified protein are also 
inactive. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As mentioned above the immunizing composition of the invention comprises a 
detoxified protein derived from a gram-negative bacterium covalently 
coupled by means of a 4-12 carbon moiety to a detoxified polysaccharide 
from said gram-negative bacterium. 
In the following description emphasis will be directed to Pseudomonas 
aeruginosa. This direction is by way of illustration only, not limitation. 
In its ambit the invention includes immunizing compositions against 
gram-negative bacteria such as Escherichia coli, Proteus sp, Serratia sp, 
Klebsiella sp et al. 
In a first step in the preparation of an immunizing composition for P. 
aeruginosa, the protein portion of the LPS-protein complexes of the P. 
aeruginosa bacteria are separated from the remaining portion of the 
complex. This may be accomplished by a variety of known chemical and 
physical methods. For example, one may use chemical methods such as 
guanadinium thiocyanate, zwitterionic detergent, lysozyme-ethylene diamene 
tetraacetic acid, sodium dodecyl sulfate, dimethyl formamide and the like. 
See, for example, Moldow et al, J. Membrane Biol., 10, 137-152 (1972), 
Hancock et al, J. of Bacteriology, 136, 381-390 (1978), Stinnett et al, 
ibid., 114, 399-407 (1973), and Robinson et al, FEMS Microbiol. Lett., 5, 
131-134 (1979). As examples of physical methods for extracting the protein 
from the protein-LPS complex, one may use such means as osmotic shock and 
sonication. Extraction of the protein may also be accomplished by a 
combination of the aforedescribed procedures. 
The extracted or isolated protein is then detoxified. To this end the 
protein is subjected to mild aqueous alkaline hydrolysis to destroy the 
toxic components such as enzymes and endotoxin. In general, the 
concentration of alkali and the temperature and duration of the treatment 
are sufficient to detoxify the protein, that is, to render the protein 
non-toxic when administered to humans. As the alkali one may use sodium 
hydroxide or potassium hydroxide, in water or solvent solution, and the 
like. Usually, 1 part of the protein is mixed with about 0.1-10.0 parts of 
0.1-3.0M alkali. The hydrolysis generally is conducted at a temperature of 
about 45.degree.-80.degree. C. for a period of about 0.5-5 hours. After 
heating water is removed from the mixture by conventional techniques such 
as ultrafiltration, lyophilization, and so forth. It is desirable to 
remove from the mixture material having a molecular weight of less than 
about 10,000. This may be accomplished by known methods such as dialysis, 
ultrafiltration, and the like. 
LPS may be isolated from P. aeruginosa bacteria by known techniques such as 
the phenol-water extraction method of Westphal et al, Z. Naturforach, 79, 
148-155 (1952), trichloroacetic acid (Staub In Meth. Carbohydrate Chem. 
Vol. 5, pp. 92-93, 1965), aqueous butanol (Leive et al, In Meth. Enzymol 
Vol. 28, pp. 254-262, 1972) and the like. In the procedure of Westphal et 
al, LPS is isolated by extraction of P. aeruginosa bacteria with a 
phenol-water mixture. The crude LPS is sonicated and digested with 
ribonuclease and deoxyribonuclease. After pronase digestion the LPS 
preparation may be subjected to diafiltration and ultrafiltration to 
remove low molecular species. 
The so-isolated LPS is next subjected to mild acid hydrolysis (Drewry et 
al, Biochem. J., 149, 93-106, 1975) to remove the lipid A moiety, i.e., to 
prepare lipid A-free, detoxified polysaccharide. For this purpose one may 
use acetic acid, hydrochloric acid and the like. Generally, the LPS is 
mixed with the acid in an aqueous medium in the proportion of about 1-4 
parts of acid per part of LPS. For instance, the LPS may be mixed with a 
0.5-3% aqueous solution of acid such that the concentration of LPS is 
about 1-5 mg per ml. The mixture is then heated at a temperature and for a 
time sufficient to remove the lipid A portion of the LPS, usually about 
1-24 hours at 60.degree.-100.degree. C. The precipitate that forms 
comprises the lipid A portion of the LPS and is separated from the lipid 
A-free polysaccharide by conventional techniques such as centrifugation, 
decantation, filtration, and so forth. To assure removal of all lipid A, 
the supernatant containing the lipid A-free polysaccharide is adjusted to 
about neutrality and extracted with a chlorohydrocarbon-alcohol mixture. 
The aqueous layer containing the lipid A-free polysaccharide is 
concentrated and the lipid A-free polysaccharide is purified by 
conventional techniques such as gel filtration, column chromatography and 
the like and then dried, e.g., by rotary evaporation or lyophilization. 
Next, the lipid A-free polysaccharide is selectively oxidized to generate 
aldehyde groups on the detoxified polysaccharide. This may be accomplished 
by known procedures such as, for example, periodate oxidation as described 
by Sanderson et al, Immunology, 20, 1061-1065, (1971). Accordingly, the 
lipid A-free polysaccharide is treated with a source of periodate ions, 
such as sodium periodate, potassium periodate, etc., in an amount and 
under conditions sufficient to selectively generate aldehyde groups on the 
detoxified polysaccharide. Generally, an aqueous solution containing 1-20 
mg/ml of detoxified polysaccharide is mixed with 1-100 mM periodate at 
ambient temperature in the dark for 10-24 hours. The reaction is stopped 
by addition of ethylene glycol and the selectively oxidized lipid A-free 
polysaccharide is purified by, for example, column chromatography or gel 
filtration and then treated to remove water by evaporation, 
lyophilization, or the like. 
The selectively oxidized lipid A-free polysaccharide is coupled to the 
detoxified protein by means of a 4-12 carbon moiety having functionalities 
reactive to the aldehyde groups of the polysaccharide and to carboxylic 
acid groups of the detoxified protein. Accordingly, the detoxified protein 
is coupled with a 4-12 carbon moiety containing at least two amino groups. 
Excess amino is generally employed. Thus, for example, about 3-20 parts of 
amine may be mixed with one part of detoxified protein in a buffered 
medium (pH 5.0-7.0) at a temperature of about 20.degree.-40.degree. C. for 
about 1-5 hours. Preferably, it is desirable to carry out the above 
coupling in the presence of an agent which will promote the coupling of 
the amino to carboxylic groups on the detoxified proteins. The generally 
preferred agent is a carbodiimide such as that described by Cuatrecasas, 
J. Biol. Chem., 245, 3059-3065 (1970). Usually, the carbodiimide is 
present in an amount of about 0.5-2 parts of amine amount. After the above 
carbodiimide-promoted amide link reaction, the mixture is treated by 
conventional means such as dialysis or diafiltration to remove unreacted 
amine compound and carbodiimide. Preferably, the reaction mixture above is 
dialyzed against a buffer system compatible with the reaction medium of 
the subsequent coupling of the derivatized detoxified protein to lipid 
A-free polysaccharide prepared as described above. Usually, the pH of the 
buffer system is about 7.0-9.0. 
The selectively oxidized lipid A-free polysaccharide is coupled via a 
Schiff's base reaction to the detoxified protein derivatized with a 4-12 
carbon moiety containing an amino group available for reaction with the 
aldehyde groups on the lipid A-free polysaccharide. In the above coupling 
it is desirable that the reaction be conducted in the presence of a 
reducing agent. For this purpose the preferred reducing agent is a 
cyanoborohydride such as that described by Borch et al, J. Am. Chem. Soc., 
93, 2897-2904 (1971). In general, about 1-5 parts of dry lipid A-free 
polysaccharide and 2-20 parts of cyanoborohydride are mixed with 0.5-3 
parts of derivatized detoxified protein in a buffer system of pH about 
7.0-9.0. The reaction mixture is then held at about 20.degree.-50.degree. 
C. for about 24-168 hours. The product comprising the detoxified protein 
covalently coupled to detoxified, lipid A-free polysaccharide by means of 
a 4-12 carbon moiety, the detoxified protein being coupled to the 4-12 
carbon moiety by means of an amide linkage and the lipid A-free 
polysaccharide being coupled to the 4-12 carbon moiety by means of amine 
linkages. This product may be purified by column chromatography, gel 
filtration, or the like. 
The immunizing compositions of this invention are free of detectable 
endotoxic activity and toxicity but are highly immunogenic. A particular 
composition may be administered to a host such as a human in an amount 
effective to induce an immune response to an organism. A particular 
composition may be administered to a host in an effective amount to 
prevent infections by gram-negative bacteria to which the composition is 
directed. The present compositions can be administered individually or in 
combination. Administration may be subcutaneous, intramuscular, or 
intracutaneous with or without adjuvant. 
The immunizing compositions can be manufactured in the usual method for 
preparing vaccines for humans and other animals. For example, the 
immunizing composition may be dissolved in a suitable solvent with or 
without adjuvant. As the solvent one may use distilled water, 
physiological saline and phosphate-buffered aqueous sodium chloride 
solution. Illustrative of adjuvants are aluminum hydroxide, aluminum 
phosphate, calcium phosphate, alum and Freund's incomplete adjuvant. The 
amount of adjuvant may be appropriately selected from the range of amounts 
being necessary and sufficient for increasing the immunoactivity. 
An immunizing dose of the present material in mice is 5 .mu.g of low 
molecular weight (5.times.10.sup.3 -4.times.10.sup.4) polysaccharide 
conjugated to 14-52 .mu.g of bovine serum albumin or bacterial protein. 
An immunizing composition may also be used to produce anti-serum against 
the gram-negative bacterium to which it is directed. Antibody can be 
collected from the anti-serum. The anti-serum and antibody can be used to 
prevent infections caused by the particular gram-negative bacterium. 
Furthermore, the blood collected from donors vaccinated with the present 
immunizing composition can be fractionated according to known techniques 
to yield a hyperimmune serum globulin having a high titer of antibody to a 
particular gram negative bacterium when compared to an immune serum 
globulin fractionated from blood obtained from a donor to whom the 
composition of the invention was not administered. Such immune serum 
globulin may be administered to a patient intramuscularly or it may be 
treated by known procedures to render it intravenously injectable. For 
example, an immunizing composition derived from P. aeruginosa may be 
administered to donors from whom blood is collected and fractionated to 
give a hyperimmune gamma globulin. The so-obtained immune serum globulin 
or gamma globulin may be administered intramuscularly to prevent infection 
by P. aeruginosa. Alternatively, the hyperimmune serum globulins may be 
rendered intravenously injectable by methods well known in the art. Such 
hyperimmune gamma globulins have a titer of antibody against P. aeruginosa 
of about 1:8,000-.gtoreq.1:64,000 as determined in the enzyme linked 
immunosorbent assay (ELISA). These hyperimmune serum globulins were 
heretofore unavailable. Thus, the invention also comprises pharmaceutical 
preparations containing an immune serum globulin with an ELISA titer of 
antibody to a gram-negative bacteria, for example, P. aeruginosa, of about 
1:8,000-.gtoreq.1:64,000. 
The term "pharmaceutical preparation" is intended in a broader sense herein 
to include preparations containing a composition in accordance with this 
invention used not only for therapeutic purposes, but also for reagent 
purposes as known in the art or for tissue culture. The pharmaceutical 
preparation intended for therapeutic use should contain a therapeutic 
amount of composition, i.e., that amount necessary for preventative or 
curative health measures. If the pharmaceutical preparation is to be 
employed as a reagent, then it should contain reagent amounts of 
composition. Similarly, when used in tissue culture or a culture medium 
the composition should contain an amount of the present composition 
sufficient to obtain the desired growth.

EXAMPLES 
The invention is demonstrated further by the following illustrative 
examples. 
EXAMPLE 1 
Isolation and Detoxification of Protein Derived from P. aeruginosa 
P. aeruginosa immunotype 3, American Type Culture Collection (ATCC) 27314 
was grown in glucose-glutamine-salts medium. Cells were killed with 0.5% 
formalin. Killed cells were suspended in 0.01M Tris [hydroxymethyl] amino 
methane hydrochloride (Tris-HCl) buffer pH 7.8 containing 5 mM ethylene 
diamine tetraacetic acid (EDTA) and 5 mM .beta.-mercaptoethanol and then 
sonicated. Disrupted cells and soluble materials were extracted with 6M 
guanidinium thiocyanate for 18 hours at room temperature. Guanidinium 
thiocyanate was removed by dialysis against 6M urea. Urea was removed by 
dialysis against saline. Saline soluble fraction contained the protein. 
The saline soluble protein from above was treated with 1M NaOH at 
56.degree. C. for 2 hours. Alkali was neutralized with acetic acid. This 
procedure removed fatty acids ester linked to lipid A, the toxic moiety of 
contaminating LPS. Detoxified protein was concentrated by ultrafiltration 
on a PM 10 filter (Amicon, Lexington, MA). Low molecular weight material 
&lt;10,000 was removed by this process. 
The detoxified protein was characterized as follows: 
1. No LPS endotoxic activity. 
2. Minimal toxicity in mice. 
3. Less than 1/1000th of activity of native protein in the Limulus 
amebocyte lysate assay. 
4. Reactive with antisera to both native and detoxified bacterial protein. 
5. By HPLC profile (TSK Gel 3000 SW) 80% of the detoxified protein is in 
the 10-30,000 MW range and 20%.gtoreq.100,000 MW. 
EXAMPLE 2 
Coupling of Diaminobutane to Detoxified Protein 
Detoxified bacterial protein (14 mg) in 5.0 ml of 0.05M phosphate buffered 
saline pH 7.2 was coupled with 1,4-diamino butane (300 mg) in the presence 
of 300 mg 1-(3-dimethyl-amino propyl)-3-ethyl carbodiimide. After 2 hours 
of gentle stirring at 21.degree. C., the reaction mixture was dialyzed 4 
times against 4 liters of 0.05M carbonate buffer pH 8.3 at 4.degree. C. to 
remove excess reagent. The aminobutyl derivatized detoxified protein was 
characterized by HPLC (TSK-Gel 4000 sw) as containing about 94% 85,000 MW 
protein. 
EXAMPLE 3 
Preparation of Lipid A-free Polysaccharide from P. aeruginosa 
LPS was isolated from P. aeruginosa cells prepared as in Example 1 by the 
phenol-water extraction procedure of Westphal et al, supra. The water 
layer containing crude LPS was dialyzed for 3-4 days against distilled 
water to remove phenol and low molecular weight bacterial substances. The 
crude LPS was further sonicated for 15 minutes to solubilize LPS micells 
and digested with ribonuclease and deoxyribonuclease in 0.1M acetate 
buffer pH 5.0 at 35.degree. C. for 18-24 hours to remove impurities of RNA 
and DNA. The contaminates of the bacterial protein, ribonuclease and 
deoxyribonuclease, in LPS were subjected to pronase digestion at pH 7.0 
for 24 hours. The LPS was purified by diafiltration and ultrafiltration 
through Amicon.RTM. hollow fiber cartridge (HIX 50) to remove low 
molecular weight nucleic acids, peptides, and amino acids. 
The purified LPS was treated in 1% acetic acid at a concentration of 2.5 
mg/ml and then heated at 87.degree. C. for 18 hours. The lipid A 
precipitate was removed by centrifugation. The acetic acid supernatant was 
adjusted to pH 7.0 with NaOH and extracted 3-5 times with two volumes of 
CHCl.sub.3 :methanol (2:1 vol./vol.) mixture to remove residual lipid A. 
The aqueous layer containing polysaccharide was concentrated by rotary 
evaporation under vacuum. The lipid A-free polysaccharide (PS) was further 
fractionated on a BioGel-A5m (2.6 cm.times.100 cm) column chromatography. 
The retarded fraction from BioGel-A5m chromatography was further purified 
on Sephadex.RTM. G-25 (2.6.times.100 cm) column. The major void volume 
fractions from Sephadex.RTM. G-25 were combined and concentrated by rotary 
evaporation or lyophilization. 
The polysaccharide was characterized as follows: 
1. Free of endotoxin (LPS) activity. 
2. Absence of toxicity in mice. 
3. Less than 1/1000th the activity of LPS in the Limulus amebocyte lysate 
assay. 
4. Reactive with antiserum to LPS in agar gel. 
5. Unable to induce antibody or resistance to infection in mice. 
6. MW of 5.times.10.sup.3 -4.times.10.sup.4. 
EXAMPLE 4 
Selective Oxidation of Polysaccharide 
Polysaccharide (2.5 mg/ml) from Example 3 was oxidized with NaIO.sub.4 (32 
mM) at room temperature in the dark for 19 hours. At the end of reaction, 
ethylene glycol was added to expend the excess NaIO.sub.4, and the 
solution was left at room temperature for an additional 3-4 hours. The 
selectively oxidized polysaccharide was further purified by gel filtration 
(Sephadex.RTM. G-25 column 1.0 cm.times.100 cm). The major 
carbohydrate-content fractions were combined and lyophilized. 
The selectively oxidized polysaccharide was characterized as follows: 
1. Increased number of aldehyde groups than native polysaccharide. 
2. Reactive with antiserum to LPS. 
3. No change in MW range. 
EXAMPLE 5 
Coupling of Selectively-oxidized Polysaccharide and Derivatized Detoxified 
Protein 
Lyophilized selectively oxidized PS (10 mg) from Example 4 and 25 mg of 
sodium cyanoborohydride were dissolved in 2.2 ml of 0.05M NaHCO.sub.3, pH 
8.3 containing 4.4 mg of derivatized detoxified protein, and the reaction 
was incubated at 37.degree. C. for 74 hours. The reaction mixture was 
further purified by gel filtration on Sephadex.RTM. G-100 column (100 
cm.times.1.0 cm). 
The so obtained product was characterized as follows: 
1. Purified polysaccharide linked covalently to non-toxic protein. 
2. Absence of toxicity in mice. 
3. Less than 1/1000th the activity of LPS in the Limulus amebocyte lysate 
assay. 
4. Reactive with antiserum to LPS and detoxified protein. 
5. Induces serum antibody and resistance to infection in mice. 
6. About 90% in 600,000 MW range. 
EXAMPLE 6 
Mouse Studies 
TABLE 1 
______________________________________ 
Immunogenicity of the components of P. aeruginosa 
immunotype 1 polysaccharide:protein conjugate vaccine 
Cumulative 
Mortality at 3 days.sup.c 
No. dead/total 
ELISA.sup.b 
Active Passive 
Immunizing Dose.sup.a 
titer Immu- Immu- 
Substance .mu.g/Mouse 
IgG nity nity.sup.d 
______________________________________ 
Free polysaccharide 
5.0 &lt;1:400 10/10 10/10 
Aminobutyl-bovine 
38.4 &lt;1:400 8/10 8/10 
serum albumin 
Polysaccharide: 
5.0: 1:2319 .sup. 3/10.sup.e 
.sup. 1/6.sup.e 
albumin 38.4 
conjugate 
Polysaccharide and 
5.0 &lt;1:400 10/10 8/10 
albumin mixture 
plus 
38.4 
Saline &lt;1:400 8/10 10/10 
______________________________________ 
.sup.a Mice immunized by subcutaneous injection on days 1, 7, 14 and 21. 
.sup.b The dilution of serum giving an A.degree. 405 nm of 0.10. 
.sup.c Mice challenged by the intraperitoneal route with 10 times the 50% 
lethal dose of P. aeruginosa immunotype 11369. 
.sup.d Serum obtained 3 days after 4th immunization. Mice passively 
immunized with 0.1 ml serum by the intraperitoneal route. 
.sup.e Statistically significant (P &lt; 0.05 Fisher's exact test) compare 
to saline controls. 
CONCLUSION: 
Resistance to infection and stimulation of passively protective antibody 
occurs only in mice immunized with the polysaccharide: protein conjugate. 
TABLE 2 
______________________________________ 
Induction of passively protective antibody with P. 
aeruginosa immunotype 1 conjugate vaccine 
Cumulative 
Day of.sup.a Mortality 
Immunization No. dead/total 
(5.0 .mu.g 
Bleeding day 
ELISA Passive immunity.sup.b 
polysaccharide/ 
after first 
titer Normal Burned 
dose) Immunization 
IgG Mice Mice 
______________________________________ 
Preimmune -1 &lt;1:400 9/10.sup. 
10/10.sup. 
1 6 &lt; 1:400 9/10.sup. 
10/10.sup. 
7 13 1:1313 6/10.sup. 
6/10.sup.d 
14 20 1:2481 0/10.sup.e 
3/10.sup.d 
21 29 1:3808 1/10.sup.e 
1/10.sup.e 
37 1:2482 4/10.sup.d 
3/10.sup.d 
______________________________________ 
.sup.a 17 mice immunized with vaccine and bled on days indicated. 
Immunized mice were challenged 72 days after 4th immunization with 10 
times the 50% lethal dose of P. aeruginosa 11369. Cumulative mortality wa 
1 of 17 and 8 of 9 in saline immunized controls (P &lt; .0005). 
.sup.b Normal and burned mice given 0.05 ml serum 3 hours before 
infection. Passively immunized normal mice were challenged with 10 
LD.sub.50. 
.sup.c Pentobarbital anesthetized mice given a 10% dorsal full thickness 
burn with a gas flame then challenged with 680 cells in 0.5 ml saline by 
subcutaneous injection in the burn site. 
.sup.d Statistically significant (P &lt; 0.05) protection compared to 
preimmune serum. 
.sup.e (P &lt; 0.001). 
CONCLUSION: 
1. Highly protective antibody is produced following 3 injections of 
conjugate vaccine. 
2. Active immunity persists for at least 21/2 months following 
immunization.