Method and kit for making a polysaccharide-protein conjugate

A method and kit for making a polysaccharide-protein Schiff base conjugate. A polysaccharide is oxidized with an oxidizing agent and combined with a protein in the presence of a macromolecular crowding agent to form a Schiff base. Preferably, the macromolecular crowding agent is a soluble linear polymer selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, dextran, nonylphenol-ethoxylates, polyvinyl alcohol, and mixtures thereof. Most preferably, the macromolecular crowding agent is a mixture of polyvinylpyrrolidone and polyethylene glycol. The microparticles or substantially dissolved microparticles are immunogenic and are useful for inducing an immune response when administered to humans or animals.

FIELD OF THE INVENTION 
This relates to the field of biochemistry and more particularly relates to 
polysaccharide-protein conjugates. 
BACKGROUND OF THE INVENTION 
Infants are routinely given a series of vaccinations within the first few 
months of life to confer protection against potentially life-threatening 
bacterial and viral diseases. Some antigens, especially the carbohydrate 
antigens of bacteria, fail to induce a protective immune response in 
infants. These antigens must be administered with a protein carrier to 
stimulate an immune response. For example, an antigen such as the capsular 
polysaccharide of Haemophilus influenzae type b (Hib), the bacteria 
primarily responsible for bacterial meningitis, must be conjugated to a 
carrier protein for successful induction of an antibody response in 
infants. 
A variety of chemical linkages have been used to prepare 
polysaccharide-protein conjugates. However, the coupling methods employed 
are time-consuming and result in linkages, such as amido linkages, that 
cause excessive crosslinking or detrimental modifications to the antigen, 
which are undesirable in human vaccines. For example, polysaccharides may 
be coupled to proteins using reductive amination as described in U.S. Pat. 
No. 4,356,170 to Jennings et al., entitled "Immunogenic 
Polysaccharide-Protein Conjugates". Conjugation is achieved by oxidizing 
the polysaccharide with an oxidizing agent, coupling the oxidized 
polysaccharide to a protein, and reducing the bond with a reducing agent 
for stability. This process requires lengthy dialysis and incubation steps 
and chromatography purification and may take up to two to three weeks for 
completion. 
What is needed is a rapid, inexpensive polysaccharide-protein conjugation 
method that results in a stable conjugate capable of conferring an immune 
response that renders protection against microbial infections, especially 
in human infants. 
SUMMARY OF THE INVENTION 
A method for preparing an immunogenic polysaccharide-protein conjugate is 
provided. In accordance with the method, a polysaccharide is first 
oxidized with an oxidizing agent. The oxidizing agent is preferably a 
glycol cleaving agent such as tetra-acetate. periodic acid, or sodium 
periodate. A protein is then added to the oxidized polysaccharide, and the 
polysaccharide and protein are coupled in the presence of a macromolecular 
crowding agent to form a Schiff base. Preferably, the macromolecular 
crowding agent is a soluble linear polymer selected from the group 
consisting of polyvinylpyrrolidone, polyethylene glycol, dextran, 
nonylphenol-ethoxylates, polyvinyl alcohol, and mixtures thereof. Most 
preferably, the macromolecular crowding agent is a mixture of 
polyvinylpyrrolidone and polyethylene glycol. The oxidized polysaccharide, 
protein, and macromolecular crowding agent are incubated together for a 
sufficient amount of time at a predetermined temperature until the 
formation of microparticles. 
The microparticles are composed of polysaccharide-protein Schiff base 
conjugates. These microparticles are immunogenic and may be administered 
to humans and other animals by methods well known to those skilled in the 
art to induce an immune response. Alternatively, the microparticles are 
removed from the solution and substantially dissolved with an alkaline 
solution, yielding a composition containing both a soluble immunogenic 
polysaccharide-protein Schiff base conjugate and a small quantity of 
inmunogenic, slow-releasing microparticles. 
The present invention also includes kits for preparing the 
polysaccharide-protein Schiff base conjugates. The kit can be in any 
configuration well known to those of ordinary skill in the art. 
Preferably, the kit contains an oxidizing agent for oxidation of the 
polysaccharide, a protein carrier, and a macromolecular crowding agent. 
The oxidizing agent is preferably a glycol cleaving agent such as 
tetra-acetate, periodic acid, or sodium periodate. The macromolecular 
crowding agent is preferably a soluble linear polymer selected from the 
group consisting of polyvinylpyrrolidone, polyethylene glycol, dextran, 
nonylphenol-ethoxylates, polyvinyl alcohol, and mixtures thereof. Most 
preferably, the macromolecular crowding agent is a mixture of 
polyvinylpyrrolidone and polyethylene glycol. 
Useful polysaccharides include, but are not limited to, those derived from 
Haemophilus influenza, pneumococci, meningococci, .beta.-hemolytic 
streptococci, Escherichia coli, Pseudomonas aeruginosa, Klebsiella, and 
Vibrio cholerae. Preferred proteins include, but are not limited to, 
tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane 
protein, nontoxic cross-reacting mutant of diphtheria toxin, a protein 
derived from bacteria, or a synthetic protein containing lysine residues. 
Accordingly, it is an object of the present invention to provide a rapid, 
inexpensive method for the preparation of polysaccharide-protein 
conjugates. 
It is a further object of the present invention to provide a method for the 
preparation of stable, immunogenic polysaccharide-protein conjugates. 
DETAILED DESCRIPTION OF THE INVENTION 
The term "Schiff base" is defined herein as any of a class of derivatives 
of the condensation of aldehydes with primary amines. 
A method for making a polysaccharide-protein conjugate by combining an 
oxidized polysaccharide with a protein in the presence of a macromolecular 
crowding agent is described herein. Preferably, the conjugate is 
immunogenic and is useful as a vaccine. Alternatively, the conjugate is 
useful for in vitro research and diagnostics wherein the protein portion 
of the polysaccharide-protein conjugate is labeled with a detectable 
label, such as fluorescein or a radiolabel, in accordance with methods 
well known to those skilled in the art, and the labeled conjugate is added 
to a biological sample or cell culture for the analysis of polysaccharide 
structure and function. Most preferably the conjugate is useful as a 
pediatric vaccine for the immunization of human infants. Alternatively, 
the conjugate may be used as a veterinary vaccine for the immunization of 
young animals. 
The polysaccharide of the protein-polysaccharide conjugate is one that 
includes an oxidizable terminal aldehyde group capable of reacting with an 
amino group of a protein to form a Schiff base. The polysaccharide is 
preferably a bacterial antigen capable of inducing an immune response when 
coupled to a protein carrier. Useful polysaccharides include, but are not 
limited to, those derived from Haemophilus influenza, pneumococci, 
meningococci, .beta.-hemolytic streptococci, Escherichia coli, Pseudomonas 
aeruginosa, Klebsiella, and Vibrio cholerae. Most preferably, the 
polysaccharide is one that is incapable of inducing an effective immune 
response in infants when administered alone, but produces a T 
lymphocyte-dependent immune response when coupled to a protein carrier and 
administered to infants. The protein may be any physiologically tolerated 
protein having a free amino group. Preferred proteins include, but are not 
limited to, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis 
outer membrane protein, nontoxic cross-reacting mutant of diphtheria 
toxin, a protein derived from bacteria, or a synthetic protein containing 
lysine residues. The protein may be derived from the same source as the 
polysaccharide. 
In accordance with the method, the polysaccharide is oxidized by incubating 
the polysaccharide with an oxidizing reagent. Preferably, the incubation 
is performed at room temperature for a sufficient amount of time to cause 
oxidation, most preferably between 15 and 45 minutes. The oxidizing agent 
may be any glycol cleaving agent capable of introducing an aldehyde. 
Preferably, the glycol cleaving agent is an oxidizing agent such as lead 
tetra-acetate, periodic acid, or sodium periodate. Most preferably, the 
glycol cleaving agent is sodium periodate. The glycol cleaving agent is 
then quenched by the addition of a quenching reagent. Preferably, the 
quenching reagent is an alcohol containing a gem-hydroxyl group such as 
ethylene glycol. 
The protein is added to the oxidized polysaccharide reaction mixture and 
incubated in the presence of a macromolecular crowding agent. The 
macromolecular crowding agent may be added to the polysaccharide either 
before or after the addition of the protein. 
A macromolecular crowding agent is defined herein as a compound that 
attracts water and allows molecules to aggregate. Preferably, the 
macromolecular crowding agent is a soluble, linear polymer such as 
polyvinylpyrrolidone or polyethylene glycol or a polymer mixture thereof. 
Such a polymer mixture may be prepared in accordance with the methods set 
forth in co-pending U.S. patent application Ser. No. 07/817,610 filed Jan. 
7, 1992 by James E. Woiszwillo, U.S. Pat. No. 5,525,519, or PCT patent 
application No. 93-00073, WOg3/14110, filed Jan. 7, 1993 by James E. 
Woiszwillo, both of which are incorporated herein by reference. It will be 
understood by those skilled in the art that other soluble, linear 
polymers, such as dextran, nonylphenol-ethoxylates, polyvinyl alcohol, and 
mixtures thereof could be used in addition to PVP and PEG or in place of 
either PVP or PEG. 
PVP is a non-ionogenic, hydrophilic polymer having a mean molecular weight 
ranging from approximately 10,000 to 700,000 and the chemical formula 
(C.sub.6 H.sub.9 NO).sub.n. PVP is also known as 
poly[1-(2-oxo-1-pyrrolidinyl)ethylene], Povidone.TM., Polyvidone.TM., RP 
143.TM., Kollidon.TM., Peregal ST.TM., Periston.TM., Plasdone.TM., 
Plasmosan.TM., Protagent.TM., Subtosan, and Vinisil.TM.. PVP is non-toxic, 
highly hygroscopic and readily dissolves in water or organic solvents. 
Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is a 
condensation polymer of ethylene oxide and water having the general 
chemical formula HO(CH.sub.2 CH.sub.2).sub.n H. 
Dextran is a term applied to polysaccharides produced by bacteria growing 
on a sucrose substrate. Native dextrans produced by bacteria such as 
Leuconostoc mesenteroides and Lactobacteria dextranicum usually have a 
high molecular weight. 
Nonylphenol-ethoxylates (NPEs) are a class of long chained compounds often 
used as surfactants. They are usually derivatized to meet the desired 
solubility requirements. 
Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates by 
replacement of the acetate groups with hydroxyl groups and has the formula 
(CH.sub.2 CHOH).sub.n. Most polyvinyl alcohols are soluble in water. 
PEG, dextran. PVA and PVP are commercially available from chemical 
suppliers such as the Sigma Chemical Company (St. Louis, Mo.). NPEs 
require custom synthesis and can be ordered from special chemical 
producers. 
Most preferably, the macromolecular crowding agent is polymer mixture 
containing an aqueous solution of PVP having a molecular weight between 
10,000 and 360,000, most preferably 40,000, and PEG having a molecular 
weight between 200 and 35,000, most preferably 3500. A polymer mixture of 
PVP having a molecular weight of 40,000 and PEG having a molecular weight 
of 3500 is the preferred macromolecular crowding agent. Preferably, the 
PVP is dissolved in an acetate buffer and PEG is added to the aqueous PVP 
solution. The concentration of each polymer is preferably between 1 and 40 
g/100 ml depending of the molecular weight of each polymer. Most 
preferably, the concentration of each polymer is 25 g/100 ml or 25%. Equal 
concentrations of PVP and PEG generally provide the most favorable polymer 
mixture for the formation of a polysaccharide-protein conjugate. The 
volume of polymer added to the polysaccharide varies depending on the 
sizes and quantities of the polysaccharide and protein. Preferably, 
approximately three volumes of the polymer mixture are added to one volume 
of a solution containing the polysaccharide and protein. The pH of the 
macromolecular crowding agent is preferably between 4 and 9, most 
preferably pH 5. 
The macromolecular crowding agent may be incubated with the oxidized 
polysaccharide and protein at a temperature between room temperature and 
58.degree. C. or at a series of different incubation temperatures within 
this range for a sufficient amount of time to allow formation of 
polysaccharide-protein microparticles. The preferred length of incubation 
time is between 30 minutes and 2 hours. 
The microparticles may be separated from the other reagents in the 
incubation mixture by conventional methods well known to those skilled in 
the art such as centrifugation, filtration or decantation in combination 
with established washing procedures. The resulting Schiff base 
microparticles may then be resuspended in a physiologically-acceptable 
buffer such as a saline solution or phosphate buffered saline. The 
polysaccharide-protein conjugate may be administered as microparticles. 
Alternatively, the microparticles may be substantially dissolved with a 
base or in a solubilizing solvent, such as an alkaline solution, to 
produce a soluble Schiff base conjugate. The conjugate may then be diluted 
with a physiologically acceptable buffer. The preferred base is sodium 
hydroxide. The solubilized conjugate microparticles are believed to be 
more immunogenic than the conjugate microparticles that have not been 
substantially dissolved and may also be administered to induce an immune 
response. The solubilized conjugate preferably contains a small amount of 
microparticles. Thus, the solubilized conjugate preferably contains a 
sufficient amount of soluble antigen to prime an immune response in a 
human or animal and contains a sufficient amount: of the slow-releasing 
antigenic microparticles, which boost the immune response. 
The polysaccharide-protein conjugate can be formulated and packaged, alone 
or in combination with other antigens, using methods and materials known 
to those skilled in he art for vaccines. The polysaccharide-protein 
conjugate is preferably added to the composite vaccine normally 
administered to infants. The polysaccharide-protein conjugate may be 
administered with an adjuvant in an amount effective to enhance the 
immunogenic response against the conjugate. At this time, the only 
adjuvant widely used in humans has been alum (aluminum phosphate or 
aluminum hydroxide). Saponin and its purified component Quil A, Freund's 
complete adjuvant and other adjuvants used in research and veterinary 
applications have toxicities which limit their potential use in human 
vaccines. However, chemically defined preparations such as muramyl 
dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those 
described by Goodman-Snitkoff et al. J. Immunol. 147:410-415 (1991) and 
incorporated by reference herein, encapsulation of the conjugate within a 
proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 
(1992) and incorporated by reference herein, and encapsulation of the 
protein in lipid vesicles such as Novasome.TM. lipid vesicles (Micro 
Vescular Systems, Inc., Nashua, N.H.) may also be useful. 
Methods of administration and dose 
In the preferred embodiment, the vaccine is packaged in a single dosage for 
immunization by parenteral (i.e., intramuscular, intradermal or 
subcutaneous) administration or nasopharyngeal (i.e., intranasal) 
administration. The conjugate is most preferably injected intramuscularly 
into the deltoid muscle. The conjugate is preferably combined with a 
pharmaceutically acceptable carrier to facilitate administration. The 
carrier is usually water or a buffered saline, with or without a 
preservative. The antigen may be lyophilized for resuspension at the time 
of administration or in solution. 
The carrier may also be a polymeric delayed release system. Synthetic 
polymers are particularly useful in the formulation of a vaccine to effect 
the controlled release of antigens. For example, the polymerization of 
methyl methacrylate into spheres having diameters less than one micron has 
been reported by Kreuter, J., Microcapsules and Nanoparticles in Medicine 
and Pharmacology, M. Donbrow (Ed). CRC Press, p. 125-148. 
Microencapsulation of the polysaccharide-protein will also give a 
controlled release. A number of factors contribute to the selection of a 
particular polymer for microencapsulation. The reproducibility of polymer 
synthesis and the microencapsulation process, the cost of the 
microencapsulation materials and process, the toxicological profile, the 
requirements for variable release kinetics and the physicochemical 
compatibility of the polymer and the antigens are all factors that must be 
considered. Examples of useful polymers are polycarbonates, polyesters, 
polyurethanes, polyorthoesters polyamides, poly (d,1-lactide-co-glycolide) 
(PLGA) and other biodegradable polymers. The use of PLGA for the 
controlled release of antigen is reviewed by Eldridge, J. H., et al. 
Current Topics in Microbiology and Immunology, 146:59-66 (1989). 
The preferred dose for the human infant is a 1 ml injection containing 
between 5 and 25 .mu.g of the polysaccharide-protein conjugate. Based on 
this range, equivalent dosages for heavier body weights can be determined. 
The polysaccharide-protein conjugate may additionally contain stabilizers 
such as thimerosal (ethyl(2-mercaptobenzoato-S)mercury sodium salt) (Sigma 
Chemical Company, St. Louis, Mo.) or physiologically acceptable 
preservatives. 
The present invention will be further understood by reference to the 
following non-limiting example.

EXAMPLE 1 
Preparation of Schiff Base Polysaccharide-protein Conjugate 
An aliquot containing 0.125 ml of purified Haemophilus influenzae type b 
polysaccharide (24.5 mg/ml) was mixed with 0.125 ml of sodium periodate (8 
mM in deionized water) (Sigma Chemical Company, St. Louis, Mo.) and 
incubated at room temperature for 30 minutes. 7.5 .mu.l of ethylene glycol 
(Sigma Chemical Company, St. Louis, Mo.) was added and incubated at room 
temperature for 30 minutes. To the mixture was added 0.460 ml of purified 
tetanus toxoid (6.9 mg/ml) and 2.13 ml of a polymer mixture containing 25% 
polyvinylpyrrolidone (PVP, molecular weight 40,000), 25% polyethylene 
glycol (PEG, molecular weight 3,500), and sodium phosphate buffer, pH 5.0, 
while vortexing. The reaction mixture was incubated either a) at room 
temperature for one hour, b) at room temperature for 30 minutes and at 
37.degree. C. for 30 minutes, or c) at room temperature for 30 minutes, at 
37.degree. C. for 30 minutes and at 58.degree. C. for 30 minutes. Schiff 
base microparticles formed. The mixture was centrifuged at 13,200 rpm for 
20 minutes to pellet the microparticles. The supernatant was decanted and 
the microparticles washed twice with 1.0 ml of 25% ethanol and 1.0 ml of 
deionized water. The resulting Haemophilus influenzae type b-tetanus 
toxoid (Hib-TT) conjugate microparticles were injected into mice as 
described below. Alternatively, microparticles were suspended in 1.0 ml of 
deionized water, substantially dissolved in 0.1N NaOH, and diluted in 
phosphate buffer to produce a soluble Schiff base Haemophilus influenzae 
type b-tetanus toxoid (Hib-TT) conjugate and injected as follows. 
A 0.2 ml aliquot of a 12.5 .mu.g/ml saline solution of the Hib-TT conjugate 
(based on polysaccharide content) microparticles or solubilized Hib-TT 
conjugate microparticles was injected subcutaneously into CD-1 female mice 
(13-15 g) (8 groups of 10 mice). A boost was injected on day 28. Blood 
samples were taken for immunogenicity analysis on days 28, 42, and 70. The 
results are shown in Table 1. Group I was injected with the Hib-TT 
conjugate microparticles, whereas Group II was injected with the 
solubilized Hib-TT conjugate. The controls contained two different doses 
of a Hib-TT conjugate prepared, by reductive amination. The Hib+TT sample 
was not conjugated. The results demonstrate that the Hib-TT Schiff base 
conjugates are highly immungenic. 
TABLE 1 
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Immunogenicity of Haemophilus Influenza Type b 
Polysaccharide (Hib)-Tetanus Toxoid (TT) Conjugates in mice 
after 1.degree. and 2.degree. doses. 
Geometric Mean Serum 
IgG Titers (.mu.g/ml) 
Dose (.mu.g) 4 wk post 1.degree. 
2 wk post 2.degree. 
Group Hib TT Hib TT Hib TT 
______________________________________ 
I 2.5 30 0.4 28 2.5 1966 
II 2.5 30 0.5 96 116.9 9199 
control 2.5 6.4 0.5 30 2.1 738 
control 11.8 30 0.9 147 2.2 225 
Hib + TT 
2.5 30 0.1 44 0.2 841 
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Modifications and variations of the method and kit for making a 
polysaccharide-protein Schiff base conjugate, will be obvious to those 
skilled in the art from the foregoing detailed description. Such 
modifications and variations are intended to come within the scope of the 
appended claims.