CA2047043A1 - Class ii protein of the outer membrane of neisseria meningitidis having immune 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 (MIEP) of Neisseria meningitidis, purified directly from the outer membrane of Neisseria meningitidis, or obtained through recombinant cloning and expression of DNA encoding the MIEP of Neisseria meningitidis, has immunologic carrier as well as immunologic enhancement, cytokine-inducing (such as interleukin-2 (I1-2)) and mitogenic properties.

Description

3~/JWW14 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 Neisseria meningitidis is used as an immunologic carrier in vaccines for human use. OMPC consists of 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 increased antibody response to the antigen results.

., . , ~ J J,~

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. inEluenzaQ, when PRP is covalently linked to OMPC.
O~PC 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, lo some potentially negative aspects of using OMPC in 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 multivalent vaccine would be very high.

OBJECTS OF THE INVENTION
It is an object of the present invention to provide substantially pure Class II protein, the major immunoenhancing protein (MIEP) dexived directly from the outer membrane of Neisseria meningtidis, free from other Neisseria menin~itidis outer membrane components. It is another object of the present invention to provide substantially pure recombinant MIEP of the outer membrane of Neisseria menin~ tidis, produced in a recombinant host cell, completely free of all other Neisseria menin~itidis 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 MIEP purified directly from the i SJ ! 3 outer membrane of Neisseria meningitidis, or recombinant MIEP of Neisseria m nin~itidis produced in a recombinant host cell. Another object of the present invention is to provide a protein which possesses immune mitogenic activity, comprising either MIEP purified directly from the outer membrane of Neisseria meningitidis, or recombinant MIEP of Neisseria menin~itidis produced in a ~ecombinant host cell. Another object of the present invention is to provide a protein which possesses the ability to induce the production of cytokines such as Il-2, comprising either MI~P purified directly from the outer membrane of Neisseria meningitidis, or recombinant MIEP of Neisseria meningitidis produced in a recombinant host cell. An additional object of the present invention is to provide vaccine compositions comprising either the recombinant MIEP, or MIEP purified directly from the outer membrane of Neisseria meningitidis.. 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 (MIEP) of the outer membrane of Neisseria meningitidis, in substantially pure form, free from other contaminating N.
meningitidis outer membrane proteins and LPS. The MIEP of the present invention, whether purified directly from the outer membrane of Neisseria menigitidis cells, or derived from a recombinant host cell producing recombinant MIEP of Neisseria meningitidis, possesses immunologic carrier activity, cytokine (Il-2) inducing activity, and mitogenic : . :

f.~ ~ .' D ~ ,3 activity. The MIEP 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 MIEP of the present invention include viral proteins, bacterial proteins and polysacharides, synthetic peptides, other immunogenic antigens, and weak or lo non-immunogenic antigens.

BRIEF DESCRIPTION OF TXE DRAWINGS
Figure 1 - Antibody responses of adoptive transfer recipients receiving spleen cells primed separately with PRP-DT and MIEP, or OMPC, or IAA-OMPC, 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 MIEP, in vitro. The increase in 3H-thymidine incorporation into cellular DNA ~as measured following exposure of the cells to bovine serum albumin (BSA), PRP-OMPC, OMPC, or MIEP.
Figure 3 - PRP-MIEP conjugates were tested for immunogenicity in mice as well as in~ant rhesus monkeys. Antibody responses were measured by ELISA
and RIA.
Figure 4 - Induction of Il-2 production by mouse spleen cells in culture following exposure to MIEP as an example of the induction of cytokine production. The results were obtained using samples from the three day culture timepoints.

` !

.

3! ` ~ ., DETAILED DESCRIPTION OF THE INVEMTION
It is known that certain substances which by themselves elicit an immune re~ponse which consists of only IgM class antibodies and no memory, can be transformed into fully immunogenic antigens which elicit IgM and IgG anithodies as well 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 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 vi~orous 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.
Helper T-lymphocytes are normally involved in the production by B-lymphocytes, of antibodies ~;

specific Eor a c~rtain type o~ 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-dependen+, 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 arltigens and not 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 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. ~hese molecules are termed "mitogens" and include plant proteins as well as bacterial products. Mitogens cause T and/or B-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.
The production of Il-2 by certain T-helper lymphocytes can cause the stimulation of growth and activity of other lymphocytes. Il-2 production by T-helper cells can be induced when the T cells are activated by certain substances or antigens presented by antigen presenting cells. The effects of Il-2 include, but are not limited to the progression to 34/JWW14 - 7 - ~8160IA

proliferation of T cells, and the proli~eration and differentiation of B cells. Many infectious disease causing agents can, by themselves, elici-t protective antibodies which can bind to and kill, render harmless, or cause to be 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.
o 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 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
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 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, MIEP, possesses the immunologic carrier and immune enhancement properties of OMPC

2~ ,' , ,,3 vesicles, and is effective in purified form, free from other N. meningitidis membrane proteins and lipopolysaccharides.
Applicants have also d:iscovered that MIEP, when chemically coupled to bacterial polysaccharide, functions as well as OMPC in inducing an antibody response to the polysaccharide. Applicants have further discovered that MIEP is the Class II protein of the outer membrane of N. meningitidis. The Class II protein of N. meningitidis is a porin protein lo [Murakami, K., et al., (1989), Infection ~nd Immunity, 57, pp.2318-23]. Porins are found in the outer membrane of all Gram negative bacteria.
While the present invention is e-~emplified by MIEP of N. meningitidis, it is readily apparent to those skilled in the 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 Gram negative bacteria include but are not limited to species of the genera Neisseria, Escherichia, Pseudomonas, ~emophilus, Salmonella, Shigella, Bordetella, Klebsiella, Serratia, Yersinia, Vibrio, and Enterobacter.
MIEP 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 non-viable, immunogenic, weakl~ 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 34/JWWl~ - 9 - 18160IA

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 lo saline solution, tissue culture fluids, or other 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 MIEP
conjugated antigen may be prepared employing 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 extracts 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, r J .. ' ~ 3 rickettsia, allergen7 poison or venom. In all cases, the antigens will be in the forrn in which their toxic or 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, i:n the case of allergens, they will aid in alleviating the symptoms of the allergy due to the specific allergen.
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 allergens, multiple proteins, multiple peptides or combinations of any of the foregoing products can be conjugated to MIEP.
Antigens of particular importance are derived from bacteria including but not limited to B.
pertussis, Lepto S~i ra pomona, and icterohaemorrhagiae, S. paratvphi A and B, .
diRhtheriae, C. tetani, C. botulinum, C. perfrin~ens, C. feseri, and other gas gangrene bacteria, B.
anthracis, Y. ~estis, P. multocida, V. cholerae, Nesseria mÇni~ , N. gonorrheae, Hemophilus influenzae, Treponema ~3~11dum, 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 ç ~

34/JWW14 - 11 - . 18160IA

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 ~irus ~SF4), Western and Eastern equi:ne encephalomyelitis virus, Japanese B. encephalomyelitis, Russian lo Spring-Summer encephalomyelitis, hog cholera virus, Newcastle disease virus, fowl pox, rabies, feline and canine distemper and the like 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 the known a~lergens, 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 are not intended to be limited to any particular types. Examples of such bacterial polysaccharides include Streptococcus ~neumoniae (pneumococcal) types 6A, ~B, lOA, llA, 18C, 19A, 19f, 20, 22F, and 23F, polysaccharides; Group B Streptococcus types Ia, Ib, 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 :
. .

polysaccharides, however, are those capsular polysaccharides selected from the group consisting of H. influenzae serotype b polysaccharides, such as described in Rosenberg et al., J. ~iol. 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 l9F polysaccharide, such as described C. J. Lee et al., Reviews o Infectious 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.
MIEP can be purified from OMPC derived from 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.
MIEP can also be obtained from recombinant DNA engineered host cells by expression of recombinant DNA encoding MIEP. The DNA encoding MIEP
can be obtained from _. menin~itidis cells [Mura~ami, K. et al., (1989), Infection And Immunity, 57, pp.
2318], or the DNA can be produced synthetically using standard DNA synthysis techniques. DNA encoding MIEP
can be expressed in recombinant host cells including but not limited to bacteria, yeast, insect, mammalian or other animal cells, yielding recombinant MIEP.

The preferred methods of the present invention ~or obtaining MIEP are purification of MIEP from OMPC and recombinant DNA expression of DNA encoding MIEP
derived from _. meningitidis.
Purified MIEP was prep~red from OMPC
vesicles by sodium dodecylsulfate (SDS) lysis of the vesicles followed by SDS polyacrylamide gel electrophoresis (PAGE). The MIEP was eluted from the gel, dialysed against a high pH buffer and concentrated. Standard methods of polyacrylamide gel electrophoresis can be utilized to purify MIEP from OMPC vesicles. Such 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, E., (1986), Purification Of Microgram Quantities Of Proteins By Polyacrylamide Gel Electrophoresis, in Methods of Protein 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.
MIEP prepared in this manner is readily suitable for conjugation to antigens derived Erom bacteria, viruses, mammalian cells, rickettsia, allergens, poisons or venoms, fungi, peptides, proteins, polysaccharides, or any other antigen.
Recombinant MIEP can be prepared by expression o:E genomic N. meningitidis DNA encoding '; '~ `
,:

-. ..." ....

MIEP in bacteria, for example E. coli or in yeast, for example S. cerevisiae. To obtain genomic DNA
encoding MIEP, 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 Imethod 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 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), 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 MIEP DNA in the N. meningitidis genomic DNA can be determined from the amino acid sequence of MIEP or by reference to the determined DNA sequence for the Class II major membrane protein of _. meningitidis [Musakami, k. et al., (1989), Infection and Immunity, 2~ 57, PP. 2318]
Recombinant MIEP can be separated from other cellular proteins by use of an affinity column made with monoclonal or polyclonal antibodies specific for MIEP. These affinity columns are made by adding the antibodies to Affigel-10 (Biorad), a gel support .

~? ,~

34/J~W14 - 15 - 1~160IA

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 HCl (pH 2.~ to removle any non-conjugated an-tibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing MIEP 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-MIEP conjugates or any other antigen-MIEP 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 allergens. The antigen-MIEP conjugates are coupled through bigeneric spacers containing a thioether group and primary amine, which form hydrolytically-labile covalent bonds with the polysaccharicle and the MIEP. Preferred conjugates according to this invention, however, are those which may be represented by the ~ormulae, Ps-A-E-S-B-Pro or Ps-A~-S-E~-B~-Pro, wherein Ps represen-ts a poly-saccharide or any other antigen; Pro represents the bacterial protein MIEP; 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-~-S-B, S is sulfur; E is the transformation product of a thiophilic group which has been reacted with a thiol lO group, and is represented by -C(CH2)pN ~ or -CCH-, wherein R is H or CH3, and p is l to 3; A is WH
-CN~CH2)mY(cH2)n wherein W is 0 or NH, m is 0 to 4, n is 0 to 3, and Y
is CH2,0,S,MR', or CHC02X, 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 0 or S, then m is greater than 1 and n is greater than l; and B is ,r~. Y,j ~ "" ~3 ~(cH2)pclH(cH2)qD- ~

Z O
wherein q is O to 2, Z is NH2, NE[CR', COOH, or H, where R~ and p are as defined above, and D is C, NR', ~ O O
or N-C(CH2)2C. Then in the spacer, A~-S-E~-B', S

is sulfur; A~ is -CNH(CH2)aR"-, wherein a is 1 to 10 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 represented by -CH-, wherein R is 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 !
. . . ..
, 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-B-Pro, accord-ing to this invention may contain spacers whose com-ponents include derivatives of, inter ~1~: carbon dioxide, 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-carbo~y-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 of, inter alia: carbon dioxide and S-carboxy-methylcysteamine; carbon dioxide and S-(~-carboxy-ethyl)cysteamine; carbon dioxide and S-carboxy-methylhomocysteamine; carbon dioxide, S-(succin-2-yl)cysteamine, and glycine; and carbon dioxide andS-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 J ~
,, ' f, , ' ,~

bis-nucleophile, and inally, if necessary, further (d) functionalizing 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 ei~her 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 procesæ of this invention also inc~udes selection of a nucleophile or bis-nucleophile which will react with the activated polysaccharide to form 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 for electrophilic reagents with the hydroxyls of water in an aqueous solution, the polysaccharide should be dissolved in non-agueous (non-hydroxylic) solvents. Suitable solvents include dimethyl-formamide, dimethylsulfoxide, dimetAylacetamide, 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 o~ H. influenzae type b, which are a 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-molecules may be replaced by large hydrophobic cations, such as tri- or tetra-(Cl- to C5)alkyl-ammonium, l-azabicyclo[2.2.2]oc~ane,1,8-diazabicyclo [5.4.0]~ndec-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 B polysaccharide has been converted into the tetrabutyl-ammonium salt, then dissolved in dimethylsul~oxide (Egan et al., J. Amer. Chem. Soc., 104, 2898 (1982)), but this product is no longer antigenic, and therefore useless for preparing 34/J~14 - 21 - 18~60IA

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 rorm, 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 activated by reaction with a bifunctional reagent at about 0-50C, while stirring for ten minutes to one hour, with the crucial weight ratio of activatlng agent to polysaccharide in the 30 range of 1:5 to 1:12. In the past, this activation 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 posslble according to the present invention, this reagent is poorly utilized in activation of polysaccharides and is not preferred.
Instead, preferred bifunctiona:L reagents for lo activating the polysaccharide include carbonic acid o 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 ls 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 H
example, HN(CH2)mY(CH2)n-NH, wherein m is 0 to 4, n .

~?, '~ C 1'`' ~ ,~

is O to 3, and Y is C~I2, 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, a~ld n is greater than 1, in a gross excess of amine (i.e., for example, 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 hour then kept for 15 minutes to one hour at about 17 to 40~C.
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., ~oppe 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.

:, ~ ;
.

?

The last step in preparing the polysaccharide, the further functionalization, if necessary, of the polysaccharide, may take the form of either reacting the nucleophile-functionalized 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 ~-haloacetyl or a-halopropionyl, OR
derivative such as XCCHX (wherein R is ~ or CH3; X is Cl, Br or I; and X' is nitrophenoxy, dinitrophenoxy, pentachlorophenoxy, pentafluorophenoxy, halide, O-(N-hydroxysuccinimidyl) or azido), particularly chloroacetic acid or ~-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, O. Keller et al, Helv. Chim. Acta., 58, 531 (1975)) to produce maleimido groups, ~( CH2) pN~l wherein p is 1 to 3; with a 2-haloacetyling agent, f,~ . -.. 3 , ".; 3 such as p-nitrophenylbromoacetate; or with an a-haloketone carboxylic acid derivative, e.g., HO2C ~CCH2Br ~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 example, acylating reagents, such as thiolactones, e.g., R4C ~ I CCH2~p, O , ~ I
S

wherein R4 is Cl- to C4-alkyl or mono- or bicyclic aryl, such as C16H5 or CloH13, and p is 1 to 3;
NHCoR5 -O3SSCH2~CH2~mCH-COX', wherein m is 0 to 4, R5 is Cl-- , ::
, , ~.

:' , : ' , !? l~

to C4-alkyl or C6H5, and X' is as defined above, followed by treatment with HSCH2CH20H; or NHCoR5 C2H5-S-S-CH2(CH2)mCHC0X', wherein m, R5 and X' are as defined immediately above, then treat-ment with dithiothreitol. Such reactions are carriedout in a nitrogen atmosphere, at about 0 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-10 derivatized polysaccharide may be reacted with ~
20to produce an appropriately-functionalized polysac-charide.
By these steps then, covalently-modified polysaccharides of the forms, Ps-A-E*- or Ps-A'-SH-, wherein E* is -CCHX or -C(C~2)P

, ` . ,~

34/J~14 - 27 - 18160IA

and A, A~, R, X and p are as defined above, are produced.
Separate functionalization of the protein to be coupled to the polysaccharicle, involves reaction of the protein with one or more reagents to generate a thiol group, or reaction of t:he protein with one or more reagents to generate an e].ectrophilic (i.e., thiophilic) center.
In preparation for conjugation with an electrophilic-functionalized polysaccharide, the lo protein is reacted in one or two steps with one or mor~ 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. Acta, 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.
When E'B-' is ~ 11 ~ N(CH2)pC~
T~
o .

,r~ p~ j ,. ) /, , ~

the conditions and method of preparing the functionalized protein are as cliscussed 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 generating an electrophilic center, such acylating agents including, for example, Il l 11 10 XCH2C-X' and XCH - CX', wherein X and X' are as defined above; and ~CO\ O
ll N (CH2)aC -X' ~`CO /
wherein X' is as defined above. Suitable proteins with.electophilic centers also include, for example, those prepared by acylation o~ the pendant lysyl amino groups with a reagent, such as activated 2s maleimido acids, for example, ~L~ o ~
NOC(CH2)nN

O O

C~ i~ n ~

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 11.
Formation of the conjugate is then merely a matter of reacting any of the covalently~modified polysaccharides having pendant electrophilic centers with of the bacterial protein MIEP having pendant thiol groups at a pH of 7 to 9, in approximate equiweight ratios, in a nitrogen atmosphere, for from six to twenty-four hours at from about 17 to 40C, to give a covalent conjugate. Eæamples of such reactions include:
OH O NHCOCH~
Ps-CNCH2CH2CH2CH2NHCCH2Br + HSCH~CH2CHCO-Pro PsCNCH2CH2CH2CH2NHCCH2SCH2CH2CHCOPro, wherein an activated polysaccharide which has been reacted with 4-bromoacetamidobutylamine is reacted with a protein which has been reacted with N-acetyl-homocysteinethiolactone, to form a conjugate, and:

' `

.
: ,;

~, ~ ,, i /,l " 3 OH
PscN~-NccH2N IJ

BCH2CH2NHCCH2CH2CPro ~CNHY"NHCCHz-N ~ / CH2CH2NHCCH2CH2CPro (where Y" is a C2-C8alkyl radical), wherein an amino-derivatized polysaccharide which has been reacted with activated maleimido acids is reacted 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 MI~P ~aving pendant electrophilic centers to give a covalent conjugate. An example of such a reaction is:
O O O H
Il 11 ll I
PsCNHC~I2CH2SH ~ ProCCH2C:EI2C-N(CH2)4NHCOCH2Br . ~, OH OH O O
PSCNC~2CH2SCH2CN~C~32~4NHCCH2CH2CPro, -f, ~

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 electrophi:Lic 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 whicll 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 20C, 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.

., .

~ f~

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 contribution of the amino acids of the protein may be removed, if necessary, by comparison with the appropriate amino acid standard for the protein lo involved, with the remaining amino acid value reflecting the covaléncy 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 15 monitor purification procedures to mark the enhancement of concentration of the biologicallyactive components. In the above examples, hydrolysis o~

20 PsCNCH2CH2CH2CH2NHCCH2SCH2CH2CHCOPro results in the release of S-carboxymethylhomocysteine, H02CCH2SCH2CH2CHC02H; hydrolysis of ~ 33 o O ~ o o PgCNHY~NHCCH N
Z ~ ~CH~CH2NHCCH2CH2CPro O s results in the release of the aminodicarboxylic acid, -EO2CCE2~HSCH2CH2NE2; and hydrolysis of ~02C
OH OH O O
PSCNCH2CH2SCH2CN(CH2)4NHCC~2CH2CPro results in the release of S-carboxymethylcysteamine, E2NCH2CE2SCH2C~2H by cleavage of the Ps-A-~-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.

r;

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 MIEP to 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 MIEP 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 MIEP or OMPC were transferred, indicating that T cell recognition of OMPC resides in the MIEP moiety.
PRP-MIEP 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.

~: . ; ..

: . .

- .':

34/JWWl~ - 35 - 18160IA

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 ~Ig) of an lo antigen (e.~. PRP) in the MIEP-conjugate form oE each of the conjugates being administered per dose. The use of an adjuvant (e.g., alum) is also intended to be within the scope o~ this invention. Passive protection may be accomplished by injecting whole antiserum obtained from animals previously dosed with the MIEP-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 stabil;ty, these conjugates may also be lyophilized in the presence of lactose ~for example, at 20 ~glmL of PRP/4 mg/mL
lactose) prior to use.
A preferred dosage level is an amount of each of the MIEP-conjugates, or derivative thereof to be administered, corresponding to between approximately 2 to 20 ~g of PRP in the MIEP-conjugate form for conjugates of H. 1 uenzae serotype B
polysaccharide, in a single administration. If necessary, an additional one or two doses of the lO MIEP-conjugate, or derivative thereof, of the H.
influenzae serotype B polysaccharide in an amount corresponding to between approximately 2 to 20 ~g of PRP in the conjugate ~orm, 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~ Fer~entation 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 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 a-t -70C. 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% C02 for 18 hours after which time the growth was harvested into 100 mL of 10% skim milk medium, aliquots were ta~en 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 a~ter 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 f. ~ t~

as an inoculum for 3 250 mL flasks of Gotschlich's yeast dialysate medium at pH 6.35. The OD660 was adjusted 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 of 1.28 resulted.
lo 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 arter 2 hours.
800-Liter Production Fermenter Approximately 40 liters of seed culture were used to inoculate a sterile 800 liter fermenter containing 568.2 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 pH 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 "; ` ~ 3 -Fraction A
L-glutamic acid 1.5 g/liter NaCl 6.0 g/liter 5 Na2HPO4.anhydrous 2.5 g/liter NH4C1 1.. 25 g/liter KCl 0.09 g/liter L-cysteine HCl 0.02 g/liter Fraction B ~Gotschlich's Yeast 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 HlOSM 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.

, ~3 :. .~ i , 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 weight of the cell paste after phenol treatment was 3.875 kg.

B. OMPC Isolation Step 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).

~ ~ Extraction An equal volume of 2X TED buffer [0.1 M TRIS
0.01 M EDTA Buffer, p~ 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 ~ *

. :

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 ree~tracted in TED buffer as described above. The supernatants were pooled and stored at 4C.

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

SteP 4. Collection and Washihg of the OMPC
The retentate from Step 3 was centrifuged at about 160,000 x 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 Buffer (190 mL buffer; 20 mL/g pellet) Step 2 and Step 4 were repeated twice (skipping Step 3).

SteP 5. Recovery of OMPC Product The washed pellets from Step 4 were suspended in 100 mL distilled water with a glass rod ~ ` ~' ` ,3 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.

_XAMPLE_2 Preparation of H. Influenzae Type b Capsular Polvsaccharide (PR~) Inoculum and Seed Development 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 37~C in a candle jar, the growth from each plate was resuspended in 1-2 mL Haemophilus inoculum medium, and pairs of slants were pooled.

`"`'"' ' . :

~ ~, J,l.

Haemophilus Inoculum Medium . . _ g/Liter Soy Peptone 10 NaCl 5 NaH2P04 3.1 Na2HP04 ~3.7 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 ~lasks (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 l.0 liter of complete Haemophilus 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

Complete Haemophilus Seed And Produc-tion Medium Per liter NaH2P04 3.1 g/L
Na2~P04 13.7 g/L
Soy Peptone 10 g/L
Yeast extract diafiltrate (1) 10 g/L
lo K2HP04 . 2.5 g/L
NaCl 5.0 g/L
Glucose (2~ 5.0 g/L
Nicotinamide adenine 2 mg/L
dinucleotide (NAD) (3~
Eemin (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 ~ , - ~ ' '' ,~`' ` ) , ,~ i ,' ,J

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 sterili7.ed by passage through a (0.22 filter) and added asepticaliy just prior to inoculation.
lo (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 fina 15 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 2~ described above) and 17 mL UCON B625 antifoam agent.
The pH at inoculation was 7.4.
D Stage: 800 Liter Production Fermenter-Approximately 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 p~ levels ~J3~

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

HARVEST AND INACl'IVATION
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 ~ f~ ~ 5,~ " '~,7 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 rpm 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 o 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% ethanol-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 CaC12.2H20 was added, and the mixture was incubated at 4C for 15 minutes. The vessel was then rinsed with 2 liters of 1 M CaC12.2H20, resulting in about 50 liters final Volume.

.~ , .

C~ r~ 7 ~

34/JWW14 - 4~ - 18160IA

23% ETHANOL PRECIPITATION
The 50 liters of CaC12 e~tract 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 mi~ture was collected by passage through a Sharples continuous flow centrifuge at 4(,. The supernatant was collected and the pellet was discarded.

38% ETHANOL PRECIPITATION AND

The 25% ethanol-soluble supernatant was brought to 38% ethanol by the addition of 13.9 liters of ~5% 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 f~ow centrifuge at 15,000 rpm (flow rate of 0.2 lite-rs 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 o~ 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 ~ ,A ~ J ~

absolute ethanol and two 2 liter portions of acetone. The material was then dried, in vac~, 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 stèp (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 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 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 cartridges, 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.

~' s ~

35/JWW15 - 50 ~ 18160IA

67~/o ETHANOL PRECIPITATION
0.81 liters of 2.0 M CaC12 was added to the 31.5 liters o~ dialysate from the previous step (final CaC12 concentration was 0.05 M) and the solution was 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 lO 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 Buchner 15 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 f dry powder. The yield from all phenol extractions was pooled resulting in a total of 212.6 grams of dry powder.

ULTRACENTRIFUGATION IN 29% ET~ANOL
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
CaCl~), 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 .~ , . ' ' :, .

.

~ J
,, ,, ~ .:: ~ ) 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.~2 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 precipltate 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 ~itu, with two fresh 0.5 liter portions and one 1 liter portions f 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, ln vacuo, at 4C (for 25 hours). The final yield of the product was 79.1 grams dry weight.

Cloning of Genomic DNA Encoding MIEP.
About 0.1 g of the phenol inactivated N.
menin~itidis cells (see Example 1) was placed in a fresh tube. T:he phenol inactivated cells were 35/JWW15 - 52 - 18~60IA

resuspended in 567 ~L of TE buffer [lOmM TRLS-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 bromide (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 centriruged 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.
15 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 centrlfuged 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. m~Din~i~idi~
genomic DNA.
Two DNA oligonucleotides were synthesized which correspond to the 5' end of the MIEP gene and to the 3' end of the MIEP gene [Murakami, E.C. et al., (1989), Infection and Immunity, 57r pp.2318-23]. I'he sequence of the DNA oligonucleotide specific for the 5' end of the MIEP gene was:

r ~ 3 5 '-ACTAGTTGCAATGAAAAAATCCCTG-3 '; and for the 3 ' end of the MIEP gene was: 5 '-GAATTCAGATTAGGAATTTGTT-3 ' .
These DNA oligonucleotides were used as primers for polymerase chain reaction (PCR) amplification of the MIEP 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 MIEP DNA was then digested 10 with the restriction endonucleases ~ and EcoRI.
The 1.3 kilobase (kb) DNA fragment, containing the complete coding region of MIEP, was isolated by electrophoresis on a 1.5% agarose gel, and recovered from the gel by electroelution [Current Protocols in 15 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 SpeI and EcoRI. The gel purified SpeI-EcoRI MIEP DNA
20 was ligated into the SpeI-EcoRI pUC-19 vector and was used to transform E. coli strain DH~. Trans~ormants containing the pUC-19 vector with the 1.3 kbp MIEP
DNA were identified by restriction endonuclease mapping, and the MIEP DNA was sequenced to ensure its identity.

EX~MPLE 4 Construction of the pcl/l.GallOp(B)AD t vector:
The Gal 10 promoter was isolated f~om plasmid YEp52 [Broach, et al., (1983) in Experimental Manipulation of Gene E~pression, Inouye, M~Ed>

' s;,'"~,)?
35/J~15 - 54 - 18160IA

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 pGAP.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 ~ . The two fragments were ligated with T4 DNA
ligase to the gel purified pucl8~Hind III vector (the Hind III site was eliminated by digesting pUC18 lo with Hind III, blunt-ending with the Klenow ~ragment 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 15 junction~
The unique ~ LIII cloning site of pGallO.tADHl was changed to a unique BamHI cloning site by digesting pGallO.tADHl with Hind III, gel purifying the cut DNA, and ligating, using T4 DNA
ligase, t~ the following Hind III-BamHI linker:
5'-AGCTCGGATCCG-3' 3'-~CCTAGGCTCGA-5'.

The resulting plasmid, pGallO(B)tADHl, has deleted the Hind III site and generated a unique BamHI cloning site.
The GallOp.tADHl fragment was isolated from pGallO(B)tAD~l by digestion with SmaI and S~hI, 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, .. ,: ;
- .

, . .

35/JI~W15 - 55 - 18160IA

pp.4642-4646] was digested with Sj~lI, 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 trans~orm 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 by digestion with HindIII and BamHI.

Construction of a Yeast MIEP Expression Vector with MIEP + Leader DNA Sequences A DNA fragment containing the complete coding region of MIEP was generated by digestion of pUC19.MIEP #7 with ~ and EcoRI, gel purification of the MIEP DNA, and blunt-ended with T4 DNA
polymerase.
The yeast internal expression vector 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 MIEP 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 was designed to be homologous with sequences overlapping the MIEP-vector junction. Preparations of DNA were made from hybridization positive transformants and digested with ~1 and SalI to verify that the MIEP 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 MIEP coding region.
Expression of MIEP by the transformants was detected by Western blot analysis. Recombinant MIEP
produced in the transformants comigrated on polyacrylamide gels with MIEP purified from OMPC
vesicles, and was immunologically reactive with 15 antibodies specific for MIEP.

Construction of yeast MIEP expression vector with a 5'-Modified MIEP DNA Sequence.
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 MIEP (beginning with Asp at position +20) and a KpnI 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 pUC19MIEP42#7 as the template and the following DNA
oligomers as primers:
5 CTM GCTT MCAAAATGGACGTTACCTTGTACGGTACAATT3 , and 5 ACGGTACCGM GCCGCCTTTCAAG3 .

.

: .

~C? ~

To remove the 5I region of the MIEP clone, plasmid pUC19MIEP42#7 was digested with K~al and ~indIII and the 3.4 Kbp vector fragment was agarose gel purified. The 280 bp PCR fragment was digested with Kpnl and ~indIII, agarose gel purified, and ligated with the 3.4 Kbp vector fragment.
Transformants of E. coll ~101 (BRL) were screened by DNA oligonucleotide hybridization and the DNA from positive transformants was analyzed by restriction lO 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, 15 and the entire open reading frame (ORF) of MIEP
beginning at the Asp codon (amino acid ~20).
The yeast MIEP expression vectors were constructed as follows. The pGAL10/pcl/1 and pGAP/pCl/l vectors [Vlasuk, G.P., et al., (1989) 20 J.B.C., 264, pp.l2,106-12,112] were digested with BamHI, flush-ended with the Klenow fragment of DNA
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 ORE of MIEP are forming pGallO/pcl/MIEP and pGAP/pGAP/pCl/MIEP.
Saccharomyces cerevisiae strain U9 (gallOpgal4-) were transformed with plasmid pGallO/p/pCl/M~[EP. Recombinant clones were isolated and examined for expression of MIEP. Clones were 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 MIEP from the GallO promoter. The cells were grown for an additional ~5 hours following galactose induction to an O.D.600 of about 9Ø The cells were then harvested by centrifugation. The cell pellet was washed wi-th distilled water and frozen.

Western Blot For Recombinant MIEP:
To determine whether the yeast was expressing MIEP, Western blot analysis was done.
Twelve percent, 1 mm, 10 to 15 well Novex Laemmli 15 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 MIEP. 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 5% bovine serum albumin in phosphate buffered saline for 1 hour, after which 15 mL of a 1:1000 dilution of rabbit anti-MI~P antiserum (generated by immunization with gel purified MIEP

using standard procedures) was added. A~ter overnight incubation at room temperature 15 mL o~ a 1:10~0 of alkaline phosphatase conjugated goa-t 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).

0 Bacterial Expression Of Recombinant MIEP
Plasmid pUC19-MIEP containing the 1.3 kilobase pair MIEP gene insert, was digested with restriction endonucleases SpeI and EcoRI. The l.lkbp fragment was isolated and purified on an agarose gel 15 using standard techniques known in the art. Plasmid pTACSD, containing the two cistron TAC promoter and a unique ECORI site, was digested with ECQRI. Blunt ends were formed on both the 1.3 kbp MIEP DNA and the pTACSD vector, using T4 DNA polymerase (Boehringer 20 Mannheim) according to the manufacturer's - directions. The blunt ended 1.3 kbp MIEP DNA was ligated into the blunt ended vector using T4 DNA
ligase (Boehringer Mannheim) according to the manufacturer's directions. The ligated D~A was used 2s to transform ~. coli strain DH5aIQMAX (BRL) according to the manufacturer's directions. Transformed cells were plated onto agar plates containing 25 ug kanamycin/mL and 50 ug penicillin/mL1 and incubated for about 15 hours at 37 C. A DNA oligonucleotide 3~ with a sequence homologous with MIEP was labelled with 32p and used to screen nitrocellulose filters ~ ~ 1 3 ~ J ~

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

1s EXAMPLE ~
Preparation of Purified MIEP from OMPC Liposomes or From Recombinant Cells by Polyacrylamide Gel Electrophoresis Acrylamide/BIS (37.5:1~ gels, 18 x 14 cm, 3 20 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 wide) and stained with Coomassie/cupric acetate (0.1%). The strip was destained until the MIEP band (about 38 Kd) became visible. Tlle strip was then placed into its original gel pos:ition and the MIEP
area was excised from the remainder of the gel using a scalpel.
The excised area was cul 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 lo 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. APter dialysis the 15 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.

Carrier activity of MIEP in covalent PRP-OMPC
conjugate Immunizations: Male C3~/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 (P~S). A second group oP
male C3H-HeN mlce, received either 17 ~g of MIEP, 17 ~g of OMPC, or 17 ~g of OMPC-IAA (OMPC derivatized , ~ :
~, 35/JWW15 - 62 - 18160.~A

with N-acetyl homocysteine thiolactone, and capped with iodoacetamide). Cell donors for acloptive transfer experiments were twice immunized IP, 21 days apart, and spleen cells were collected 10 days after the second immuni2ation. Adoptive transfer recipients were male C3H/HeN mice given 500R total body gamma-irradiation ~rom a 137Cs source and immediately reconstituted by intravenous injection of 8 x 107 spleen cells from each of two syngeneic lo donors separately primed with PRP-DT, and OMPC, MIEP, or OMPC-I M . Control mice received 8 x 107 spleen cells from one donor mouse primed with PRP-OMPC alld an equal number of spleen cells from an unprimed donor mouse.
ELISA for anti-PRP antibody: Reactive amines were introduced into purified E. _nfluenzae PRP by treatment with carbonyldiimidazole and reaction with butanediamine as described by Marburg et al., U.S. Patent 4,882,317. This derivati~ed PRP
20 was chromatographed on Sephadex G-25 in 0.lM sodium bicarbonate buffer, pH 8.4. N-hydro~y-succinimidobiotin (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 2s for 4 hours at ambient temperature (about 25-28C).
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, ,fT` ~

overnight at ambient temperature and 100% humidi-ty.
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 T~S-T plus 0.1% gelatin (blocking buffer) at ambient temperature and 100% humidity for 1 hour. Plates were blotted without washing, 50 ~glwell 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 T~S-T prior to sample lO addition. Samples were added in two-fold serial dilutions in blocking buffer, and incubated for 2 hours at ambient temperature and 100~/o humidity. The plates were then washed 3 times with T~S-T, and appropriate alkaline-phosphatase conjugated 15 anti-immunoglobulins diluted in blocking buf~er 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) (~RL), IgG2b (gamma) (Southern ~iotechnology Associates, ~irmingham, 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
diethanolamine, Kirkegaard and Perry, Gaithersburg, ~). ~ilutions 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 ', : .

' r7 ~

which gave a positive reaction in the ELISA as described above. Logarithms of reciprocal titers were compared between treatment groups by two-way analysis of variance [Lindeman, R.~. et al., (1980~, Introduction to Bivariate and Multivariate Analysis, Scott Foresman (pub.), New York].
RIA for anti-PRP antibo~y~uantitation.
The experimental samples of serum to be tested for the amount of anti-PRP antibodies were lo diluted 1:2, 1:5, and 1:20, using fetal calf serum as the diluent. 25 ~L o~ each diluted serum sample was 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 15 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 20 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 ~ntiserum containing a known guantity of anti-PRP antibodies was prepared as described above and were assayed concomitantly with the experimental serum samples. The quantity of anti-PRP antibodies in the standard curve was between 14 ~g/mL as the highest quantity of antibodies and .

0.056 ~g/mL as the lowest quantity o~ 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.
Antibody responses of adoptive transfer recipients: Recipients o~ spleen cells primed separately with PRP-DT, and either MIEP or OMPC or 10 IAA-OMPC, responded to immunization with PRP-OMPC by production of equivalent amounts o~ serum IgGl and IgG2a anti-PRP antibody within 4 days (see Figure 1). Irradiated mice reconstituted with spleen cells which were carrier-primed with MIEP or OMPC or 15 IAA-OMPC, had significantly higher IgG~ (p<O.OOl) and IgG2a (p<0.04) anti-PRP antibody titers after immunization with PRP-OMPC than control mice, given PRP-DT-primed but not OMPC-primed spleen cells. The serum antibody responses to immunization with 20 PRP-OMPC in mice given spleen cells primed separately with PRP-DT and either MIEP or OMPC or IAA-OMPC we~e 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 2s antibody response was seen when irradiated mice reconstituted with PRP-DT-primed and either MIEP or OMPC-primed spleen cells were immunized with PRP
without a protein carrier. Statistical analysis was done by two-was analysis of variance ~ANOVA~
[Lindeman, R.H. et al., Introduction to ~ivariate and Multivariate Analysis, (1980), Scott Foresman, New York].

:, ~. ' These results demonstrate that MIEP
functioned in mice as well as OMPC to induce a carrier T helper cell response for the generation of anti-PRP IgG antibodies.
s Mitogenic Activity~ of MIEP
MIEP purified from N. meningitidis OMPC was lO 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 15 passed through a sterile, fine mesh screen to remove the stromal debris, and suspended in K medium [RPMI
1640 (GIBCO) plus 10% fetal calf serum (Armour), 2 mM
Glutamine (GIBCO), 10 mM Hepes (GIBCO), 100 u/mL
penicillin/100/~g/mL streptomycin (GI~CO), and 50 ~M
20 ~-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), p~
2~ 7.2] at room temperature, 0.1 mL cells/mL buffer for two minutes. Cells were underlayered with 5 mL o~
fetal calf serum and centrifuged at 4,000 x g for 10 minutes, then washed with K medium two times and resuspended in K medium at 5 x 106 cells/mL. These cells were plated (100 ~L/well) into 96 well plates along with 100 ~L of protein or peptide sample, in triplicate.

The MIEP of N. meningitidis was purified as previously described in Example 7. Control proteins included bovine serum albumin, PRP-OMPC and OMPC
itself, and lipopolysaccharide (endoto~in). 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 o 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 3~-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, MIEP 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 MIEP was free of detectable LPS, measured by rabbit pyro~enicity 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.

. ' ~ , ....
.

:' '.. ~J ~1,3 '.`~ ) _XAMPLE 11 Conjugation of _. influenzae type-b PRP
polvsaccharide to N. meningitidis MIEP
Chemical conjugations were conducted according to the method disclosed in U.S. Patent number 4,882,317.
10 mg of MIEP in 3 mL of 0.1 M borate buffer, pH 11.5, was mixed with 10 mg of ethylenediamine tetraacetic acid disodium salt (~DTA, 10 Sigma chemicals~ and 4 mg of dithiothreitol (Sigma Chemicals). The protein solution was flushed thoroughly with N2. 125 mg of N-acetylhomocysteinethiolactone (Aldrich Chemicals) was added to the MIEP solution, and the mixture was 15 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).
~or conjugation of MIEP to PRP, a 1.5 fold excess (wt/wt) of bromoacetylated H. influenzae serotype b PRP was added to the MIEP solution and the pH was 25 adjusted to 9 - 9.5 with 1 N NaOH. The mixture was allowed to incubate under N2 for 6 to 8 hours at room 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 18 hours under N2 at room temperature. The conjugate solution was acidified to between pH 3 to 4 with 1 N HCl, and ~ r? ~ 3 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 2 50 cm, Pharmacia) and the conjugate was eluted with PBS. The void volume peak which contains the polysaccharide-protein conjugate (PRP-MIEP), was pooled. The con~jugate solution was then filtered through a 0.22 ~ filter for sterilization.

Demonstration of Immunogenicitv of PRP-MIEP conjugates Immunizations: Male Balb/c mice (Charles River~ Wilmington, MA) were immunized IP with PRP
15 covalently conjugated to MIEP 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 [Anderson, M.E. et al., (1985), J. Pediatrics, 107, pp. 346-351] (2.5 ~g 20 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, 25 were immunized with PRP-MIEP 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 30 weeks.
Antibody responses were measured by the 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-MIEP conjugates are shown in Figure 3.
The results show that PRP-MIEP conjugates are capable of generating an immune response in mice consisting of IgG anti-PRP antibody and a memory lo response. This is in contrast to the PRP-CRM and PRP-DT which do not elict measurable anti-PRP
antibody. Thus, MIEP functions as an immunologic carrier protein for PRP and is capable of engendering an anti-PRP antibody response when covalently lS conjugated to the PRP antigen. Purified MIEP is therefore an effective immunologic carrier protein replacing the heterogeneous OMPC in construction of bacterial polysaccharide conjugate vaccines.

Interleukin-2-induction following exposure to MIEP.
CTLL cells (commerically available from the American Type Culture Collection designated ATCC TIB
214)[Gillis, S., and Smith, K. (1977) Nature, 268, pp.154-156] were maintained in RPMI 1640 (GIBCO), supplemented with 10% Defined Fetal Bovine Serum (HyClone), L-glutamine 2 ~M (GIBCO), 100 u/mL
Pennicillin, 100 ~M Streptomycin (GIBCO), 2-Mercaptoethanol 20 ~M (BioRad), 10% Rat T-Cell Polyclone (Collaborative Research, Inc.). CTLL cells were seeded every 2 to 3 days at 1-2 X 104 cells/mL.

i ,f " j ,' Supernatants to be tested for IL-2 were prepared by culturing peripheral blood lymphocytes at 4 X 106 cells/mL with MIEP at 50 ~g/mL, :PRP-OMPC at 50 ~g/mL, Bovine Serum Albumin (Pierce) at 50 ~g/mL, or medium alone in a 6-well plate (CoStar), containing equal volumes of each. Supernatants were harvested at intervals of 2, 3, or 4 days in culture and frozen until assay. The assay for IL-2 was performed as described by Gillis et al., J. Immunol. 120 lO pp.2027-2032 (1978). Two-fold serial dilutions of supernatants ~ere prepared, and 100 ~L placed into wells containing 4,000 CTLL cells which had been starved for one hour in the above medium without Rat T-Cell Polyclone. All samples were prepared in 15 triplicate in a 96-well plate ~CoStar). Plates were incubated overnight at 37C, 8% CO2. Each well was pulsed with l~Ci 3H-Thymidine (Amersham) for 10 hours, harvested onto a ~etaPlate filtermate (Pharmacia/LK~), and read in a ~etaPlate 1205 ~LKB
Instruments). An IL-2 standard curve was determined simultaneously using Recombinant Xuman IL-2 (Cellular Products1 Inc.). Quantitative determinations of IL-~were calculated using the methodology of Gillis et al., J. Immunol. 120: pp.2027-2032 (1978). Probit analysis using the Recombinant IL-2, defined 1 U/mL
of IL-2 as 50% of maximal 3H-Thymidine incorporation. Test supernatants were also expressed as a percent of maximum 3H-Thymidine incorporation from which the U/mL of IL-2 could be determined.
Figure 4 shows the result of this experiment demonstrating the ability of MIEP to induce an increase in Il-2 concentration. The results shown in Figure 4 were derived from the 3 day timepoint.

Claims (15)

1. A method for increasing the immune response to antigens comprising the administration of a protein, in substantially pure form, purified from the outer membrane of a Gram-negative bacterium.

2. The method according to Claim 2, comprising the administration of the Class II protein of the outer membrane of Neisseria meningitidis, serogroup B, in substantially pure form.

3. The method according to Claim 2 which also comprises the administration of an antigen or antigens.

4. The method according to Claim 3 wherein the antigens are derived from bacteria, viruses, mammalian cells, fungi, rickettsia, allergens.
poisons or venoms, synthetic peptides, and polypeptide fragments.

5. A method for increasing the immune response to antigens comprising the administration of a recombinant protein of the outer membrane of a Gram-negative bacterium, produced in a recombinant hose cell.

6. The method according to Claim 5, comprising the administration of a recombinant Class II protein of the outer membrane of Neisseria meningitidis serogroup B, produced in a recombinant host cell.

7. The method according to Claim 6, which also comprises the administration of an antigen or antigens.

8. The method according to Claim 7, wherein the antigens are derived from bacteria, viruses, mammalian cells, fungi, rickettsia, allergens, poisons or venoms, synthetic peptides and polypeptide fragments.

9. A method for increasing the level of cytokines in a mammal comprising the administration of a protein, in substantially pure form, purifed from the outer membrane of a Gram-negative bacterium.

10. The method according to Claim 9, comprising the administration of the Class II protein of the outer membrane of Neisseria meningitidis, serogroup B, in substantially pure form.

11. The method according to Claim 9 comprising the administration of a recombinant Class II protein of the outer membrane of Neisseria meningitidis.
serogroup B, produced in a recombinant host cell.

12. A method for increasing the level of interleukin - 2 in a mammal comprising the administration of a protein in substantially pure form, purified from the outer membrane of a Gram-negative bacterium.

13. The method according to Claim 12 comprising the administration of a recombinant protein of the outer membrane of a gram-negative bacterium, produced in a recombinant host cell.

14. A method for increasing the level of interleukin-2 in a mammal, comprising the administration of the Class II protein of the outer membrane of Neisseria meningitidis, serogroup B, in substantially pure form.

15. The method according to Claim 14 for increasing the level of interleukin-2 in a mammal, comprising the administration of a recombinant Class II protein of the outer membrane of Neisseria meningitidis serogroup B, produced in a recombinant host cell.