CA2047030A1 - Class ii protein of the outer membrane of neisseria meningitidis having immunologic carrier and enhancement properties - Google Patents

Abstract

TITLE OF THE INVENTION
THE CLASS II PROTEIN OF THE OUTER MEMBRANE OF
NEISSERIA MENINGITIDIS HAVING IMMUNOLOGIC CARRIER AND
ENHANCEMENT PROPERTIES

ABSTRACT OF THE INVENTION
The Class II major immuno-enhancing protein (MIP) of Neisseria meningitidis, purified directly from the outer membrane of Neisseria meningitidis, or obtained through recombinant cloning and expression of DNA encoding the MIP of Neisseria meningitidis, has immunologic carrier as well as immunologic enhancement and mitogenic properties.

Description

20~3~

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lO TITLE OF THE INVENTION
THE CLASS II PROTEIN OF THE OUTER MEMBRANE OF
NEISSERIA MENINGITIDIS HAVING IMMUNOLOGIC CARRIER AND
ENHANCEMENT PROPERTIES

BACKGROUND OF THE INVENTION
The outer membrane protein complex (OMPC) of Neisæeria meningitidis is used as an immunologic carrier in vaccines for human use. OMPC consists of 20 liposomes containing a variety of proteins as well as membranous lipids, including lipopolysaccharide (LPS
or endotoxin).
OMPC has the property of immune enhancement, and when an antigen is chemically coupled to it, an : 2s increased antibody response to the antigen results.

2~7~3a OMPC is currently used in vaccines for human infants against infectious agents such as HaemoPhilus influenzae, and renders the infants capable of mounting an IgG and memory immune response to polyribosyl ribitol phosphate (PRP) of H. influenzae, when PRP is covalently linked to OMPC.
OMPC is a mixture of a variety of proteins and lipids, and it was not known which component or components of OMPC bestows the beneficial immune enhancing effect to the coupled antigens. However, some potentially negative aspects of using OMPC in lO human vaccines include LPS related reactions.
Furthermore, OMPC-antigen conjugates are quite heterogeneous in that the antigen may become conjugated to any of the protein moieties which make up OMPC, and the total protein load per dose of a 15 multivalent vaccine would be very high.

OBJECTS OF THE INVENTION
It is an object of the present invention to 20 provide substantially pure Class II protein, the major im~unoenhancing protein (MIP~ derived directly from the outer membrane of Neisseria meningtidis, free from other Neisseria meningitidis outer membrane components. It is another object of the present invention to provide substantially pure recombinant, MIP of the outer membrane of Neisseria meningitidis, produced in a recombinant host cell, completely free of all other 2 ~ 3 ~

25/JWW - 3 - 1~110 Neisseria meningitidis proteins. A further object of the present invention is to provide an efficient immunocarrier protein for the enhancement of an immune response to antigens, comprising either MIP
purified directly from the outer membrane of Neisseria meningitidis, or recombinant MIP of Neisseria meningitidis produced in a recombinant host cell. Another object of the present invention is to provide a protein which possesses immune mitogenic activity, comprising either MIP purified directly from the outer membrane Neisseria meningitidis, or 10 recombinant MIP of Neisseria menin~itidis produced in a recombinant host cell. An additional object of the present invention is to provide vaccine compositions containing either the recombinant MIP, or MIP
purified directly from the outer membrane of 15 Neisseria menin~itidis. These and other objects will be apparent from the following description.

SUMMARY OF THE INVENTION
The present invention relates to the Class II major immunoenhancing protein (MIP> of the outer membrane of Neisseria meningitidis, in substantially pure form, free from other contaminating _.
meningitidis outer membrane proteins and LPS. The 25 MIP of the present invention, whether purified directly from the outer membrane of Neisseria menigitidis cells, or derived from a recombinant host cell producing recombinant MIP of Neisseria - meningitidis, possesses immunologic carrier and 30 mitogenic activity. The MIP

2~7030 of the present invention, when coupled to an antigen, is capable of immune enhancement in that the antibody response to the coupled antigen is augmented or the antigen is transformed to a T-dependent antigen which ensures that immunoglobulins of the IgG class are produced. The antigens which may be coupled to the MIP of the present invention include viral proteins, bacterial proteins and polysacharides, synthetic peptides, other immunogenic antigens, and weak or non-immunogenic antigens.

BRIEF DESCRIPTION OF THE DRAWING
Figure 1 - Antibody responses of adoptive transfer recipients receiving spleen cells primed separately with PRP-DT and MIP, or OMPC, or IAA-OMPC, 15 were measured by ELISA in blood samples taken on the indicated days post-immunization with PRP-OMPC.
Figure 2 - Lymphocyte proliferation assay for mitogenic activity of MIP, in vitro. The increase in 3H-thymidine incorporation into cellular 20 DNA was measured following exposure of the cells to bovine serum albumin (BSA), PRP-OMPC, OMPC, or MIP.
Figure 3 - PRP-MIP conjugates were tested for immunogenicity in mice as well as infant rhesus monkeys. Antibody responses were measured by ELISA
and RIA.

2~i~703a D~TAILED DESCRIPTION OF THE INVENTION
It is known that certain substances which by themselves elicit an immune response which consists of only IgM class antibodies and no memory, can be transformed into fully immunogenic antigens which elicit IgM and IgG anitbodies as we~l as memory, by chemical coupling to an antigenic moiety which can elicit T lymphocyte help for the immunoglobulin production. This immunologic phenomenon is termed the "carrier effect~, while the weak or non-immunogenic moiety, and the strongly antigenic substance are termed "hapten~ and "carrier", respectively.
Injection of the hapten-carrier or ploysaccharide-carrier complex into an animal will result in the formation of antibodies by lS B-lymphocytes, some of which will be specific for, and bind to the hapten, and others which will be specific for, and bind to the carrier. An additional aspect of the carrier effect is that upon a subsequent exposure to the hapten-carrier complex, a vigorous antibody response to the hapten ensues.
This is termed a memory, or anamnestic response.
The carrier effect appears to involve functions mediated by certain T-lymphocytes, called "helper T-lymphocytes~. The carrier molecule stimulates the helper T-lymphocytes to assist, in some way, formation of anti-hapten IgG class antibody-producing B-lymphocytes and a memory response.

20~703~
~5/JWW - 6 - 18110 Helper T-lymphocytes are normally involved in the production by B-lymphocytes, of antibodies specific for a certain type of antigens, termed "T-dependent~ antigens, but not for other antigens termed "T-independent~' antigens. A carrier molecule can convert a T-independent, weak or non-immunogenic hapten into a T-dependent, strongly antigenic molecule. Furthermore, a memory response will follow a subsequent exposure to the hapten-carrier complex and will consist primarily of IgG, which is characteristic of T-dependent antigens and not lo T-independent antigens.
The utility of carrier molecules is not - limited to use with T-independent antigens but can also be used with T-dependent antigens. The antibody response to a T-dependent antigen may be enhanced by 15 coupling the antigen to a carrier, even if the antigen can, by itself, elicit an antibody response.
Certain other molecules have the ability to generally stimulate the overall immune system. These molecules are termed ~mitogens" and include plant 20 proteins as well as bacterial products. Mitogens cause T and/or ~-lymphocytes to proliferate, and can broadly enhance many aspects of the immune response including increased phagocytosis, increased resistance to infection, augmented tumor-immunity, and increased antibody production.
Many infectious disease causing agents can, by themselves, elicit protective antibodies which can bind to and kill, render harmless, or cause to be ~0 2~47338 killed or rendered harmless, the disease causing agent and its byproducts. Recuperation from these diseases usually results in long lasting immunity by virtue of protective antibodies generated against the highly antigenic components of the infectious agent.
Protective antibodies are part of the natural defense mechanism of humans and many other animals, and are found in the blood as well as in other tissues and bodily fluids. It is the primary function of most vaccines to elicit protective ~antibodies against infectious agents and/or their lo byproducts, without causing disease.
OMPC from N. meningitidis has been used successfully to induce antibody responses in humans when OMPC is chemically coupled to T-cell independent antigens, including bacterial polysaccharides. OMPC
15 contains several bacterial outer membrane proteins as well as bacterial lipids. In addition, OMPC has a liposomal three dimensional structure.
The efficacy of OMPC as an immunologic carrier was thought to depend on one or more of the 20 bacterial membrane proteins, bacterial lipids, the liposomal three dimensional structure, or a combination of bacterial proteins, lipids, and liposomal structure. Applicants have discovered that one of the proteins, MIP, possesses the immunologic carrier and immune enhancement properties of OMPC
vesicles, and is effective in purified form, free from other N. memingitidis membrane proteins and lipopolysaccharides.

703~

Applicants have also discovered that MIP, when chemically coupled to bacterial polysaccharide, functions as well as OMPC in inducing an antibody response to the polysaccharide. Applicants have further discovered that MIP is the Class II protein of the outer membrane of N. meningitidis. The Class II protein of N. meningitidis is a porin protein [Murakami, K., et al., (1989), Infection And Immunity, 57, pp.2318-23~. Porins are found in the outer membrane of all Gram negative bacteria.
While the present invention is exemplified lo by MIP of N. meningitidis, it is readily apparent to those skilled in thé art that any outer membrane protein from any Gram negative bacterium, which has immunologic carrier and immune enhancement activity, is encompassed by the present invention. Examples of 15 Gram negative bacteria include but are not limited to species of the genera Neisseria, Escherichia, Pseudomonas, Eemophilus, Salmonella, Shigella, Bordetella, Klebsiella, Serratia, Yersinia, Vibrio, and Enterobacter.
MIP may be employed to potentiate the antibody response to highly antigenic, weakly antigenic, and non-antigenic materials. The term "antigen~ and "antigenic material" which are used interchangeably herein include one or more 25 non-viable, immunogenic, weakly immunognic, non-immunogenic, or desensitizing ~antiallergic) agents of bacterial, viral, or other origin. The antigen component may consist of a dried powder, an aqueous phase such as an aqueous solution, or an 2~47033 aqueous suspension and the like, including mixtures of the same, containing a non-viable, immunogenic, weakly immunogenic, non-immunogenic, or desensitizing agent or agents.
The aqueous phase may conveniently be comprised of the antigenic material in a parenterally acceptable liquid. For example, the aqueous phase may be in the form of a vaccine in which the antigen is dissolved in a balanced salt solution, physiological saline solution, phosphate buffered saline solution, tissue culture fluids, or other lo media in which an organism may have been grown. The aqueous phase also may contain preservatives and/or substances conventionally incorporated in vaccine preparations. Adjuvant emulsions containing MIP
conjugated antigen may be prepared employing 15 techniques well known to the art.
The antigen may be in the form of purified or partially purified antigen including but not limited to antigens derived from bacteria, viruses, mammalian cells and other eukaryotic cells ~including parasites~, fungi, rickettsia; or the antigen may be an allergen including but not limited to pollens, dusts, danders, or e~tracts of the same; or the antigen may be in the form of a poison or a venom including but not limited to poisons or venoms derived from poisonous insects or reptiles. The antigen may also be in the form of a synthetic peptide, or a fragment of a larger polypeptide, or any subportion of a molecule or component derived from bacteria, mammalian cell, fungi, viruses, 2 ~ 3 ~

rickettsia, allergen, poison or venom. In all cases, the antigens will be in the form in which their toxic ~r virulent properties have been reduced or destroyed and which when introduced into a suitable host will either induce active immunity by the production therein of antibodies against the specific proteins, peptides, microorganisms, extract, or products of microorganisms used in the preparation of the antigen, poisons, venoms, or, in the case of allergens, they will aid in alleviating the symptoms o the allergy due to the specific allergen.
lo The antigens can be used either singly or in combination, for example, multiple bacterial antigens, multiple viral antigens, multiple mycoplasmal antigens, multiple rickettsial antigens, multiple bacterial or viral toxoids, multiple 15 allergens, multiple proteins, multiple peptides or combinations of any of the foregoing products can be conjugated to MIP.
Antigens of particular importance are derived from bacteria including but not limited to B.
pertussis, Leptospira pomona, and icterohaemorrhagiae, S. paratyphi A and B, C.
di~htheriae, C. tetani, C. botulinum, C. perfringens, C. feseri, and other gas gangrene bacteria, _.
anthracis, Y. pestis, P. multocida, V. cholerae, Nesseria meningitidis, N. gonorrheae, Hemo~hilus _fluenzae, Treponema Pallidum, and the like; from mammalian cells including but not limited to tumor cells, virus infected cells, genetically engineered cells, cells grown in culture, cell or tissue 20~7~3~
25/JWW - ll - 18110 extracts, and the like; from viruses including but not limited to human T lymphotropic virus (multiple types), human immunodeficiency virus (multiple variants and types), polio virus (multiple types), human papilloma virus (multiple types) adeno virus (multiple types), parainfluenza virus (multiple types), measles, mumps, respiratory syncytial virus, influenza virus (various types), shipping fever virus (SF4), Western and Eastern equine encephalomyelitis virus, Japanese B. encephalomyelitis, Russian Spring-Summer encephalomyelitis, hog cholera virus, lo Newcastle disease virus, fowl pox, rabies, feline and canine distemper and the li~e viruses, from rickettsiae including but not limited to epidemic and endemic typhus or other members of the spotted fever group, from various spider and snake venoms or any of 15 the known allergens, including but not limited to those from ragweed, house dust, pollen extracts, grass pollens, and the like.
The polysaccharides of this invention may be any bacterial polysaccharides with acid groups, but 20 are not intended to be limited to any particular types. Examples of such bacterial polysaccharides include Streptococcus pneumoniae (pneumococcal> types 6A, 6B, lOA, llA, 18C, 19A, 19f, 20, 22F, and 23F, polysaccharides; Group B Streptococcus types Ia, Ib, 2s II and III; Haemophilus influenzae serotype b polysaccharide; Neisseria meningitidis serogroups A, B, C, X, Y, W135 and 29E polysaccharides; and Escherichia coli Kl, K12, K13, K92 and K100 polysaccharides. Particularly preferred .
.

2 ~ 3 polysaccharides, however, are those capsular polysaccharides selected from the group consisting of ~[. influenzae serotype b polysaccharides, such as ctescribed in Rosenberg et al., J. Biol. Chem., 236, 2845-2849 (1961) and Zamenhof et al., J. Biol. Chem., 203, 695-704 ~1953). Streptococcus pneumoniae (pneumococcal) type 6B or type 6A polysaccharide, such as described in Robbins et al., Infection and Immunity, 26, No. 3 1116-1122 (Dec., 1979);
pnemococcal type 19F polysaccharide, such as described C. J. Lee et al., Reviews of Infectious lO Diseases, 3, No. 2, 323-331 (1981); and pneumococcal type 23F polysaccharide, such as described in O. Larm et al., Adv. Carbohyd Chem and Biochem., 33, 295-321, R. S. Tipson et al., ed., Academic Press 1976.
MIP can be purified from OMPC derived from 15 cultures of N. meningitidis grown in the usual manner as described in U.S. Patent number 4,459,286 and U.S.
Patent number 4,830,852. OMPC purification can be done according to the methods described in U.S.
Patent number 4,271,147, 4,459,286, and 4,830,852.
MIP can also be obtained from recombinant DNA engineered host cells by expression of recombinant DNA encoding MIP. The DNA encoding MIP
can be obtained from _. meningitidis cells [Murakami, K. et al., (1989), Infection And Immunity, 57, pp.
25 2318], or the DNA can be produced synthetically using standard DNA synthysis techniques. DNA encoding MIP
can be expressed in recombinant host cells including but not limited to bacteria, yeast, insect, mammalian or other animal cells, yielding recombinant MIP. The 2047a33 preferred methods of the present invention for obtaining MIP are purification of MIP from OMPC and recombinant ~NA expression of DNA encoding MIP
derived from _. meningitidis.
Purified MIP was prepared from OMPC vesicles by sodium dodecylsulfate (SDS) lysis of the vesicles followed by SDS polyacrylamide gel electrophoresis (PAGE). The MIP was eluted from the gel, dialysed against a high pH buffer and concentrated. Standard methods of polyacrylamide gel electrophoresis can be utilized to purify MIP from OMPC vesicles. Such lO methods are described in Molecular Cloning: A
Laboratory Manual, Sambrook, J. et al., (1989), Cold Spring Harbor Laboratory Press, New York, and Current Protocols In Molecular Biology, (1987) Ausubel F.M.
et al., editors, Wiley and Sons, New York.
Standard methods of eluting proteins from SDS-polyacrylamide gels are described in Hunkapiller, M.W., and Lujan, ~., (1986), Purification Of Microgram Quantities Of Proteins By Polyacrylamide Gel Electrophoresis, in Methods of Protein 20 Microcharacterization (J. Shively editor) Humanna Press, Clifton N.J., and Current Protocols In Molecular Biology (1987), Ausubel, F.M., et al., editors, Wiley and Sons, New York.
MIP prepared in this manner is readily 25 suitable for conjugation to antigens derived from bacteria, viruses, mammalian cells, rickettsia, allergens, poisons or venoms, fungi, peptides, proteins, polysaccharides, or any other antigen.
Recombinant MIP can be prepared by e~pression of genomic N. meningitidis DNA encoding 20~703a MIP in bacteria, for example E. coli or in yeast, for example S. cerevisiae. To obtain genomic DNA
encoding MIP, genomic DNA is extracted from N.
meningitidis and prepared for cloning by either random fragmentation of high molecular weight DNA
following the technique of Maniatis, T. et al., ~1978~, Cell, 15, pp. 687, or by cleavage with a restriction endonuclease by the method of Smithies, et al., (1978), Science, 202, pp. 1248. The genomic DNA is then incorporated into an appropriate cloning vector, for example lambda phage ~see Sambrook, J. et lO al., (1989), Molecular Cloning, A Laboratory Manual.
Cold Spring Harbor Press, New York]. Alternatively, the polymerase chain reaction (PCR) technique (Perkin Elmer) can be used to amplify specific DNA sequences in the genomic DNA [Roux, et al., (1989), 15 Biotechniques, 8, pp. 48]. PCR treatment requires a DNA oligonucleotide which can hybridize with specific DNA sequences in the genomic DNA. The DNA sequence of the DNA oligonucleotides which can hybridize to MIP DNA in the N. meningitidis genomic DNA can be determined from the amino acid sequence of MIP or by reference to the determined DNA sequence for the Class II major membrane protein of N. meningitidis [Musakami, k. et al., (1989), Infection and Immunity, 57, pp. 2318].
Recombinant MIP can be separated from other cellular proteins by use of an affinity column made with monoclonal or polyclonal antibodies specific for MIP. These affinity columns are made by adding the antibodies to Affigel-10 ~Biorad), a gel support 2~703~

which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with lM ethanolamine HCl (pH
8). The column is washed with water followed by 0.23 M glycine ~Cl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing MIP are slowly passed through the column.
The column is then washed with phosphate buffered saline until the optical density (A280) falls to background, then the protein is eluted with 0.23 M
glycine-HCl (pH 2.6). The protein is then dialyzed against phosphate buffered saline.
The conjugates of the present invention may be any stable polysaccharide-MIP conjugates or any other antigen-MIP conjugates, including synthetic peptide antigens. The synthetic peptides may possess one or more antigenic determinants of any antigen including antigenic determinants from bacteria, rickettsia, viruses (including human immunodeficiency viruses), mammalian cells or other eukaryotic cells including parasites, toxins or poisons, or 2S allergens. The antigen-MIP conjugates are coupled through bigeneric spacers containing a thioether group and primary amine, which form hydrolytically-labile covalent bonds with the polysaccharide and the MIP. Preferred conjugates according to this invention, however, are those which may be represented by the formulae, Ps-A-E-S-B-Pro or 204~io3n Ps-A~-S-E~-B~-Pro, wherein Ps represents a poly-saccharide or any other antigen; Pro represents the bacterial protein MIP; and A-E-S-B and A~-S-E~-B~
constitute bigeneric spacers which contain hydrolytically-stable covalent thioether bonds, and which form covalent bonds (such as hydro-lytically-labile ester or amide bonds) with the macromolecules, Pro and Ps. In the spacer, A-E-S-B, S is sulfur; E is the transformation product of a thiophilic group which has ~een reacted with a thiol group, and is represented by O

wherein R is H or CH3, and p is 1 to 3; A is W~
-CN~CH2)mY(CH2)n~NH ' wherein W is 0 or NH, m is 0 to 4, n is O to 3, and Y
is CH2,O,S,NR~, or CHCO2H, where R~ is H or Cl- or C2-alkyl, such that if Y is CH2, then both m and n cannot equal zero, and if Y is O or S, then m is greater than 1 and n is greater than 1; and B is :`

~, .

20~7~a ~(CH2)pClH(CH2)qD~~

wherein q is O to 27 Z is NH2, NHCR', COOH, or H, o where R~ and p are as defined above, and D is C, NR', H O
or N-C(CH2)2C. Then in the spacer, A'-S-E'-B', S
W
is sulfur; A~ is -CNH(CH2)aR"-, wherein a is 1 to HOY' 4, and R" is CH2, or NCCH(CH2)p, where Y~ is NH2 or NHCOR', and W, p and R~ are as defined above, and E' is the transformation product of a thiophilic group which has been reacted with a thiol group, and is R
represented by -CH-, wherein R is q as defined above, and B' is -C-, or E' is ~
N -, o B~ is -(CH2)pC-, wherein p is 1 to 3. Further, of the bigeneric spacers, A-E-S-B and A'-S-E'-B', the E-S-B and A'-S-E' components are determinable and quantifiable, with this identification reflecting the covalency of the conjugate bond linking the side of 2~47~3a the thioethersulfur which originates from the covalently-modified polysaccharide with the side of the spacer which originates from the functionalized protein.
Then the conjugates, Ps-A-E-S-~-Pro, accord-ing to this invention may contain spacers whose com-ponents include derivatives of, inter alia: carbondiGXide 7 1,4-butanediamine, and S-carboxymethyl-N-acetylhomocysteine; carbon dioxide, 1,5-pentanedia-mine, and S-carboxymethyl-N-acetylhomocysteine, carbon dioxide, 3-oxa-1,5-pentanediamine, and S-carbo2y-methyl-N-acetylhomocysteine; carbon dioxide, 1,4-butane-diamine, and S-carboxymethyl-N-acetyl-cysteine; carbon dioxide, 1,3-propanediamine, and S-carboxymethyl-N-benzoylhomocysteine; carbon dioxide, 3-aza-1,5-pentanediamine, and S-carboxy-methyl-N-acetylcysteine; and carbon dioxide, 1,2-ethanediamine, glycine, and S-(succin-2-yl)-N-acetylhomocysteine. The conjugates, Ps-A'-S-E'-B'-Pro, according to this invention, may contain spacers whose components include derivatives f. inter alia: carbon dioxide and S-carboxy-methylcysteamine; carbon dioxide and S-(a-carboxy-ethyl)cysteamine; carbon dioxide and S-carboxy-methylhomocysteamine; carbon dioxide, S-(succin-2-yl)cysteamine, and glycine; and carbon dioxide and S-carboxymethylcysteine.
In the process of the present invention, the polysaccharide is covalently-modified by (a) solubilizing it in a non-hydroxylic organic solvent, then (b) activating it with a bifunctional reagent, (c) reacting this activated polysaccharide with a 2~7~3~

bis-nucleophile, and finally, if necessary, further (d) Eunctionalizing this modified polysaccharide by either reaction, (i) with a reagent generating electrophilic (e.g., thiolphilic) sites or, (ii) with a reagent generating thiol groups. The protein is conversely either reacted (i) with a reagent generating thiol groups or (ii) with a reagent generating thiolphilic sites, then the covalently-modified polysaccharide and the functionalized protein are reacted together to form the stable covalently-bonded conjugate and the final mixture is purified to remove unreacted polysaccharides and proteins.
The process of this invention also includes selection of a nucleophile or bis-nucleophile which will react with the activated polysaccharide to form lS a covalently-modified polysaccharide with pendant electrophilic sites or pendant thiol groups, thereby ; obviating the need to further functionalize the bis-nucleophile-modified polysaccharide prior to reacting the covalently-modified polysaccharide with the covalently-modified protein. Also, the functionalization of the protein to either moiety form may be accomplished in more than one step . according to the selection of reactants in these steps.
In the first step toward covalently-modifying the polysaccharide, the solid poly-saccharide must be solubilized.
Since the nucleophilic alcoholic hydroxyl groups of a polysaccharide cannot compete chemically 2~7~3~

for electrophilic reagents with the hydroxyls of water in an aqueous solution, the polysaccharide should be dissolved in non-aqueous (non-hydroxylic) solvents. Suitable solvents include dimethyl-formamide, dimethylsulfoxide, dimethylacetamide, formamide, N,N'-dimethylimidazolidinone, and other similar polar, aprotic solvents, preferably dimethylformamide.
In addition to the use of these solvents, converting the polysaccharides (e.g., the capsular polysaccharides of H. influenzae type b, which are a lo ribose-ribitol phosphate polymers), which have acid hydrogens, such as phosphoric acid mono- and diesters, into an appropriate salt form, causes the polysaccharides to become readily soluble in the above solvents. The acidic hydrogens in these macro-15 molecules may be replaced by large hydrophobiccations, such as tri- or tetra-(Cl- to C5)alkyl-ammonium, l-azabicyclo[2.2.2]octane,1,8-diazabicyclo t5-4 0]undec-7-ene or similar cations, particularly tri- or tetra-(Cl- to C5)alkylammonium, and the resultant tri- or tetraalkylammonium or similar salts of phosphorylated polysaccharides readily dissolve in the above solvents at about 17-50C, while being stirred for from one minute to one hour.
Partially-hydrolyzed H. influenzae serotype ~ polysaccharide has been converted into the tetrabutyl-ammonium salt, then dissolved in dimethylsulfoxide (Egan et al., 1- Amer. Chem. Soc., 104, 2898 (1982)), but this product is no longer antigenic, and therefore useless for preparing 20~7~

vaccines. By contrast, Applicants accomplish the solubilization of an intact, unhydrolyzed polysaccharide by passing the polysaccharide through a strong acid cation exchange resin, in the tetraalkylammonium form, or by careful neutralization of the polysaccharide with tetraalkyl-ammonium hydroxide, preferably by the former procedure, and thereby preserve the viability of the polysaccharide for immunogenic vaccine use.
Subsequent steps are then directed to overcoming the other significant physico-chemical limitation to making covalent bonds to poly-saccharides, that being the lack of functional groups on the polysaccharides, other than hydroxyl groups, which are reactive enough with reagents commonly or practically used for functionalization of units with which bonding is desired. Activation of the polysaccharide to form an activated polysaccharide, reaction with bis-nucleophiles to form a nucleophile-functionalized polysaccharide, and functionalization with reagents generating either electrophilic sites or thiol groups, are all directed to covalently-modifying the polysaccharide and developing functional groups on the polysaccharide in preparation for conjugation.
In the next step, the solubilized polysaccharide is ac~ivated by reaction with a bifunctional reagent at about 0-50C, while stirring for ten minutes to one hour, with the crucial weight ratio of activating agent to polysaccharide in the range of 1:5 to 1:12. In the past, this activation 20~7~33 has been accomplished by reaction of the polysaccharide with cyanogen bromide. However, derivatives activated with cyanogen bromide, which has a ~proclivity~ for vicinal diols, have shown transient stability during dialysis against a phosphate buffer. Therefore, while activation with cyanogen bromide is still possible according to the present invention, this reagent is poorly utilized in activation of polysaccharides and is not preferred.
Instead, preferred bifunctional reagents for activating the polysaccharide include carbonic acid ~0 derivatives, R2-C~R3, wherein R2 and R3 may be independently, azolyl, such as imidazolyl;
halides; or phenyl esters, such as ~-nitrophenyl, or polyhalophenyl.
Carbonyldiimidazole, a particularly preferred reagent, will react with the hydroxyl groups to form imidazolylurethanes of the polysaccharide, and arylchloroformates, including, for example, nitrophenylchloroformate, will produce mixed carbonates of the polysaccharide. In each case, the resulting activated polysaccharide is very susceptible to nucleophilic reagents, such as amines, and is thereby transformed into the respective urethanes.
In the next stage, the activated polysaccharide is reacted with a nucleophilic reagent, such as an amine, particularly diamines, for H
example, HN(CH2)mY(CH2)n-N~, wherein m is 0 to 4, n 2 ~ 3 ~

25/JWW - 23 - 1~110 is O to 3, and Y is CH2, O, S, NR~, CHC02H, where R~
is H or a Cl- or C2-alkyl, such that if Y is CH2, then both m and n cannot equal zero, and if Y is O or S, then m is greater than 1, and n is greater than 1, in a gross excess of amine (i.e., for eæample, a 50-to 100-fold molar excess of amine vs. activating agent used). The reaction is kept in an ice bath for from 15 minutes to one hou~ then ~ept for 15 minutes to one hour at about 17 to 40C.
An activated polysaccharide, when reacted with a diamine, e.g., 1,4-butanediamine, would result in a urethane-form polysaccharide with pendant amines, which may then be further functionalized by acylating. Mixed carbonates will also readily react with diamines to result in pendant amine groups.
Alternatively, the activated polysaccharide may be reacted with a nucleophile, such as a monohaloacetamide of a diaminoalkane, for example, 4-bromoacetamidobutylamine ~see, W. B. Lawson et al., Hoppe Seyler~s Z. Physiol Chem., 349, 251 (1968)), to generate a covalently-modified polysaccharide with pendant electrophilic sites. Or, the activated polysaccharide may be reacted with an aminothiol, such as cysteamine (aminoethanethiol~ or cysteine, examples of derivatives of which are well-known in the art of peptide synthesis, to produce a polysaccharide with pendant thiol groups. In both cases, no additional functionalization is necessary prior to coupling the covalently-modified polysaccharide to the modified bacterial ~carrier~' protein.

20~7~3~

The last step in preparing the polysaccharide, the further functionalization, if necessary, of the polysaccharide, may take the form of either reacting the nucleophile-functional-ized polysaccharide with a reagent to generate electrophilic (i.e., thiophilic) sites, or with a reagent to generate thiol groups.
Reagents suitable for use in generating electophilic sites, include for example, those for acylating to a-haloacetyl or ~-halopropionyl, 1l 1 derivative such as XCCHX (wherein R is H or CH3; X is Cl, Br or I; and X~ is nitrophenoxy, dinitrophenoxy, pentachlorophenoxy, pentafluorophenoxy, halide, 0-(N-hydroxysuccinimidyl) or azido), particularly chloroacetic acid or a-bromopropionic acid, with the reaction being run at a pH of 8 to 11 (maintained in this range by the addition of base, if necessary) and at a temperature of about 0 to 35C, for ten minutes to one hour. An amino-derivatized polysaccharide may be acylated with activated maleimido amino acids (see, 0. Keller et al, Eelv. Chim. Acta., 58, 531 (1975)) to produce maleimido groups, O
- c(CH2) wherein p is l to 3; with a 2-haloacetyling agent, 2~47~

.

such as p-nitrophenylbromoacetate; or with an a-haloketone carbo~ylic acid derivative, e.g., HO2C~CH2Br (Ber., 67, 1204, (1934)) in order to produce appropriately functionalized polysaccharides susceptible to thio substitution.
Reagents suitable for use in generating thiol groups include, for exam-ple, acylating reagents, such as thiolactones, e.g., R4CNH ~ IH2)p, S

wherein R4 is Cl- to C4-alkyl or mono- or bicyclic aryl, such as C6Hs or ClOH13~ and p is NHCoR5 -O3SSCH2(CH2)mCH-COX', wherein m is 0 to 4, R5 is Cl-to C4-alkyl or C6H5, and X~ is as defined abovc, followed by treatment with HSCH2CH20H; or NHCoR5 C2H5-S-S-CH2(CH2)mCHC0X', wherein m, R5 ~ ;

2~7~3~

and X~ are as defined immediately above, then treat-ment with dithiothreitol. Such reac~ions are carried out in a nitrogen atmosphere, at about 0O to 35C
and at a pH of 8 to 11 (with base added, as necessary, to keep th pH within this range), for one to twenty-four hours. For example, an amino-derivatized polysaccharide may be reacted with ~
O~\ S

to produce an appropriately-functionalized polysac-15 charide By these steps then, covalently-modified polysaccharides of the forms, Ps-A-E*- or Ps-A~-SH-, wherein E* is -CCHX or O

C(CH2)pN~3 and A, A~, R, X and p are as defined above, are produced.
Separate functionalization of the protein to ~7~3~

be coupled to the polysaccharide, involves reaction of the protein with one or more reagents to generate a thiol group, or reaction of the protein with one or more reagents to generate an electrophilic ~i.e., thiophilic) center.
In preparation for conjugation with an electrophilic-functionalized polysaccharide, the protein is reacted in one or two steps with one or more reagents to generate thiol groups, such as those acylating reagents used for generating thiol groups on polysaccharides, as discussed on pages 15-17 above. Thiolated proteins may also be prepared by aminating carboxy-activated proteins, such as those shown in Atassi et al., Biochem et Biophvs. Act_, 670, 300, (1981), with aminothiols, to create the thiolated protein. A preferred embodiment of this process step involves the direct acylation of the pendant amino groups (i.e., lysyl groups) of the protein with N-acetylhomocysteinethiolactone at about 0 to 35C and pH 8-11, for from five minutes to two hours, using equiweights of reactants.
20When E'B' is O O
~4 11 25~ N(CH2) 20~703a the conditions and method of preparing the functionalized protein are as discussed above for preparing the counterpart polysaccharide by reaction with activated maleimido acids.
In preparing for conjugation with a covalently-modified bacterial polysaccharide with pendant thiol groups, the protein is acylated with a reagent gener-ating an electrophilic center, su~h acylating agents including, for example, XCH2C-XI and XCH - CX', wherein X and X' are as defined above; and ~CO\ O
N (CH2)aC -X' CO/

wherein X~ is as defined above. Suitable proteins with electophilic centers also include, for example, those prepared by acylation of the pendant lysyl amino groups with a reagent, such as activated maleimido acids, for example, O O

~ NOC(CH2)n - O O

2047~

or by reacting the carboxy-activated protein with monohaloacetyl derivatives of diamines. In both preparation reactions, the temperature is from 0 to 35C for from five minutes to one hour and the pH is from 8 to 1-1.
Formation of the conjugate is then merely a 5 matter of reacting any of the covalently-modified polysaccharides having pendant electrophilic centers with of the bacterial protein MIP having pendant thiol groups at a pH of 7 to 9, in approximate equiweight ratios, in a nitrogen atmosphere, for from lO six to twenty-four hours at from about 17 to 40C, to give a covalent conjugate. Examples of such reactions include:

Ps-CNCH2CR2CH2CH2NHCCH2Br + HSCH2CH2CHCO Pro >
OHI 1l NHCOCH3 PscNcH2cH2cH2cH2NHccH2scH2cH2cHcopro ' wherein an activated polysaccharide which has been reacted with 4-bromoacetamidobutylamine is reacted 20 with a protein which has been reacted with N-acetyl-homocysteinethiolactone, to form a conjugate, and:

20~7~3a OH ~I~
PF. CNY '--~CH2 ,~
~r ~

HSCH2CH2N~CCH2C~2CPrc) >
10 PgCNlIY" NHCCH2- N~ ~cH2cH2NHccH2cH2cpro o S

lS

(where Y" i8 a C2-C8alkyl radical), wherein an amino-derivatized polysaccharide which has been reacted with activated maleimido acids is reacted 20 with a carboxy-activated protein which has been aminated with an aminothiol, to form a conjugate.
Similarly, any of the covalently-modified polysaccharides with pendant thiol groups may be reacted with the bacterial protein MIP having pendant i 25 electrophilic centers to give a covalent conjugate.
An example of such a reaction is:

PSCNHCH2CH2SH + ProCCH2CH2C-N~CH2)4NHCOCH2Br >

PscNcH2cH2scH2cN(cH2)4NHccH2cH2cpro ' , 20~7~3~

wherein an activated polysaccharide which has been reacted with an aminothiol is reacted with a carboxy-activated protein which has been reacted with monohaloacetyl derivatives of a diamine, to form a conjugate.
Should the electrophilic activity of an excess of haloacetyl groups need to be eliminated, reaction of the conjugate with a low molecular weight thiol, such as n-acetylcysteamine~ will accomplish this purpose. Use of this reagent, n-acetylcysteamine, also allows confirmation accounting of the haloacetyl moieties used (see Section D), because the S-carboxymethylcysteamine which is formed may be uniquely detected by the method of Spackman, Moore and Stein.
These conjugates are then centrifuged at about 100,000 x g using a fixed angle rotor for about two hours at about 1 to 20OC, or are submitted to any of a variety of other purification procedures, including gel permeation, ion exclusion chromatography, gradient centrifugation, or other differential adsorption chromatography, to remove non-covalently-bonded polysaccharides and proteins, using the covalency assay for the bigeneric spacer ~see below) as a method of following the desired biological activity.
The further separation of reagents may be accomplished by size-exclusion chromatography in a column, or in the case of very large, non-soluble proteins, separation may be accomplished by ultracentrifugation.

2~7~3a Analysis of the conjugate to confirm the covalency, and hence the stability of the conjugate, is accomplished by hydrolyzing (preferably with 6N
HCl at 110C for 20 hours) the conjugate, then quantitatively analyzing for the amino acid of the hydrolytically-stable spacer containing the thioether bond and constituent amino acids of the protein. The contributio~ of the amino acids of the protein may be removed, if necessary, by comparison with the appropriate amino acid standard for the protein involved, with the remaining amino acid value reflecting the covalency of the conjugate, or the amino acid of the spacer may be designed to appear outside the amino acid standard of the protein in the analysis. The covalency assay is also useful to monitor purification procedures to mar~ the enhancement of concentration of the biologicallyactive components. In the above examples, hydrolysis of OHI 1l NHCOCH3 PsCNCHzCH2CH2CH2NHCCH2SCH2CH2CHCOPro results in the release of S-carboxymethylhomocysteine, H02CCH2SCH2CH2CHC02H; hydrolysis of 2~7~3~

PsCNHY~ NEICCH2 N~~
~\ /CE~2CH2NHCCH2CH2CPro o results in the release of the aminodicarbo~ylic acid, H02CCH2CHSCH2CH2NH2; and hydrolysis of OH OH O O
PSCNCH2CH2SCH2CN(CH2)4NHCCH2CH2CPro results in the release of S-carboxymethylcysteamine, H2NCH2CH2SCH2C02H by cleavage of the Ps-A-E-S-B-Pro molecule at peptide linkages and other hydrolytically-unstable bonds. Chromatographic methods, such as those of Spackman, Moore, and Stein, may then be conveniently applied and the ratio of amino acid constituents determined.
Optimal production of IgG antibody requires collaboration of B and T lymphocytes with specificity for the antigen of interest. T lymphocytes are - incapable of recognizing polysaccharides but can provide help for anti-polysaccharide IgG antibody responses if the polysaccharide is covalently linked to a protein which the T cell is capable of recognizing.

2~ ~7~3~

In mice this requirement exists for secondary, as well as primary, antibody responses and is carrier-specific, i.e. a secondary antibody response occurs only if the T helper cells have previously been sensitized with the carrier protein used for the secondary immunization. Therefore, the ability of a mouse to make a secondary antibody response to a PRP-protein conjugate is dependent on the presence of primed T lymphocytes with specificity for the carrier protein.
Demonstration of the ability of MIP to 10 provide carrier priming for anti-PRP antibody responses was done in mice adoptively primed with PRP
covalently linked to a heterologous carrier, diphtheria toxoid ~DT). Adoptive transfer was used in order to determine whether the administration of lymphocytes primed with MIP alone was sufficient to generate effective helper-T cell activity for anti-PRP antibody formation in response to PRP-OMPC.
Comparable secondary anti-PRP antibody responses were elicited by PRP-OMPC when lymphocytes primed with MIP
or OMPC were transferred, indicating that T cell recognition of OMPC resides in the MIP moiety.
PRP-MIP conjugates were tested for immunogenicity in mice as well as infant Rhesus monkeys. The immune response in both of these animal models share, with infant humans, a deficiency in their ability to generate antibody responses against T-independent antigens such as bacterial polysaccharides. These animals are commonly used as models for assessment of the immune response of infant humans to various antigens.

2 ~ 3 ~

One or more of the conjugate vaccines of this invention may be used in mammalian species for either active or passive protection prophylactically or therapeutically against infectious agents including bacteria, rickettsia, parasites, and viruses, or against toxins or poisons, allergens, and mammalian cells or other eukaryotic cells. Active protection may be accomplished by injecting an effective quantity capable of producing measurable -amounts of antibodies ~e.g., 2 to 50 ~g) of an antigen (e.g. PRP) in the MIP-conjugate form of each Of the conjugates being administered per dose. The use of an adjuvant (e.g., alum) is also intended to be within the scope of this invention. Passive protection may be accomplished by injecting whole antiserum obtained from animals previously dosed with the MIP-conjugate or conjugates, or globulin or other antibody-containing fractions of said antisera, with or without a pharmaceutically-acceptable carrier, such as sterile saline solution. Such globulin is obtained from whole antiserum by chromatography, salt or alcohol fractionation or electrophoresis. Passive protection may be accomplished by standard monoclonal antibody procedures or by immunizing suitable mammalian hosts.
In a preferred embodiment of this invention, the conjugate is used for active immunogenic vaccination of humans, especially infants and children, immunocompromised individuals (such as asplenic persons and post-chemotherapy patients) and the elderly. For additional stability, these 2~ ~703~

conjugates may also be lyophilized in the presence of lactose (for example, at 20 ~g/mL of PRP/4 mg/mL
lactose) prior to use.
A preferred dosage level is an amount of each of the MIP-conjugates, or derivative thereof to be administered, corresponding to between approximately 2 to 20 ~g of PRP in the MIP-conjugate form for conjugates of H. influenzae serotype B
polysaccharide, in a single administration. If necessary, an additional one or two doses of the MIP-conjugate, or derivative thereof-, of the ~.
influenzae serotype B polysaccharide in an amount corresponding to between approximately 2 to 20 ~g of PRP in the conjugate form, may also be administered.
The invention is further defined by reference to the following examples, which are intended to be illustrative and not limiting.

Preparation of Neisseria meningitidis Bll Serotype 2 OMPC

A. Ferme~tation 1. Neisseria meningitidis Group Bll A tube containing the lyophilized culture of Neisseria meningitidis (obtained from Dr. M.
Artenstein, Walter Reed Army Institute of Research (WRAIR), Washington, D.C.) was opened and Eugonbroth (BBL) was added. The culture was streaked onto 20~7~3~

Mueller Hinton agar slants and incubated at 37C with 5% C2 for 36 hours, at which time the growth was harvested into 10% skim milk medium (Difco), and aliquots were frozen at -70OC. The identity of the organism was confirmed by agglutination with specific antiserum supplied by WRAIR, and typing serum supplied by Difco.
A vial of the culture from the second passage was thawed and streaked onto 10 Columbia Sheep Blood agar plates (CBAB-BBL). The plates were incubated at 37C with 5~/O C02 for 18 hours after which time the growth was harvested into lO0 mL of 10% skim milk medium, aliquots were taken in 0.5 mL
amounts and frozen at -70C. The organism was positively identified by agglutination with specific antiserum, sugar fermentation and gram stain.
A vial of the culture from this passage was thawed, diluted with Mueller-Hinton Broth and streaked onto 40 Mueller-Hinton agar plates. The plates were incubated at 37C with 6% C02 for 18 hours after which time the growth harvested into 17 mL of 10% skim milk medium, aliquotted in 0.3 mL
amounts and frozen at -70C. The organism was positively identified by Gram stain, agglutination with specific antiserum and oxidase test.
2. Fermentation and collection of cell paste a. Inoculum Development- The inoculum was grown from one frozen vial of Neisseria memingitidis Group B, B-ll from above (passage 4). Ten Mueller-Hinton agar slants were inoculated, and six were harvested approximately 18 hours later, and used 2~7~3 as an inoculum for 3 250 mL flasks of Gotschlich's yeast dialysate medium at pH 6.35. The OD660 was ad~usted to 0.18 and incubated until the OD660 was between 1 and 1.8. 1 mL of this culture was used to inoculate each of 5 2L. Erlenmeyer flasks (each containing 1 liter of medium; see below) and incubated at 37C in a shaker at 200 rpm. The O.D.
was monitored at hourly intervals following ,inoculation. 4 liters of broth culture, at an O.D.660 f 1.28 resulted.
70 Liter Seed Fermenter- Approximately 4 liters of seed culture was used to inoculate a sterile 70-liter fermenter containing about 40 liters of complete production medium ~see below).
The conditions for the 70-liter fermentation included 37C, 185 rpm with 10 liters/minute air sparging and constant pH control at about pH 7.0 for about 2 hours. For this batch, the final O.D.660 was 0.732 after 2 hours.
800-Liter Production Fermenter Approximately 40 liters of seed culture were used to inoculate a sterile 800 liter fermenter containing S68.2 liters of complete production medium (see below). The batch was incubated at 37C, 100 rpm with 60 liters/minute air sparging and constant pH control at p~ 7Ø For this batch, the final O.D.
was 5.58 thirteen hours after inoculation.

3. Complete Medium for Erlenmeyer flasks and 70-and 800-liter fermenters 20~7~3a Fractio~ A
L-glutamic acid 1.5 g/liter NaCl 6.0 g/liter Na2HPO4.anhydrous 2.5 g/liter NH4C1 1.25 g/liter KCl 0.09 g/liter L-cysteine HCl 0.02 g/liter .

Fractio~ B ~Gotschlich's ~east Dialysate):
1280 g of Difco Yeast Extract was dissolved in 6.4 liters of distilled water. The solution was dialyzed in 2 Amicon DC-30 hollow fiber dialysis units with three H10SM cartridges. 384 g MgSO4.7-H2O
and 3200 g dextrose were dissolved in the dialysate and the total volume brought to 15 liters with distilled water. The pH was adjusted to 7.4 with NaOH, sterilized by passage through a 0.22 ~ filter, and transferred to the fermenter containing Fraction A.
For the Erlenmeyer flasks: 1 liter of Fraction A and 25 mL of Fraction B were added and the pH was adjusted to 7.0-7.2 with NaOH.
For the 70 liter fermenter: 41.8 liters of Fraction A and 900 mL of Fraction B were added and the pH was adjusted to 7.0-7.2 with NaOH.
For the 800 liter fermenter: 553 liters of Fraction A and 15.0 liters of Fraction B were added and the pH was adjusted to 7.1-7.2 with NaOH.

.

~7~3~

d. Harvest and Inactivation After the fermentation was completed, phenol was added in a separate vessel, to which the cell broth was then transferred, yielding a final phenol concentration of about 0.5%. The material was held a room temperature with gentle stirring until the culture was no longer viable (about 24 hours).
e. Centrifugation After about 24 hours at 4C, the 614.4 liters of inactivated culture fluid was centrifuged through Sharples continuous flow centrifuges. The lO weight of the cell paste after phenol treatment was 3.875 kg.

B. OMPC Isolatio~

Ste~ 1. Concentration and diafiltration The phenol inactivated culture was concentrated to about 30 liters and diafiltered in sterile distilled water using 0.2 ~ hollow fiber filters (ENKA).

$te~ 2. Extraction An equal volume of 2X TED buffer [0.1 M TRIS
0.01 M EDTA Buffer, pH 8.5, with 0.5% sodium deoxycholate] was added to the concentrated diafiltered cells. The suspension was transferred to a temperature regulated tank for OMPC extraction at 56 C with agitation for 30 minutes.
The extract was centrifuged at about 18,000 , 20~703~

rpm in a Sharples continuous flow centrifuge at a flow rate of about 80 mL/minute, at about 4C. The viscous supernatant was then collected and stored at 4C. The extracted cell pellets were reextracted in TED buffer as described above. The supernatants were pooled and stored at 4C.

Ste~ 3. Concentration by Ultrafiltration The pooled extract was transferred to a temper~ature regulated vessel attached to AG-Tech 0.1 micron polysulfone filters. The temperature of the extract was held at 25~C in the vessel throughout the concentration process. The sample was concentrated tenfold at an average transmembrane pressure of between 11 and 24 psi.

Ste~ 4. Collection and Washing of the OMPC
The retentate from Step 3 was centrifuged at about 160,000 ~ g (35,000 rpm) at about 70C in a continuous flow centrifuge at a flow rate between 300 to 500 mL/minute, and the supernatant was discarded.
The OMPC pellet was suspended in TED ~uffer (190 mL buffer; 20 mL/g pellet) Step 2 and Step 4 were repeated twice (s~ipping Step 3).

Ste~ 5. ~ecovery of OMPC Product : The washed pellets from Step 4 were suspended in 100 mL distilled water with a glass rod ~ .
.

.
., 2~03~

and a Dounce homogenizer to insure complete suspension. The aqueous OMPC suspension was then filter sterilized by passage through a 0.22 ~ filter, and the TED buffer was replaced with water by diafiltration against sterile distilled water using a 0.1 ~ hollow fiber filter.

Preparation of H. Influenzae Type b Capsular Polvsaccharide (PRP) Inoculum and Seed De~elopme~t A Stage: A lyophilized tube of Haemophilus influenzae type b, (cultured from Ross 768, received from State University of New York) was suspended in 1 mL of sterile Haemophilus inoculum medium (see below) and this suspension was spread on 9 Chocolate Agar slants (BBL), The pH of the inoculum medium was adjusted to 7.2 + 0.1 (a typical value was pH 7.23) and the medium solution was sterilized prior to use by autoclaving at 121C for 25 minutes. After 20 hours incubation at 37C in a candle jar, the growth from each plate was resuspended in 1-2 mL Haemophilus inoculum medium, and pairs of slants were pooled.

2~7n3~

Haemophilus Inoculum Medium g/Liter Soy Peptone 10 NaCl 5 NaH2P04 3.1 Na2HP04 13.7 K2HP04 2.5 Distilled Water To Volume The resuspended cells from each pair of slants was inoculated into three 250 mL Erlenmeyer flasks containing about 100 mL of Haemophilus Seed and Production medium. The 250 mL flasks were incubated at 37C for about 3 hours until an D660 f about 1.3 was reached. These cultures were used to inoculate the 2 liter flasks (below~.
B Stage: 2 Liter non-baffled Erlenmeyer flasks- 5 mL of culture from ~A stage~ (above) were used to inoculate each of five two-liter flasks, each containing about 1.0 liter of complete ~aemophilus seed and production medium (see below). The flasks were then incubated at 37C on a rotary shaker at about 200 rpm for about 3 hours. A typical OD660 value at the end of the incubation period was about 1Ø

:.

2~70~

Complete Haemophilus Seed And Production Medium Per liter NaH2P04 3.1 g/L
Na2HP04 13.7 g/L
Soy Peptone lO g/L
Yeast e~tract diafiltrate (1) 10 g/L
K2HP04 2.5 g/L
NaCl 5.0 g/L
Glucose (2) 5.0 g/L
Nicotinamide adenine 2 mg/L
dinucleotide (NAD) (3) Remin (4) 5 mg/L

The salts and soy peptone were dissolved in small volumes of hot, pyrogen-free water and brought to correct final volume with additional hot, pyrogen-free water. The fermenters or flasks were then sterilized by autoclaving for about 25 minutes at 121C, and after cooling yeast extract diafiltrate (1), glucose (2), NAD (3), and hemin (4) were added aseptically to the flasks or fermenters prior to inoculation.
(1) Yeast extract diafiltrate: 100 g brewers~ yeast extract (Amber) was dissolved in 1 liter distilled water and ultrafiltered using an ,, 2~7~3a Amicon DC-30 hollow fiber unit with H10 x 50 cartridges with a 50 kd cutoff. The filtrate was collected and sterilized by passage through a 0.22 filter.
(2) Glucose was prepared as a sterile 25%
solution in distilled water.
(3) A stock solution of NAD containing 20 mg/mL was sterilized by passage through a (0.22 filter) and added aseptically just prior to inoculation.

(4) A stock solution of Hemin 3X was prepared by dissolving 200 mg in 10 mL of 0.1 M NaOH
and the volume adjusted with distilled, sterilized water to 100 mL. The solution was sterilized for 20 minutes at 121C and added aseptically to the final medium prior to inoculation.
C Stage: 70 Liter Seed Fermenter- Three liters of the product of B Stage was used to inoculate a fermenter containing about 40 liters of Complete Haemophilus Seed And Production medium (prepared as described above) and 17 mL UCON B625 antifoam agent.
The pH at inoculation was 7.4.
D Stage: 800 Liter Production Fermenter-Appro~imately 40 liters of the product of "C Stage"
was used to inoculate an 800 liter fermenter containing 570 liters of Haemophilus Seed and Production medium (prepared as described above), scaled to the necessary volume, and 72 mL of UCON
LB625 antifoam agent was added.
The fermentation was maintained at 37C with 100 rpm agitation, with the O.D.660 and pH levels ~7~3~

measured about every two hours until the OD660 was stable during a two-hour period, at which time the fermentation was terminated (a typical final OD660 was about 1.2 after about 20 hours).

XARVEST AND INACTIVATION
Approximately 600 liters of the batch was inactivated by harvesting into a ~kill tank~
containing 12 liters of 1% thimerosal.

CLARIFICATION
After 18 hours of inactivation at 4C, the batch was centrifuged in a 4-inch bowl Sharples ; contiuous flow centrifuge at a flow rate adjusted to maintain product clarity (variable between 1.3 and 3.0 liters per minute). The supernatant obtained after centrifugation (15,000 rpm) was used for product recovery.

ISOLATION AND CONCENTRATION BY ULTRAFILTRATION
The supernatant from two production fermentations was pooled and concentrated at 2 to 8C
in a Romicon XM~50 ultrafiltration unit with twenty 50 kd cut-off hollow fiber cartridges ~4.5 m2 membrane area; 2.0 Lpm air flow and 20 psi).
: Concentration was such that after approximately 4.5 hours, about 1,900 liters had been concentrated to 57.4 liters. The filtrate was discarded.

48% AND 61% ETHANOL PRECIPITATION
To the 57.4 liters of ultrafiltration retentate, 53 liters of 95% ethanol was added 2~ia3~

dropwise over 1 hour with stirring at 4C to a final concentration of 48% ethanol by volume. The mixture was stirred two additional hours at 4C to insure complete precipitation, and the supernatant was collected following passage through a single 4-inch Sharples continuous flow centrifuge at 15,000 rp~ at a flow rate of about 0.4 liters per minute. The pellet was discarded and the clarified fluid was brought to 82% ethanol with the addition of 40.7 liters of 95% ethanol over a one hour period. The mixture was stirred for three additional hours to insure complete precipitation.

RECOVERY OF THE SECOND PELLET
The resulting 48% ethanol-soluble-82%
ethanol-insoluble precipitate was collected by centrifugation in a 4 inch Sharples continuous flow centrifuge at 15,000 rpm with a flow rate of about 0.24 liters per minute and the 82% ethanol supernatant was discarded. The crude product yield was about 1,4 kg of wet paste.

CALCIUM CHLORIDE EXTRACTION
The 1.4 kg grams of 82% etha~ol-insoluble material, was mixed in a Daymax dispersion vessel 2-8C with 24.3 liters of cold, distilled water. To this mixture, 24.3 liters of cold 2M CaC12.2H2O was added, and the mixture was incubated at 4C for 15 minutes. The vessel was then rinsed with 2 liters of 1 M CaC12.2H2O, resulting in about 50 liters final volume.

2~7~3~

23% ETHANO~ PR~CIPITATION
The 50 liters of CaC12 extract was brought to 25% ethanol by adding 16.7 liters of 95% ethanol dropwise, with stirring, at 4C over 30 minutes.
After additional stirring for 17 hours, the mixture was collected by passage through a Sharples continuous flow centrifuge at 4C. The supernatant was collected and the pellet was discarded.

38% ETHANOL PRECIPITATION AND
COLLECTION OF CRUDE PRODUCT PASTE
The 25% ethanol-soluble supernatant was brought to 38% ethanol by the addition of 13.9 liters of 95% ethanol, dropwise with stirring, over a 30 minute period. The mixture was then allowed to stand with agitation for one hour, then without agitation for 14 hours, to insure complete precipitation. The resulting mixture was then centrifuged in a 4 inch Sharples continuous flow centrifuge at 15,000 rpm (flow rate of 0.2 liters per minute) to collect the precipitated crude H. influenzae polysaccharide.

TRITURATION
The pellet from the centrifugation was transferred to a 1 gallon Waring Blender containing 2 to 3 liters of absolute ethanol and blended for 30 seconds at the highest speed. Blending was continued for 30 seconds on, and 30 seconds off, until a hard white powder resulted. The powder was collected on a Buchner funnel with a teflon filter disc and washed sequentially, in situ, with two 1 liter portions of 20~7~3~

absolute ethanol and two 2 lite r po rtions of acetone. The material was then dried, in vacuo, at 4C for 24 hours, resulting in about 337 g (dry weight) of product.

PHENOL EXTRACTION
About 168 grams of the dry material from the trituration step (about half of the total) was resuspended in 12 liters of 0.488 M sodium acetate, pH 6.9, with the aid of a Daymax dispersion vessel.
The sodium acetate solution was immediately extracted 10 with 4.48 liters of a fresh aqueous phenol solution made as follows: 590 mL of 0.488 M sodium acetate, pH
6.9, was added to each of eight 1.5 kg bottles of phenol (Mallinckrodt crystalline) in a 20 liter pressure vessel and mixed into suspension. Each ,j 15 phenol extract was centrifuged for 2.5 hours at 30,000 rpm and 4C in the K2 Ultracentrifuge (Electronucleonics). The aqueous effluent was extracted three additional times with fresh aqueous phenol solution as described above. The phenol phases were discarded.

ULTRAFILTRATION
The aqueous phase from the first phenol extraction above (12.2 liters) was diluted with 300 liters of cold, distilled water and diafiltered at 4C on an Amicon DC-30 ultrafiltration apparatus using 3 HlOP10, 10 kd cutoff cartrid~,es, to remove the carryover phenol. The Amicon unit was rinsed and : the rinse added to the retentate, such that the final volume was 31.5 liters. The ultrafiltrate was discarded.
.

. ' ' .; .

~O~L7~3a 67% ETHANOL PRECIPITATION
0.81 liters of 2.0 M CaC12 was added to the 31.5 liters of dialysate from the previous step (final CaC12 concentration was 0.05 M) and the solution wa~ brought to 82% ethanol with dropwise addition and rapid stirring over one hour, of 48.5 liters of 95% ethanol. After 4 hours of agitation, then standing for 12 hours at 4C, the supernatant.
was siphoned off and the precipitate was collected by centrifugation in a 4 inch Sharples continuous flow centrifuge (15,000 rpm), at 4C for 45 minutes. The resulting polysaccharide pellet was triturated in a 1 gallon Waring blender using 30 second pulses with 2 liters of absolute ethanol, collected on a ~uchner funnel fitted with a teflon filter disc, and washed, in situ, with four 1 liter portions of absolute ethanol followed by two 1 liter portions of acetone.
The sample was then dried in a tared dish, in vacuo, at 4C for 20 hours. The yield was about 102 grams of dry powder. The yield from all phenol extractions was pooled resulting in a total of 212.6 grams of dry powder ULTRACENTRIFUGATION IN 29% ETHANOL
AND COLLECTION OF FINAL PRODUCT
The 212.6 grams of dry powder from above was dissolved in 82.9 liters of distilled water, to which was added 2.13 liters of 2 M CaC12.2H2O, (0.05M
CaC12), 2.5 mg polysaccharide/mL), and the mixture was brought 29% ethanol with the dropwise addition of 29.86 liters of 95% ethanol over 30 minutes. The 20~3~

mixture was clarified immediately by centrifugation in a K2 Ultracentrifuge containing a K3 titanium bowl and a Kll Noryl core (30,000 rpm and 150 mL per minute flow rate) at 4C. The pellet was discarded and the supernatant was brought to 38% ethanol by the addition of 17.22 liters of 95% ethanol over 30 minutes with stirring. After stirring 30 additional minutes the mixture was allowed to stand without agitation at 4C for 17 hours and the precipitate was collected using a 4 inch Sharples continuous flow centrifuge at 15,000 rpm (45 minutes was required).
The resulting paste was transferred to a l-gallon Waring blender containing 2 liters of absolute ethanol and blended at the highest speed by 4 or 5 cycles of 30 seconds on, 30 seconds off, until a hard, white powder formed. This powder was collected on a Buchner funnel fitted with a Zitex teflon disc and rinsed sequentially, in situ, with two fresh 0.5 liter portions and one 1 liter portions of absolute ethanol, and with two 1 liter portions of acetone. The product was removed from the funnel and transferred to a tared dish for drying, in vacuo, at 4C (for 25 hours). The final yield of the product was 79.1 grams dry weight.

Cloning of &enomic DNA Encoding MIP.
About 0.1 g of the phenol inactivated _.
meningitidis cells ~see Example 1) was placed in a fresh tube. The phenol inactivated cells were ~0~703~

resuspended in 567 ~L of TE buffer [lOmM TRIS-HCl, lmM EDTA, pH 8.0]. To the resuspended cells was added 30 ~L of 10% SDS, and 3 ~L of 20 mg/mL
proteinase K (Sigma). The cells were mixed and incubated at 37C for about 1 hour, after which 100 ~L of 5 M NaCl was added and mixed thoroughly. 80 ~L
of 1% cetyltrimethylamonium bromi-de (CTAB) in 0.7 M
NaCl was then added, mixed thoroughly, and incubated at 65C for 10 minutes. An equal volume (about 0.7 to 0.8 mL) of chloroform/isoamyl alcohol (at a ratio of 24:1, respectively) was added, mixed thoroughly and centrifuged at about 10,000 x g for about 5 minutes. The aqueous (upper) phase was transferred to a new tube and the organic phase was discarded.
An equal volume of phenol/chloroform/isoamyl alcohol (at a ratio of 25:24:1, respectively> was added to the aqueous phase, mixed thoroughly, and centrifuged at 10,000 x g for about 5 minutes. The aqueous phase (upper) was transferred to a new tube and 0.6 volumes (about 420 ~L) of isopropyl alcohol was added, mixed thoroughly, and the precipitated DNA was centrifuged at 10,000 x g for 10 minutes. The supernatant was discarded, and the pellet was washed with 70%
ethanol. The DNA pellet was dried and resuspended in 100 ~L of TE buffer, and represents N. menin~itidis genomic DNA.
Two DNA oligonucleotides were synthesized which correspond to the 5' end of the MIP gene and to the 3' end of the MIP gene [Murakami, E.C. et al., (1989), Infection and Immunity, 57, pp.2318-23]. The sequence of the DNA oligonucleotide specific for the 5' end of the MIP gene was:

20~7~3~3 5'-ACTAGTTGCAATGAAAAAATCCCTG-3'; and for the 3' end of the MIP gene was: 5'-GAATTCAGATTAGGAATTTGTT-3'.
These DNA oligonucleotides were used as primers for polymerase chain reaction (PCR) amplification of the MIP gene using 10 nanograms of N. meningitidis genomic DNA. The PCR amplification step was performed according to the procedures supplied by the manufacturer ~Perkin Elmer).
The amplified MIP DNA was then digested with the restriction endonucleases S~I and EcoRI. The 1.3 kilobase (kb) DNA fragment, containing the complete coding region of MIP, was isolated by electrophoresis on a 1.5% agarose gel, and recovered from the gel by electroelution tCurrent Protocols in Molecular Biology, (1987), Ausubel, R.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidman, J.G. and Struhl, K., eds., Greene Publishing Assoc.]
The plasmid vector pUC-19 was digested with ~ and EcoRI. The gel purified SpeI-EcoRI MIP DNA
was ligated into the ~peI-Eco~I pUC-19 vector and was used to transform E. coli strain DH5. Transformants containing the pUC-19 vector with the 1.3 kbp MIP DNA
were identified by restriction endonuclease mapping, and the MIP DNA was sequenced to ensure its identity.

Construction of the pcl/l.GallOp(B)ADHlt vector:
The Gal lO promoter was isolated from plasmid YEp52 [Broach, et al., (1983) in Experimental Manipulation of Gene Expression, Inouye, M(Ed) ~703~

Academic Press pp. 83-117] by gel purifying the 0.5 kilobase pair (Kbp) fragment obtained after cleavage with Sau 3A and Hind III. The ADHl terminator was isolated from vector p&AP.tADH2 [Kniskern, et al., (1986), Gene, 46, pp. 135-141] by gel purifying the 0.35 Kbp fragment obtained by cleavage with Hind III
and SpeI. The two fragments were ligated with T4 DMA
ligase to the gel purified pucl8~Hlnd III vector ~the Hind III site was eliminated by digesting pUC18 with Hind III, blunt-ending with the Klenow fragment of E. coli DNA polymerase I, and ligating with T4 DNA
ligase) which had been digested with BamHI and SphI
to create the parental vector pGallo-tADHl. This has a unique Hind III cloning site at the GallOp.ADHlt junction.
The unique Hind III cloning site of pGallO.tADHl was changed to a unique Bam~I cloning site by digesting pGallO.tADHl with Hind III, gel purifying the cut DNA, and ligating, using T4 DNA
ligase, to the following Hind III-BamHI linker:
5'-AGCTCGGATCCG-3' 3'-GCCTAGGCTCGA-5~.

The resulting plasmid, pGallO(B)tADHl, has deleted the Hind III site and generated a unique BamHI cloning site.
2s The GallOp.tADHl fragment was isolated from pGallO(B)tADHl by digestion with SmaI and blunt-ended with T4 DNA polymerase, and gel purified. The yeast shuttle vector pCl/l [Brake et al., (1984), Proc. Nat'l. Acad. Sci. USA, 81, 20'~70~

pp.4642-4646] was digested with SphI, blunt-ended with T4 DNA polymerase, anpurified. This fragment was ligated to the vector with T4 DNA ligase. The ligation reaction mixture was then used to transform E. coli HB101 cells to ampicillin resistance, and transformants were screened by hybridization to a single strand of the 32P-labelled HindIII BamHI
linker. The new vector construction, pcill.GallOp~B)ADHlt was confirmed hy digestion with HindIII and BamHI.

EXAMPL~ 5 Construction of a Yeast MIP Expression Vector with MIP + Leader DNA Se~uences A DNA fragment containing the complete coding region of MIP was generated by digestion of pUC19.MIP #7 with SpeI and EcoRI, gel purification of the MIP DNA, and blunt-ended with T4 DNA polymerase.
The yeast internal expression vec~or pCl/l.GallOp(B)ADHlt was disgested with Bam HI, dephosphorylated with calf intestinal alkaline phosphatase, and blunt-ended with T4 DNA polymerase.
The DNA was gel purified to remove uncut vector.
The 1.1 Kbp blunt-ended fragment of MIP was ligated to the blunt-ended pcl/l.GallOp(B)ADHlt vector, and the ligation reaction mixture was used to transform competent E. coli DH5 cells to ampicillin resistance. Transformants were screened by hybridization to a 32P-labelled DNA oilgoncleotide:
5'... AAGCTCGGATCCTAGTTGCAATG...3', which ~7~

was designed to be homologous with sequences overlapping the MIP-vector junction. Preparations of DNA were made from hybridization positive transformants and digested with KpnI and SalI to verify that the MIP fragment was in the correct orientation for expression from the GallO promoter.
Further confirmation of the DNA construction was obtained by dideoxy sequencing from the GallO
promoter into the MIP coding region.
Expression of MIP by the transformants was detected by Western blot analysis. Recombinant MIP
produced in the transformants comigrated on polyacrylamide gels with MIP purified from OMPC
vesicles, and was immunologically reactive with antibodies specific for MIP.

Construction of yeast MIP expression vector with a 5'-Modified MIP DNA Se~uence.
A DNA oligonucleotide containing a HindIII
site, a conserved yeast 5' nontranslated leader (NTL), a methionine start codon (ATG~, the first 89 codons of the mature MIP (beginning with Asp at position +20) and a KRnI site (at position +89) was generated using the polymerase chain reaction (PCR) technique. The PCR was performed as specified by the manufacturer (Perkin Elmer Cetus) using the plasmid pUC19MIP42#7 as the template and the following DNA
oligomers as primers:
5 CTAAGCTTAACAAAATGGACGTTACCTTGTACGGTACAATT3 , and 5 ACGGTACCGAAGCCGCCTTTCAAG3 .

20~703~

To remove the 5' region of the MIP clone, plasmid pUC19MIP42#7 was digested with Kpnl and HindIII and the 3.4 Kbp vector fragment was agarose gel purified. The 280 bp PCR fragment was digested with Kpnl and HindIII, agarose gel purified, and ligated with the 3.4 Kbp vector fragment.
Transformants of E. coli HB101 (~RL) were screened by DNA oligonucleotide hybridization and the DNA from positive transformants was analyzed by restriction enzyme digestion. To ensure that no mutations were introduced during the PCR step, the 280 bp PCR
generated DNA of the positive transformants was sequenced. The resulting plasmid contains a HindIII
- EcoRI insert consisting of a yeast NTL, ATG codon, and the entire open reading frame (ORF) of MIP
beginning at the Asp codon (amino acid +20).
The yeast MIP expression vectors were constructed as follows. The pGAL10/pcl/1 and pGAP/pCl/l vectors [Vlasuk, G.P., et al., (1989) J.B.C., 264, pp.l2,106-12,112] were digested with BamHI, flush-ended with the Klenow fragment of DMA
polymerase I, and dephosphorylated with calf intestinal alkaline phosphatase. These linear vectors were ligated with the Klenow treated-and gel purified HindIII - EcoRI fragment described above, which contains the yeast NTL, ATG and ORF of MIP are 5 forming pGallO/pcl/MIP and pGAP/pGAP/pCl/MIP.
Saccharomyces cerevisiae strain U9 (gallOpgal4-) were transformed with plasmid pGallO/p/pCl/MIP. Recombinant clones were isolated and examined for expression of MIP. Clones were ~7~

grown at 37C with shaking in synthetic medium (leu-) containing 2% glucose (w/v~ to an O.D.660 of about 6Ø Galactose was then added to 2% (w/v) to induce ~expression of MIP from the GallO promoter. The cells were grown for an additional 45 hours following galactose induction to an O.D.600 of about 9Ø The cells were then harvested by centrifugation. The cell pellet was washed with distilled water and frozen.

Western Blot For Recombinant MIP:
To determine whether the yeast was expressing MIP, Western blot analysis was done.
Twelve percent, 1 mm, 10 to 15 well Novex Laemmli gels are used. The yeast cells were broken in H20 using glass beads (sodium dodecylsulfate (SDS) may be used at 2% during the breaking process). Cell debris was removed,by centrifugation for 1 minute at 10,000 x g.
The supernatant was mixed with sample running buffer, as described for polyacrylamide gel purification of MIP. The samples were run at 35 mA, using OMPC as a reference control, until the bromophenol dye marker runs of the gel.
Proteins were transferred onto 0.45 ~ pore size nitrocellulose paper, using a NOVEX transfer apparatus. After transfer the nitrocellulose paper was blocked with 5a/0 bovine serum albumin in phosphate buffered saline for 1 hour, after which 15 mL o~ a 1:1000 dilution of rabbit anti-MIP antiserum (generated by immunization with gel purified MIP

2~7~3a using standard procedures) was added. After overnight i~cubation at room temperature 15 mL of a 1:1000 of alkaline phosphatase conjugated goat anti-rabbit IgG was added. After 2 hours incubation the blot was developed using FAST RED TR SALT (Sigma) and Naphthol-AS-MX phosphate (Sigma).

Bacterial Expression Of Recombinant MIP
Plasmid pUC19-MIP contain-ng the 1.3 kilobase pair MIP gene insert, was digested with restriction endonucleases ~ and EcoRI. The l.lkbp fragment was isolated and purified on an agarose gel using standard techniques known in the art. Plasmid pTACSD, containing the two cistron TAC promoter and a l5 unique ECORI site, was digested with ECORI. Blunt ends were formed on both the 1.3 kbp MIP DNA and the pTACSD vector, using T4 DNA polymerase (Boehringer Mannheim) according to the manufacturer's directions. The blunt ended 1.3 kbp MIP DNA was ligated into the blunt ended vector using T4 DNA
ligase (Boehringer Mannheim) according to the manufacturer's directions. -The ligated DNA was used to transform E. coli strain DHSaIQMAX (BRL) according to the manufacturer's directions. Transformed cells were plated onto agar plates containing 25 ug kanamycin/mL and 50 ug penicillin/mL, and incubated for about 15 hours at 37 C. A DNA oligonucleotide with a sequence homologous with MIP was labelled with 32p and used to screen nitrocellulose filters : 30 ~Q~7~3~

containing lysed denatured colonies from the plates of transformants using standard DNA hybridization techniques. Colonies which were positive by hybridization were mapped using restriction endonucleases to determine the orientation of the MIP
gene.
Expression of MIP by the transformants was detected by Western blot analysis. Recombinant MIP
produced in the transformants comigrated on polyacrylamide gels with MIP purified from OMPC
vesicles, and was immunologically reactive with antibodies specific for MIP.

Preparation of Purified MIP from OMPC Liposomes or From Recombinant Cells by Polyacrylamide Gel ElectrophoreSis Acrylamide/BIS (37.5:1) gels, 18 x 14 cm, 3 mm thick were used. The stacking gel was 4%
polyacrylamide and the separating gel was 12%
polyacrylamide. Approximately 5 ~g of OMPC protein, or recombinant host cell protein, was used per gel.
To 1 mL of OMPC was added 0.5 mL of sample buffer (4%
glycerol, 300 mM DTT, 100 mM TRIS, 0.001% Bromophenol blue, pH 7.0). The mixture was heated to 105C for 20 minutes and allowed to cool to room temperature before loading onto the gel. The gel was run at 200-400 milliamps, with cooling, until the Bromophenol blue reached the bottom of the gel. A
vertical strip of the gel was cut out (about 1-2 cm 20'~7~30 wide) and stained with Coomassie/cupric acetate (0.1%). The strip was destained until the MIP band (about 38 Kd) became visible. The strip was then placed into its original gel position and the MIP
area was excised from the remainder- of the gel using a scalpel.
The excised area was cut into cubes (about 5 mm) and eluted with 0.01 M TRIS-buffer, pH 0.1.
After 2 cycles of elution the eluate was evaluated for purity by SDS-PAGE. The eluate was combined with a common pool of eluates and dialysed for 48 hours against 60 mM ammonia-formic acid, pH 10.
Alternatively, the eluted protein can be dialyzed against 50% acetic acid in water. After dialysis the eluted protein was evaporated to dryness. The material was further purified by passage through a PD10 sizing column (Pharmacia, Piscataway, NJ), and was stored at room temperature.

EXAMPLE20 Carrier activitY of MIP in covalent PRP-OMPC conjuga~
Immunizations: Male C3H/HeN mice (Charles River, Wilmington, MA) were immunized intraperitoneally ~IP) with PRP covalently linked to OMPC (PRP-OMPC; comprising 2.5 ~g PRP and 17 ~g OMPC) or PRP coupled to DT (PRP-DT; containing 2.5-7.5 ~g PRP and 1.8-5.4 ~g DT) (Connaught Laboratories, Willowdale, ONT), suspended in 0.5 mL of 0.01 M
phosphate-buffered saline (PBS). A second group of male C3H-HeN mice, received either 17 ~g of MIP, 17 ~g of OMPC, or 17 ~g of OMPC-I M (OMPC derivatized 20~7~3~
25/JWW - 62 - 18~10 with N-acetyl homocysteine thiolactone, and capped with iodoacetamide). Cell donors for adoptive transfer experiments were twice immunized IP, 21 days apart, and spleen cells were collected l0 days after the second immunization. Adoptive transfer recipients were male C3H/HeN mice given 500R total body gamma-irradiation from a 137Cs source and immediately reconstituted by intravenous injection of~
8 x 107 spleen cells from each of two syngeneic donors separately primed with PRP-DT, and OMPC, MIP, or OMPC-IAA. Control mice received 8 x 107 spleen cells from one donor mouse primed with PRP-OMPC and an equal number of spleen cells from an unprimed donor mouse.
E1ISA for anti-PRP antibodv: Reactive amines were introduced into purified H. influenzae PRP by treatment with carbonyldiimidazole and reaction with butanediamine as described by Marburg et al., U.S. Patent 4,882,317. This derivatized PRP
was chromatographed on Sephadex G~25 in 0.lM sodium bicarbonate buffer, pH 8.4.
N-hydroxysuccinimidobiotin (Pierce Chemical, Rockford, IL) in dimethylsulfoxide was added to the eluate to a final concentration of 0.3 mg/mL and reacted in the dark for 4 hours at ambient temperature (about 25-28OC). Unreacted N-hydroxysuccinimidobiotin was removed by gel filtration over Sephadex G-25 in PBS. Costar (Cambridge, MA) polyvinyl chloride ELISA plates were coated with 50 ~g/well of avidin (Pierce Chemical) at - 10 ~g/mL in 0.1 M sodium bicarbonate buffer, pH 9.5, 20~703~

overnight at ambient temperature and 100% humidity.
Plates were washed 3 times with 0.05 M TRIS-buffered saline, pH 8.5, containing 0.05% Tween-20 (TBS-T), and blocked with TBS-T plus 0.1% gelatin (blocking buffer) at ambient temperature and 100% humidity for 1 hour. Plates were blotted without washing, 50 ~g/well PRP-biotin in PBS at 15-40 ~g/mL was added, and the plates were incubated for 1 hour. Plates were washed 3 times with TBS-T prior to sample addition. Samples were added in two-fold serial dilutions in blocking buffer, and incubated for 2 lO hours at ambient temperature and 100% humidity. The plates were then washed 3 times with TBS-T, and appropriate alkaline-phosphatase conjugated anti-immunoglobulins diluted in blocking buffer were added. The antibodies used were goat anti-mouse IgM
(Jackson Immunoresearch, West Grove, PA), IgG (Fc) (Jackson Immunoresearch), IgGl (gamma) (BRL, Gaithersburg, MD), IgG2a (gamma) (BRL), IgG2b (gamma) (Southern Biotechnology Associates, Birmingham, AL), IgG3 (gamma) (Southern Biotechnology Associates), and goat anti-rabbit IgG (Jackson Immunoresearch).
Plates were incubated for 2 hours at ambient temperature and 100% humidity, washed with blocking buffer, and substrate development was carried out using p-nitrophenyl phosphate (1 mg/mL in 1 M
2S diethanolamine, Kirkegaard and Perry, Gaithersburg, MD). Dilutions were considered positive if the sample absorbance exceeded the mean absorbance plus 3 times the standard deviation of 8 reagent blanks, and the difference in absorbance between successive dilutions was 0.01 or greater. Endpoint titers were defined as the reciprocal of the highest dilution 2!0~703a which gave a positive reaction in the LLISA as described above. Logarithms of reciprocal titers were compared between treatment groups by two.-way analysis of variance [Lindeman, R.H. et al., (1980), Introduction to Bivariate and Multivariate Analysis, Scott Foresman (pub.), New York].
RIA for anti-PRP antibodv quantitation:
The experimental samples o~serum to be tested for the amount of anti-PRP antibodies were diluted 1:2, 1:5, and 1:20, using fetal calf serum as the diluent. 25 ~L of each diluted serum sample was lo transferred, in duplicate, to 0.5 mL RIA vials (Sarstedt~. A solution of PRP labelled with 125I was diluted to yield between 300 and 800 counts per minute (cpm) per 50 ~L, using phosphate buffered saline as the diluent. 50 ~L of diluted 125I-PRP was transferred to each RIA vial, mixed thoroughly and incubated for about 15 hours at 4C. 75 ~L of a saturated solution of ammonium sulfate at 4C was added to each RIA vial, mixed thoroughly and incubated at 4C for 1 hour. The RIA vials were then centrifuged for 10 minutes at 10,000 x g, the supernatant was discarded and the cpm in the pellet was measured in a gamma counter (LKB).
A standard curve consisting of serial two-fold dilutions of an antiserum containing a known quantity of anti-PRP antibodies was prepared as described above and were assayed concomitantly with the experimental serum samples. The quantity o~
anti-PRP antibodies in the standard curve was between 14 ~g/mL as the highest quantity of antibodies and ,.

.

2~7~3~

25/J~ - 65 - 18110 0.056 ~g/mL as the lowest quantity of antibodies.
All samples were run in duplicate.
The average CPM of the duplicate samples was compared with the standard curve to calculate the amount of anti-PRP antibodies present in the experimental serum samples.
Antibodv responses of adoptive transfer recipients: Recipients of spleen cells primed separately with PRP-DT, and either MIP or OMPC or IAA-OMPC, responded to immunization with PRP-OMPC by production of equivalent amounts of serum IgGl and 10 IgG2a anti-PRP antibody within 4 days (see Figure 1). Irradiated mice reconstituted with spleen cells which were carrier-primed with MIP or OMPC or IAA-OMPC, had significantly higher IgGl (p<O.OOl~ and IgG2a (p<0.04) anti-PRP antibody titers after immuniæation with PRP-OMPC than control mice, given PRP-DT-primed but not OMPC-primed spleen cells. The serum antibody responses to immunization with PRP-OMPC in mice given spleen cells primed separately with PRP-DT and either MIP or OMPC or IAA-OMPC were comparable to those in mice given spleen cells primed with PRP-OMPC (p>0.12 for IgGl antibody on days 6-13, and p>O.5 for IgG2a antibody on days 9-13). No antibody response was seen when irradiated mice reconstituted with PRP-DT-primed and either MIP or OMPC-primed spleen cells were immunized with PRP
without a protein carrier. Stasticial analysis was done by two-was analysis of variance (ANOVA) [Lindeman, R.H. et al., Introduction to Bivariate and Multivariate Analysis, (1980), Scott Foresman, New Yor~].

, ~47V3~

These results demonstrate that MIP
functioned in mice as well as OMPC to induce a carrier T helper cell response for the generation of anti-PRP IgG antibodies.

E~AMPLE 10 - Mitogenic Activitv ~f MIP
MIP purified from N. meningitidis OMPC was tested for mitogenic activity in a lymphocyte proliferation assay. Murine splenic lymphocytes were obtained from C3H/HeN, C3H/FeJ, C3H/HeJ, or Balb/c mice. The mice were either naive or had previously been vaccinated with PRP-OMPC. The spleen cells were passed through a sterile, fine mesh screen to remove the stromal debris, and suspended in K medium ~RPMI
16~0 (GIBCO) plus 10% fetal calf serum (Armour), 2 mM
Glutamine (GIBCO), 10 mM Hepes (GIBCO), 100 u/mL
penicillin/100/~g/mL streptomycin (GIBC0), and 50 ~M
~-mercaptoethanol (Biorad)]. Following pipetting to disrupt clumps of cells, the suspension was centrifuged at 300 x g for 5 minutes, and the pellet was resuspended in red blood cell lysis buffer [90%
0.16 M NH4Cl (Fisher), 10% 0.7 TRIS-HCl (Sigma), pH
7.2] at room temperature, 0.1 mL cells/mL buffer for two minutes. Cells were underlayered with 5 mL of fetal calf serum and centrifuged at 4,000 ~ g for 10 minutes, then washed with K medium two times and resuspended in K medium at 5 ~ 106 cells/mL. These cells were plated (100 ~L/well) into 96 well plates along with ].00 ~L of protein or peptide sample, in triplicate.
-2~703~

The ~IP of N. meningitidis was purified as previ.ously described in Example 7. Control proteins included bovine serum albumin, PRP-OMPC and OMPC
itself, and lipopolysaccharide (endotoxi.n). All samples were diluted in K medium to concentrations of 1, 6.5, 13, 26, 52, 105, and 130 ~g/mL, then plated as described above such that their final concentrations were one-half of their original concentrations. Triplicate wells were also incubated for each type of cell suspended in K medium only, to determine the baseline of cell proliferation.
On day 3, 5, or 7 in culture, the wells were pulsed with 25 ~L of 3H-thymidine (Amersham~
containing 1 mCi/25 ~L. The wells were harvested 16-18 hours later on a Skatron harvester, and counts per minute (CPM) was measured in a liquid scintillation counter. The net change in cpm was calculated by subtracting the mean number of cpm taken up per well by cells in K medium alone, from the mean of the experimental cpm. The stimulation index was determined by dividing the mean experimental cpm by the mean cpm of the control wells.
As shown in Figure 2, MIP as well as OMPC
and PRP-OMPC vaccine resulted in proliferation of lymphocytes from previously vaccinated mice. This mitogenic activity did not appear to be due to lipopolysaccharide (LPS) since the MIP was free of detectable LPS, measured by rabbit pyrogenicity assays, and the proliferative effect was greater than that which could have been caused by LPS present in amounts below the level of detectability on silver stained polyacrylamide gels.

2~7~3~

Conjugation of _. influenzae type-b PRP
~olvsaccharide to N. meningitidis MIP
Chemical conjugations were conducted according to the method disclosed in U.S. Patent number 47882,317.
10 mg of MIP in 3 mL of 0.1 M borate buffer, p~ 11.5, was mixed with 10 mg of ethylenediamine tetraacetic acid disodium salt (EDTA, Sigma chemicals) and 4 mg of dithiothreitol (Sigma Chemicals). The protein solution was 1ushed thoroughly with N2. 125 mg of N-acetylhomocysteinethiolactone (Aldrich Chemicals) was added to the MIP solution, and the mixture was incubated at room temperature for 16 hours. It was then twice dialyzed under N2 against 2 L of 0.1 M
borate buffer, pH 9.5, containing 4 mM EDTA, for 24 hours at room temperature. The thiolated protein was then assayed for thiol content by Ellman's reagent (Sigma Chemicals) and the protein concentration was determined by Bradford reagent (Pierce Chemicals).
For conjugation of MIP to PRP, a 1.5 fold excess (wt/wt) of bromoacetylated _. influenzae serotype b PRP was added to the MIP solution and the pH was adjusted to 9 - 9.5 with 1 N NaOH. The mixture was ~ allowed to incubate under N2 for 6 to 8 hours at room ; 25 temperature. At the end of the reaction time, 25 ~L
`- of N-acetylcysteamine (Chemical Dynamics) was added to the mixture, and was allowed to stand for lB hours under N2 at room temperature. The conjugate solution was acidified to between pH 3 to 4 with 1 N HCl, and 2~703~

centrifuged at 10,000 x g for 10 minutes. 1 mL of the supernatant was applied directly onto a column of FPLC Superose 6B (1.6 x 50 cm, Pharmacia) and the conjugate was eluted with PBS. The void volume peak which contains the polysaccharide-protein conjugate (PRP-MIP), was pooled. The conjugate solution was then filtered through a 0.22 ~ filter for sterilization.

EXAMPL~ 12 Demonstration of Immunogenicity of PRP-MIP coniugates Immunizations: Male Balb/c mice (Charles River, Wilmington, MA) were immunized IP with PRP
covalently conjugated to MIP as described in Example 11, using 2.5 ~g PRP in 0.5 mL of preformed alum.
Control mice were immunized with equivalent amounts of PRP given as PRP-CRM tAnderson, M.E. et al., (1985), J. Pediatrics, 107, pp. 346-351] (2.5 ~g PRP/6.25 ~g CRM; 1/4 of the human dose), PRP-DT (2.5 ~g PRP/1.8 ~g DT; 1/10 of the human dose such that constant amounts of PRP were used), and PRP-OMPC (2.5 ~g PRP/35 ~g OMPC; 1/4 of the human dose).
Infant Rhesus monkeys, 6-13.5 weeks of age, were immunized with PRP-MIP conjugates adsorbed onto alum. Each monkey received 0.25 mL of conjugate at two different sites of injection, for a total dose of 0.5 mL. The monkeys were immunized on day 0, 28, and 56, and blood samples were taken every two to four weeks.
Antibody responses were measured by the ;

~L~ 7 ~ ~ ~

ELISA described in Example 9, which distinguishes the class and subclass of the immunoglobulin response.
An RIA which quantitates the total anti-PRP antibody (see Example 9) was also used to evaluate the monkey response. Antibody responses of recipients of PRP-MIP conjugates are shown in Figure 3.
The results show that PRP-MIP conjugates are capable of generating an immune response in mice consisting of IgG anti-PRP antibody and a memory response. This is in contrast to the PRP-CRM and PRP-DT which do not elict measurable anti-PRP
antibody. Thus, MIP functions as an immunologic carrier protein for PRP and is capable of engendering an anti-PRP antibody response when covalently conjugated to the PRP antigen. Purified MIP is therefore an effective immunologic carrier protein replacing the heterogeneous OMPC in construction of bacterial polysaccharide conjugate vaccines.

.

.

Claims (17)

1. A protein in substanitally pure form purified from the outer membrane of a Gram-negative bacterium, which possesses immunologic enhancement and mitogenic activity in mammals.

2. The protein, according to Claim 1, which possesses immunologic enhancement and mitogenic activity in adult and infant humans.

3. The protein, according to Claim 2, wherein the Gram-negative bacterium is of the genus Neisseria.

4. The protein, according to Claim 3, wherein the bacterium is Neisseria meningitidis.

5. The protein, according to Claim 4, which corresponds to the Class II protein of the outer membrane of the bacterium Neisseria meningitidis.

6. The protein according to Claim 5, which corresponds to the Class II protein of the outer membrane of Neisseria meningitidis serogroup B .

7. The protein, according to Claim 6, which possesses immunological carrier activity.

8. The protein according to Claim 6, which possesses immunological mitogenic activity.

9. A recombinant protein in substantially pure form, produced in a recombinant host cell, which corresponds to an outer membrane protein of a Gram-negative bacterium, and which possesses immunologic enhancement and mitogenic activity in mammals.

10. The recombinant protein of Claim 9 wherein the recombinant host is either yeast cells or bacterial cells.

11. The recombinant protein, according to Claim 10, which possesses immunologic enhancement and mitogenic activity in adult and infant humans.

12. The recombinant protein, according to Claim 11, wherein the Gram-negative bacterium is of the genus Neisseria.

13. The recombinant protein, according to Claim 12, wherein the bacterium is Neisseria meningitidis.

14. The recombinant protein, according to Claim 13, which corresponds to the Class II protein of the outer membrane of the bacterium Neisseria meningitidis.

15. The recombinant protein, according to Claim 14, which corresponds to the Class II protein of the outer membrane of Neisseria meningitidis, serogroup B.

16. The recombinant protein, according to Claim 15, which possesses immunological carrier activity.

17. The recombinant protein, according to Claim 15, which possesses immunological mitogenic activity.