PT98381A - METHOD FOR INCREASING THE RESPONSE IMMUNE TO NEISSERIA MENINGITIDIS EXTERNAL MEMBRANE CLASS II PROTEIN ANTIGENS - Google Patents

Description

The outer membrane protein complex (PCP) of Heisssria meninqitidis is used as an immunological vehicle in vaccines for use in humans. "OMPC consists of liposomes containing a variety of proteins as well as membran-505 lipids, including lipopolysaccharides (LPS or andotoxin)"

OMPC has the property of immune enhancement, and when an antigen is chemically coupled to it, results in an increased antibody scavenging to the antigen. OMPC is currently used for vaccines for human neonates against infectious agents such as Haemophilus influenzae, and for newborns capable of assembling an IgG immune response to polyribosyl-ribotol-phosphate (CPRP) of influences, when the PRP is covalently bound to the OMPC. OMPC is a mixture of a variety of proteins and lipids, and it has not been known which. the component or components of OMPC which confer the beneficial effect of immune enhancement on the coupled antibodies. However, some of the potential negative aspects of using OMPC in human vaccines include LPS-related reactions.

In addition, the DMPC-antigen conjugates are heterogeneous based since the antigen may become conjugated to any of the portions of the protein constituting the OMPC, and the total protein load per dose of a mutated vaccine would be quite long a1 ta.

In one embodiment of the present invention there is provided a substantially pure Class II protein, the major immuno-enhancer protein (PIII-1 directly derived from

outer membrane of Meisseria meninqitidis ,; free of other outer membrane components of Meisseria boyitidis. It is a further object of the present invention to provide substantially pure MIEP of the outer membrane of Meisseria meningitidis, produced in a recombinant host cell, completely free of all other Meisseria meninitidis proteins, A further object of the present invention is to provide an ethical immunobetic protein for the enhancement of an antigen immune response comprising either purified MIEP directly from the outer membrane of Meisseria meninitidis or Meisseria boyitidis recombinant MIEP produced in a host cell A further object of the present invention is to provide a protein having immunogenic mitogenic activity, comprising either MIEP purified directly from the outer membrane of Heisseria roaninqitidis, or recombinant MIEP from Meisseria ineningltiriis produced in a cell. hostess rsco / nbinsnte. It is a further object of the present invention to provide a protein having the ability to induce the production of cytokines such as 11-2, comprising either MIEP purified airtically from the outer membrane of Meisseria mymenitidis

The invention relates to a composition comprising a recombinant MIEP and a recombinant MIEP produced in a recombinant host cell. A further object of the present invention is to provide vaccine compositions comprising either recombinant MIEP or MIEP purified directly from the outer membrane of Meisseria boyitidis. are apparent from the following description =

SUMMARY OF THE INVENTION The present invention relates to the main immunosensor of Cyste II CHIEP of the outer membrane of Neisseria meninitidis in the substantially pure form free of other contaminating outer membrane proteins of Neisseria meninitidis and LPS A. MI activity of the present invention, either purified directly from the outer membrane of Neisseria meninitidis cells or derived from a recombinant host cell producing Neisseria meninitidis recombinant MIEP, has immunological vehicle activity, cytokine inducing activity <11 -2), and mitogenic activity. The MIEP of the present invention, when coupled to an antigen, is capable of immunoinfensitization since the response of the antibody to the coupled antigen is enhanced, or the antigen is transformed into a T-dependent antigen which assures the production of immunoglobulins of class I-S-1. Antigens which can be coupled to The present invention includes viral proteins, phagemid proteins and polysaccharides, synthetic peptides, other immunogenic antigens, and non-immunogenic or weak antigens.

Figure 1 - Responses from adoptive transfer vessel antibodies, which received spleen cells inoculated separately with PRP-DT and MIEP, or OMPC, or ΪΑΑ-GMPC, were measured by ELISA on blood samples taken after immunization with PRP-OMPC.

Figure 2 - Lymphocyte proliferation assay for the mitogenic activity of MIEP, in vitro. The increase of 3 h -timithin incorporation in the cellular DMA was measured following exposure of the cells to bovine serum albumin (CBSA), PEP-OHPC, OMPC, or MIEP *

The PRP-MIEP conjugates were tested in mice as well as in monkeys. Ehesus antibody responses were measured by ELI Sf and RI HB Figure - Induction of pf QH is / - S r \ .4 W. V and W. W Ho '' '' '' '' '' '' '' '' '' '' '' '' CltdP U faith? D ote of ra tines in u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u The induction of the production of rhodium δ 5% by weight was obtained by the use of the following equation: Ϋ r *: p. -: n; r] .¾ cj tab; in culture) of JL ... L? dx • as »&gt;

Figure 3 shows recessive immunogenicity. At

DETAILED DESCRIPTION Ώ0 antigé ™ INVENTION

It is known that certain substances that by themselves induce a. immune response consisting only of class Igli antibodies and no memory can be transformed into fully immunogenic IgM and IgII antibodies that induce asbestos IgB antibodies as memory by chemical coupling to a single portion. which may induce the help of T-lymphocytes for the production of if-immunoglobulin. This imanological phenomenon is called the "vehicle effect", while the π portion to the immunogenic or. weak, and strongly antigenic SUBSTATION is present in osteoarthritis. The invention relates to the use of a compound of the formula: ## STR1 ## in which the pharmaceutically acceptable carrier is selected from the group consisting of pharmaceutically acceptable carriers, pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, pharmaceutically acceptable excipients, pharmaceutically acceptable excipients, liss. In the present invention, the compounds of formula (I) are in the form of a compound of the formula: ## STR1 ## in which R 1 is H, i. t X cos for 5 s- i Xcf ar- • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • - • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • a hot water at the BK r? hapt in ο- V síc UID? "I'll tell you what to do. u8 Θ. η † †. to ιΊ iapt.e; ηο, Σ sto called one. memory or anamnestic response.

The vehicle effect appears to involve functions mediated by certain γ-lymphocytes, called helper lymphocytes. The vehicle molecule stimulates the helper T-lymphocyte to somehow aid in the formation of B-lymphocytes producing antibodies. anti-hapten IgS class and a memory response »

Auxiliary T-lymphocytes are typically involved in the production of B-specific X-cell antibodies specific for a certain type of antigens, termed &quot; T-dependent &quot; antigens, but not for other antigens &quot; T-indent &quot; antigens &quot; A molecule of the carrier can convert an unsatisfactory, weak or non-immunogenic T hapten into a strongly antigenic T-dependent molecule. In addition, a memory response will follow a subsequent exposure to the hapten vehicle complex and will consist primarily of IgS, which is characteristic of T-dependent antigens and not of T-indenting antigens. The use of the carrier molecules is not limited to use with T-antigen dependent antigens but may also be used with T-dependent antigens. The antibody response to a T-dependent anion can be enhanced by coupling the antigen to a carrier, even though the anion can itself induce an antibody response.

Some other molecules have the ability to stimulate, generally, the whole immune system. These molecules are termed &quot; mitogens &quot; and include plant proteins as well as bacterial products. Mitogens proliferate B-and / or T-lymphocytes and can greatly enhance many aspects of immune response including increased phagocytosis, increased resistance to infestation, increased tumor immunity, and production of increased antibodies »

The production of 11-2 T-helper cells can be induced when T-cells are activated by certain substances or cells, and the T-helper cells can be stimulated to stimulate the growth and activity of other lymphocytes. antigens presented by antigen presentation cells. The effects of 11-2 include, but are not limited to, the progression of T cell proliferation, and proliferation and differentiation of B cells. Many causative agents of infectious diseases may, for them induce protective antibodies that can bind and kill, render them harmless, or may cause death or render harmless the causative agent of diseases and their by-products. Recovery from these diseases usually results in long-term immunity by virtue of the protective antibodies raised against the highly antigenic components of the infectious agent.

Protective antibodies are part of the natural defense mechanism of humans and 'many other animals, and are found in the blood as well as in other tissues and body fluids' is the main function of most vaccines is to induce protective antibodies against infectious agents and / or their by-products without causing disease '

Oligocyanurate has been successfully used to induce antibody responses in humans when OMPC is chemically coupled to T-cell independent antigens including bacterial polysaccharides. The OMPC contains various bacterial outer membrane proteins as well as bacterial lipids. Additionally, OMPC has a three dimensional liposomal structure. The efficacy of OMPC as an immunological vehicle was thought to be dependent upon one or more of the bacterial membrane proteins.

bacterial lipids of the tri-dimensional iiposomal structure of a combination of liposomal proteins, lipids, and liposomal structure. The applicants will find that one of the proteins, MIEP, possesses the immunological carrier and immunointensifiable properties of OMPC vesicles, and is effective in the purified form, free of other membrane proteins of __ __ __ men men men men men men men men men men men men men men men men men men men men men men e e e.

Applicants have also found that MIEP, when chemically coupled to a bacterial polysaccharide, works as well as OMPC in inducing antibody response to the polysaccharide. Applicants have further discovered that MIEP is the Class II protein of the outer membrane of N-meninitidis. Class II protein of N. meninqitidis is a porcine protein EMurakami, K ", et al. (1989), Infection And Immunity, 37, pgs. 2318-233 = Porines are found on the outer membrane of all Gram-negative bacteria '

Although the present invention is exemplified by the MIEP of N, meninqitidis ·, it is readily apparent to those skilled in the art that any outer membrane protein of any Gram-negative bacterium bearing an immunological carrier and immunostimulatory activity is encompassed by the present invention. of Gram-negative bacteria include but are not limited to species of the genus Meisseria, Escherichia, Psiformesi, Haemophiius, Salmonella, Shiaella. Edge! Klsbslella, Serrphytia, Yersinia, Vigricus Entergbacter. MIEP may be employed to target the antibody response to highly antigenic, weakly antigenic, and non-antigenic materials. The terms &quot; antigen &quot; and &quot; antigen material &quot; which are used interchangeably herein include other non-viable, immunogenic, weakly immunogenic, non-immunogenic, non-immunogenic,

The antigenic component may consist of a dry powder, an aqueous phase such as an aqueous solution, or an aqueous suspension and the like, including mixtures thereof, containing an agent (s) or agent (s). non-viable, immunogenic, weakly immunogenic, non-immunogenic, or desensitizing agents.

The aqueous phase may, for example, be in the form of a vaccine in which the antigen is dissolved in a balanced salt solution, physiological saline, phosphate buffered saline, tissue culture fluids, or other media on which an organism may have grown, the aqueous phase may also contain preservatives and / or substances conventionally incorporated into vaccine preparations. The adjuvant emulsions containing HIEP conjugated antigen may be prepared employing well known techniques in art,

The antigen may be in the form of the purified antigen or. partially purified, including, but not limited to, antigens derived from bacteria, viruses, mammalian cells and other eukaryotic (including parasite) cells, fungi, rickettsiae or the antigen may be an allergen including, but not limited to, pollens, dust , slags, or strata thereof, or the antigen may be in the form of a toxic or poison including, but not limited to, poisons or poisons derived from reptiles or poisonous insects. The antigen may also be in the form of a synthetic peptide, or of a fragment of a larger polypeptide, or any subportion of a molecule or component derived from bacteria, mammalian cell, fungus, virus, rickettsia, allergen, poison or poison. In all cases, the antigens will be in the form in which their toxic and virulent properties were

reduced or destroyed, and which when introduced into a suitable host will, or induce, active immunity by producing therein antibodies against the specific proteins, proteins, microorganisms, excipients, organochlorines used in antigen preparation, toxins, poisons, or , in the case of allergens, will aid in alleviating the allergy symptoms due to the specific allergen,

Antigens may be used either as a combination, e.g., multiple bacterial antigens, multiple viral antigens, multiple mycoplasma antigens, multiple virus antigens, multiple bacterial or viral toxoids, multiple allergens, multiple proteins, multiple peptides or may be conjugated to MIEP combinations of any of the background products *

Particularly important antigens are derived from bacteria including, but not limited to, B. pertussis, L., P., P., B., B., and C. et al., S, paratyphi A and B, C. diisopropylcarboxamide. ............. .. botulinum, C. perf rinciens. Crabs, and other bacteria from gas gangrene, B. anthracis, Y. pestis, V &amp; bgl-grag. Naissaria meninqjtidis. M ........ gpnprrheae,

Haemophilus influenzae, Treponema pallidium, and the like? of mammalian cells including, but not limited to, tumor cells, virus infected cells, genetically engineered cells, cultured cells, fabrics, and the like? of viruses including but not limited to human T lymphocyte virus (multiple types), human immunodeficiency virus (multiple variant types), poliovirus (multiple types), human papilloma virus (multiple types), -adenovirus (multiple types), parainfluenza viruses (multiple types), measles, parotitis, respiratory virus, virus viruses (various types), typhus virus (SF), Western and Eastern equine encephalomyelitis, encephalomyelitis B, Japanese encephalomyelitis Russian purpura virus, Wewcastle's disease virus, varicella, rabies, canine and feline synostosis, and similar viruses of rickettsiae including but not limited to endemic and endemic typhus or other members of the spotted fever group (meningitis cerebrospinal), various venoms of spider and snake or any of known slergenes, including but not limited to those of santiago (tasnsira), house dust, strata of pollen, pollen, grass, and the like »

The palissaccharides of this invention may be any acidic bacterial polysaccharides, but are not intended to be limited to any particular types. Examples of such bacterial polysaccharides include polymethaccharides of Streotococcus pneumoniae pneumoniae types 6A, 6B, 1α, 11α, 18α, 19α , 19, 23, 22F, and 23F? Streptococcus Brupo B types Ia, Ib, II and III? polysaccharide of Haemophilus infiusnsas serotype B. polysaccharides of Neisseria meninqitidis serogroups A, B, C, X, V, W, and E. The polysaccharides of Escherichia coli

However, the particularly preferred polysaccharides are those capsular polysaccharides selected from the group consisting of serotype B influences polysaccharides, such as described in Rosenberg et al., BJ, Bioi. Chem., 29, 2845-2849 (1961) and Zamenhot et al., J. Biol. Chem., 2, 3, 695-7, 34, 1953), Streptococcus pneumoniae polysaccharide (pneumococci) type 6A or type 6B, as described in Robbins et al., Infection and Immunity, 26 , No. 3, 1116-1122 (December, 1979)? pneumococcal polysaccharide of the type 9F, as described in C.J. Lee et al., Reviews of Infectious Diseases, 3, Vol. 2, 323-331 (1981); and 23F pneumococcus polysaccharide, as described in, Larm et al., Adv, Carbohyd Chem

and Biochsm, 33, 95-321, R, S, Tipson et al., ed., Academic

The IMEP can be purified from the DMPC derived from cultures of N, N-menincitidis which grow in the usual manner as described in U.S. Patent 4,495,236 and U.S. Patent No. 483,3852. Purification of OMPC can be made in accordance with the process described in U.S. Patent No. 4,177,447, 4,485,928 and 4,830,852.

MIEP can also be obtained from recombinant DNA constructed in host cells by expressing the redoxing DNA encoding MIEP. The DMA encoding MIEP can be obtained from NMS men cells, Et al., (1989), Infection And Immunity, 57, pgs. 23183, or DMA may be produced synthetically using standard DMA synthetase techniques. The DMA encoding MIEP may be expressed in recombinant host cells including but not limited to bacteria, yeast, insect, mammalian or other animal cells, giving MIHR rewinding. Preferred processes of the present invention for obtaining MIEP are and purification of MIEP from OMPC ε expression of recombinant DNA encoding DMA encoding MIEP derived from N, meningitidis, &quot; purified MIEP fox prepared from OHPC vesicles by sodium dodecylsulfate (<SDS) lysis of the vesicles followed by electrophoresis on SDS-polyacrylamide gel (PAGE). MIEP was eluted from the gel, dialyzed against a pH buffer high and concentrated. Standard procedures for electrophoresis were polyacrylamide gel can be used to purify MIEP from OHPC vesicles. Such processes are described in Molecular Cloning A Labor Manual, Sambrook, J. et al., (1989), Cold Spinning Harbor Laboratory Press , New York, and Currerit Protocols In -13--

nolscuiar Bioloqy, (198 /) Ausubel F M. editors Wilev and Sons, New York. The pathogenic processes of protein elution to the partition of S-DS gels. 1 amide are described in Hunkaper, M. W, and Luj an, E., &lt; 198%), Purification of Microgram Quantities of Proteins by Pol and Acetylcholines, on rlethods of

Protein Mierocharacterisation (editor of Shively) Humanna Press, Cliffcon N.J., Current Protocole in Molecular Biology &lt; 19875, Ausubel, F. M. et al. , Niiey and Sons, New York editors. The MIEP prepared in this manner is suitably suitable for conjugation to antigens derived from bacteria, viruses, mammecaris, rxcKsttsis, fungi, tc-micelles or poisons, fungi, peptides, proteins, polysaccharides , or any other analogue. The recombination MIEP may be prepared by expression of genomic N. meningitidis DNA encoding MIEP in bacteria, for example E. coli or in yeast, for εκ in p.o., erev ss ss »&gt;

To obtain the genomic DNA encoding MIEP, the genomic DNA is extracted from N. iaininitidis and prepared for clone-gera by either random fragmentation of the high molecular weight DNA followed by the Maniatis, T ", et al. (1978), 11, q-pτι pg. 08, or by clxvayem with a restriction endonuclease by the method of Smithies, et al., &Lt; 19785, Science, 242 pg. 1248. The genomic DNA is then incorporated into an appropriate cloning vector, for example, phage lambda Lver Sambrook, J. St. Al., 1939), Molecular Cloni.nqr. Alternatively, the polymerase chain reaction (PCR) technique may be used to amplify the specific DMA sequences in the genomic DMA lRoux, et al. (1989), Biatechniques, 8, pg. 483. The PCR treatment requires a DMA oligonucleotide that can hybridize to the specific DMA sequences in the genomic DMA. The sequence is in the range of one that can enhance CD m DMA dB B ΗI t.P in the DMA that in N ψΡ i ndi i ndi s can be u ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ ΓΊΤ. the sequence of the MI of the EPO or of the DMA demineralized for the B major. (1) (1) (1) (1) (1) (1) (1). Immunization and Immunity 57, pgs. 183. Immunization can be carried out in the same way as a plow of three or more prophylactic agents. and the acylation of a co-mole. of af i X X d Λ made with specific monoclonal or palielonal antibodies to MIEP. These affinity matrices are made by adding the antibodies to AffigeI-ΊΘ (Biorad), a gel carrier that is preactivated with N-hydroisosuccinimide sterols so that the antibodies form covalent bonds with the carrier gel beads agaross. Antibodies are then coupled to the gel via ligations with the spacer arm. The remaining activated esters are then destroyed with ethanolamine 1N HCl &lt; The column is then flushed with glycine HCl, 23 M H 2 H 2) to remove any unconjugated antibody or foreign protein. The column is then equilibrated in phosphate buffered saline <pH 7.3 ) and the stj ibrsnadant. is of the cul ture of DU 03 t cells. The cells of the cells are in the form of cells in the cell, and the cells of the cells are in the form of a cell. The salt is cooled to room temperature until the optical insensitivity (Α. = Οα) drops to the desired temperature, so the propionate is treated with glycine-HCl. pH 2.6) The pro tein is so smoothed with the solvent and the pH of the phosphate. 0 C FUNCTIONS OF THE PRES. .e x π v in x. the polyolefin moiety may be any of the polysaccharides of the present invention. x dc or other. the antigen-M1 EP, in vacuums of antigenic peptides. Peptides 1 Vw. In addition, X is one or more anionic determinants of any species including antigenic determinants of bacteria, rickettsia, viruses (including human immunodeficiency virus), mammalian cells or other sucaryl cells including parasites, toxins or toxins. The antigen-HIEP conjugates are coupled through bigeners containing a thioether group and a primary amine, which form hydrolytically labile covalent bonds with the polysaccharide and MIEP. Preferred conjugates according to this invention, however, are those which may be represented by the formulas, Ps-A-E-Q-B-Pro or Ps-AZ-S-E'-B'-Pro, wherein Ps represents a polysaccharide or any other antigen; Pro represents the bacterial protein MIEP; and AE-8-B and A-S-E '' -B 'are bigeneic spacers which contain hydrolytically stable covalent thioether bonds, and which form covalent bonds (such as amide bonds or hydrolytically labile ester) with macromolecules , Pro and Ps-Mo spacing, A-E- SB, S is sulfur; E is the transformation product of a thiophyl group which has been reacted with a thiol group, s is represented by wherein κ is H or CH 2, ε p is 1 to .55 A is

W H

wherein W is 0 or NH, m is 4 to 4, n is 3 to 3, and Y is 0 to 0; 0.3, NR% or CHCOOH, where R 'is H or C 1 -C 6 alkyl, such that if Y is

then both m and n can not be equal to zero? and if Y is 0 or S, then m is greater than 1 and n is greater than and B is CH 2 (CH 2) NH 2 -, NH 4 ', R 4, or H, where K p and p are as defined above, and D is C 5 -alkyl;

W

H in the spacer, A '-S-E &quot; -Ei &quot; , S is sulfur A &quot; is -CNH (Cl-f ') -, in Η 0 Y' which is è 1 s. 4, and R '' '' is CH-, or -CH-CH (Cl-Cl, where R 'is N--U or - defined above, ε E 'is the transformation product of a thiopholy group which has been reacted with a thiois group and is represented by -uH-, wherein H is as cretinit

II above, and B 'is -C-5 or E1 is

O )

N,

O 0

H 2 'is 1 to 3, which is 1 to 3. A, B and A'-S-E'-B ', the components ES-B s A' - b> -E 'P are determinable and when they are feasible, f 1 and the identification of the covariance of the conjugate binding by the side oo

sulfur thioether having the covalently modified polysaccharide with the spatter side which originates from the fungal protein i. i layed

Thus, the conjugates, Ps-AEBB-Pro, according to this invention may contain spellers whose components include, inter alia, carbon dioxide, 1,4-butanediamine, and S-carbomethyl-N-acetyl homocysteine carbon dioxide, 1,5-pentanediamine, and S-carboxymethyl-N-acetylhomocysteine, carbon dioxide, 3-ota-1,5-pentanediamine, ε-carboxymethyl-N '- acetyl homodexide carbon dioxide, 1,4-butanediamine, and S-carbymethyl-N-acetylcysteine, carbon dioxide, 1,3-prapano-diamine, and B-carboxymethyl-N-bensoyl-homocysteine; dioxide) (0.11 g, 0.13 mol) in hexane. 2-yl) -jy-. and 8-carboxymethyl methacrylate, carbonate, 1,2-ethanediamine, glycine, and S-1-N-acetyl -B '· -Pro, according to this invention, may contain compounds whose components include derivatives of, inter alia, carbon dioxide and S-carbocyclic-distelamide-dioxide carbon &amp; B- (Î ± -C-arboxyethyl) cysteamine β-carbon dioxide and S-carboxymethyl homocysteamine; carbon dioxide. B- (succinate: 4-fluorophenyl) -4-fluorophenyl) cysteamine, and glycine; and

In the process of the present invention, the polysaccharide is cavitated by &lt; a) solubilisation in a non-organic organic solvent, then (b) activation with a. (b) reacting this polysaccharide with the disulfonate, and finally, if necessary, (d) reacting the polysaccharide with the desired reactive polysaccharide, (i) reacting the polysaccharide and (iii) with a thiol group-generating reagent. The compounds of the present invention may be prepared by reacting the compound of formula (I) with a thiol grouping reagent. Protein è. The reaction is carried out in the presence of a reagent generating a thiol group or (iii) with a

thiolyl sites, then the covalently modified polysaccharide and the functionalized protein are &quot; reacted together to form the stable, covalently bound conjugate, and the final mixture is purified to remove unreacted polysaccharides and proteins.

The process of this invention also includes the selection of a nucleophile or bis-nucleophile which will react with the acylated polysaccharide to form a covalently modified polysaccharide with pendant electrophilic sites or pendant thio groups, thus preventing the need to further functionalize the modified bis-nucleophilic polysaccharide prior to reacting the covalently modified polysaccharide with the covalently modified protein. The functionalization of the protein for any one of the portion forms can also be SS " the results of the pSSSS-O were determined in accordance with the selection of reagents in these passages

In the first step in order to covalently modify the polysaccharide, the solid polysaccharide must be solubile.

In one embodiment, the hydroxyl groups are alcohols. A nucleophilic polysaccharide can compete with the hydrophilic acids of the water in a solution, the polysaccharide of which is to be dissolved in aqueous aqueous solutions (inert solvents). Suitable solvents include dimethylformamide, dimethylsulfoxide, dimethylacetamide, formamide, N, N'-dimethylimidacolidinone, and other aprotic, polar solvents, preferably dimethylformamide.

Add 1 mind, è ·. use of these solvents, the convention of the polysaccharides (for example, the capsular polysaccharides of Hexaparin and type b, which are ribose-ribitol phosphate polymers), which are acidic hydrogens, such as mono- and -19 -

T 32 diesters of phosphoric acid in a suitable salt meal, provided that the polysaccharides readily become soluble in the above solvents. The acidic hydrogens in these macromolecules may be replaced by large hydrophobic cations such as trihydro- or tetra-C1-4 alkylamino, 1- azabicyclo [2.2.1] octane, diazabicyclo [3.3.1] oct-J-ene or the like, particularly tri- or tetraalkyl CC, - a) ammonium, and tri- or tri-

Resulting from the resulting tetraalkyl ammonium salts or similar salts of phosphorylated polysaccharides readily dissolved in the solvents above at about 0Â ° C. / hour. while stirring for from one minute to one. The partially hydrolyzed H, influenzae, serotype B polysaccharide was converted to the tetrabutylammonium salt, then dissolved in dimethyl sulfoxide CEgan et al., J. Org. Chem., Chem. Soc., 4, 2898 (1982)), but this product is no longer active, and therefore is useless. In contrast, in the presence of a tetrahydronaphthyridine derivative or a pharmaceutically acceptable salt thereof. The title compound was prepared according to the procedure described above, and the polyacrylamide viability of the polysaccharide was maintained. use in the immunogenic vaccine are then performed to overcome physiochemical to make xdos, which are the ones that are used, others that in reactive groups with reagents use a functionalization of u , which is desired to bind. Activation of the polysaccharide to form an activated polysaccharide reaction with bis-nucleophiles to form a polysaccharide and functionalisation with reactants generating both electron polysiloxane and indole groups are all directed to covalently modify the polysaccharide and to provide functional groups in the

This is not the case. CO 1 'C, while agitating with a cross-linking weight of 1 to X in a bifunctional reagent is inert to the? minutes at one hour with the activator of polysaccharide In the past, this action was carried out by reaction of the polysaccharide with cyanogen bromide. However, the activated derivatives were cyanogen bromide, which filtered a The present invention relates to a process for the preparation of the compounds of the present invention in the presence of a phosphate buffer in the presence of a phosphate buffer. This reagent is useful for the activation of polysaccharides and is not otherwise suitable for polysaccharide activations include carbonic acid derivatives, wherein R &quot; and R &quot; May be independently asolyl, such as imidazolyl halides, or phenyl esters, such as D-nitrophenyl, or polyhalophenyl. carbonyldiimidazole? a particularly preferred reagent will react with hydroxyl groups to form imidazolyl urethanes of the polysaccharide, and the aryl chloroformate including, for example, nitrophenyl chlorophosphate will produce mixed carbonates of the polysaccharide,

For each case,

1 &gt; 1 &lt; / RTI &gt; &lt; / RTI &gt; &lt; / RTI &gt; by consistently transforming the respective urethanes. 4-amine, H, partite ... NH, in? wherein in the case of K * is H or an ε η n is not more than 1% of the amine (i.e., and in the latter case a reaction bath is reacted with a &quot; in 1 ο ο i cá cá cá **** ****

In the next phase. For example, HN (CH3) yCCH2) -NH, where m is 1 · m ··· r> 4 to 4, n is 3 to 3, and Y is CH3 O, NH2, CH3 OH, where K2 is H or C1-4 alkyl. or C.-, so that if Y is plus m and n can not. aL. * j. be equal to 70 °, 0 if Y is 0 or S, then m is greater than 141-1 ct X in a large ex CBS SD tDt for example, a molar ratio of V to ° 10 vs activating agent used K The reaction is maintained ice for 15 minutes and then one hour after one minute at one hour at about 17 DEG-4 DEG C., for example, tetrahydrofuran is obtained in the form of urethane with amines The mixed carbonates will also readily react with diamines to give the pending amines.

Alternatively, the activated palissaccharide may be

was used as an amide in the form of a compound of formula (I). that of in situ dihydrochloride. 4-b romoacetamidobut-1-amine (see, W., et al., Lawson et al., P. (19eS) for a 5% polysaccharide modi fi ed in the same manner as the elec- tional cells. D is not activated can be reacted with an aminothiol, such as cysteamine (aminnoethanethiol) or cystine, examples of derivatives which are well known in the art of peptide synthesis to produce a pendant thiol group polysaccharide. In both cases ,

Further functionalization is required prior to coupling of the covalently modified polysaccharide to the carrier moiety. The last step in the preparation of the polysaccharide further functionalisation, if necessary, of the polysaccharide may take the form of either reacting the functionalized nucleophilic polysaccharide with a reagent to generate cyst sites Cyst, thiophylic) or with a reagent to generate thiol groups

Reagents suitable for use in the production of? electrophilic sites include, for example, those for acylation of G-haloacetyl or halopropionyl, derivatives

OR

and X 'is nitrophenyl, dinophenophenoxy, pentachlorophenoxy, pentafluorophenoxy, isoste, u- (N-hydroxysuccinimidyl), or the like, wherein R is H or CH-T% X is Cl, Br or 1 and X' is Nitrofe- acid), paractic acid, α-oracetic acid or α-bromopiopropionic acid, the reaction is carried out at a pH of 8 to 11 (maintained therein by the addition of base, if necessary) at a temperature of about 0 ° to 35 ° C The amino-derivative polysaccharide may be acylated with activated maleimido amino acids (see, Keller et al., Helv. Chem. Acta., 8, 531 (1975)) to afford groups roaleimide. 0

-OCH2) pN

The 3-position of the 3s with a 2-haloacetylating agent, such as ρ-nitrophenyl bromoacetate or a carboxylic acid Î ± -haloketone derivative, for example. (1934)) so as to produce 1 x ssau ã r. 1 d properly functionalized susceptible to substitution

Suitable reagents for use in the production of thial groups include, for example, acylating reagents, such thiolation beds, for example.

- (CH2) p, wherein R 'is C1 to C4 alkyl or mono- or bicyclic aryl, such as I, A, Cr, WHCOR, and X is 1, -COX ', wherein m is 3-COOID of TminDQ-SCXH-1; R 2 is alkyl, G 1, G 2, C 1 -C 4 alkyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, (S-S-CH 2) C (O) C (CH 2) 2 -CH 2 -CH 2 -O- 1, 2, 3, 4, 5, 6, 7, 8, 10, 10, 10, 10, 10, 10, 10, is added to a pH of 10, the base is added, when necessary, to maintain the pH within this range) for one to twenty five minutes. they are reacted with a ρi

* sji2> at-the-wheel

to produce a polyolefin suitably works 1 iza do =

By these steps, polysaccharides covalently modified are then produced, Ps-A-Et- or Ps-A "-SH- 5 wherein Et is -CCHX or

Oo

II

C (O) p N

The method of claim 1, wherein the compound of formula (I) the reaction of a compound of the formula: ## STR1 ## in which R 1 is as defined in formula (I). A 1, A 2, R 3, X and R 4 are bed defined above in the form of a polysaccharide, to generate a group t: one or more reactants to generate a THYTOL:

In preparing for conjugation with a functionalized electrophilic polysaccharide, the protein is reacted in one or two steps with one or more reagents to generate thiol groups, such as those acylating reagents used for. in the form of a blank, as shown in the preceding pages.

Thiolated proteins may also be prepared by aminating activated carboxy proteins such as Atassi et al. (N-acetyl-homocysteinathiolactone groups at about 0Â ° for five minutes to two hours, using N-acetyl-homocysteinathiolactone groups).

iS shown in i.e., W i. the direct transport of the protein at 0 ° C to 3 ° C is pH and is equal to, In order to create the protein of the peptide,

When E'B 'is

O

The conditions and the process for the preparation of the functionalized protein are as discussed above for the preparation of the complement polysaccharide by reaction with activated monosodium acids.

In the preparation for the covalently modified bacterial conjugation with g? protein is acylated with a reagent generating the formula U CH η | for example, XCH3 C-X 'and XCH as defined above. 0 with an outstanding thiol polysaccharide, at an electrophilic center

CX ', wherein X and X the

Ζ &amp;

HELLO . For example, those prepared by acylation of the pendulum groups with a reactant, such as activated maleimido acids, such as, for example, for example,,

O 0

or by the action of 5, p. The reaction is carried out in the presence of a solution of a compound of formula (I). bacillus ana ΜIEP: * C for five minutes at a time. hour and

The electrophilic polymers are pendant with the pendant thiol groups at a pH of 7: 9 in approximate equivalent weight ratios in a nitrogen atmosphere for from six to twenty-four hours at about of 17 ° to 4 ° C, to give a covalent conjugate. Examples of such reactions are Ti

Oh

-CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH

fCOCH, XBCO-P

TO

The reaction mixture is reacted with 4-bromoacetamidobutylamine and is reacted with a protein which has been reacted with N-acetyl-hammoconitriloyl ar t on a "to form a conjugate, and; 3 ro. wherein a polysaccharide

OH III

PSCNY &quot; -NCCH2 N

O

II i

HSCH2CH2NHCCH2CH2CPro PsCNHY &quot; NHCCH, -N

O O II II. , CH2CH2NHCCH2CH2CPro where Y '&quot; is a C1 -C4 -alkyl radical, wherein a polysaccharide which is reacted with activated maleimido acids is reacted with a carbovti-activated protein which has been aminated with an aminothiol to form a conjugate »

Similarly, either the polysaccharides covalently modified with pendant thiol groups may be reacted with the MIEP bacterial protein having pendant slectrophil centers to give a covalent conjugate. An example of such a reaction is ο ο Ο Η

NHCOCHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3CH2CH2CH2CH2CH

wherein an activated ροϊ.isaccharide which has been reacted with an aminothiol is reacted with a carboxy-activated protein which has been reacted with mono-haloacetyl derivatives of a diamine to form a conjugate.

As the electrophilic activity of an excess of haloacetyl groups needs to be eliminated, the reaction of the conjugate with a low molecular weight thiol, such as n-acetylcysteamine, will accomplish this purpose. The use of this reagent, n- cysteamine, also allows for the confirmation count of the haloacetyl moieties used (see Section D), the β-alkoxy-cysteamine moiety that is formed can only be detected by the Spackman, Hoore and Stein method.

These conjugates are then spun at about Ϊ &amp; Using a fixed angle rotor for about two hours at about 1Â ° to 2Â ° C, or are subjected to any of a variety of ds; other purification procedures, including gel permeation, ion exclusion chromatography, gradient centrifugation, or other differential adsorption chromatography, to remove non-covalently attached proteins and polysaccharides using the covalence assay for. the generic Pi spacer (see below) as a process of following the desired biological activity. Further separation of the reactants may be carried out by size exclusion chromatography on a column, or. in case

of very large non-soluble proteins, separation can be carried out by ultracentrifugation. The conjugate conjugate assay to confirm cavity and hence the stability of the conjugate is performed by hydrolysis of the conjugate preferably with 6N HCl at 1 ° C for 2 hours), then quantitative analysis for the amino acid of the hydrolytically stable spacer containing the thioether linker and amino acid constituents of the protein. The amino acid contribution of the protein can be removed. by comparison with the standard appropriate amino acid sequence for the protein involved, reflecting the remaining amino acid value at the covalent linkage of the species of the species may be brought to appear outside the standard amino acid of the protein in the analysis covalent assay is also useful for monitoring purification procedures to mark the concentration enhancement of the biologically active components.

Uh n

NHCOCH gives PsCNCH2 CH3 CH3 CH3 CH3 CH3 CH3, CH3 CH3, CH3 CH

Accordingly, the invention provides a process for the preparation of a compound of the formula: ## STR1 ## in which R 1 is H,

Hello

Hello

Ps CNHY &quot; NHCCH? C H 2 CH 2 NHC H CH 2 CH 2 CO 2 yields the release of the aminodicarboxylic acid.

CH 2 CHCH 3 CHCH 3 CH, NHo; and the hydrolysis of ho2c OH OH 0 0 IS | yl | s

(CH 2) 3, NHCCH 2 CH 3 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 CH

S-carboxymethyl-cysteamine, H₂O-CH,, -CH₂CH X, XO HH by cleavage of

to the peptide bonds and other hydrolytically unstable bonds. Chromatographic processes, such as those of Spackman, Moore, ε Stein, can then be conveniently applied and the ratio of amino acid constituents determined. The optimal production of IgS antibodies requires the collaboration of the T and B lymphocytes with specificity for the antigen of interest. The β-lymphocytes are unable to recognize the polysaccharides but may provide support for IgB antibody responses to polysaccharide if the polysaccharide is covalently bound to a protein that the T cell is able to recognize. i

In mice this requirement exists for secondary, as well as primary, antibody responses, and is vehicle specific, i.e., a secondary antibody response occurs only if the helper T cells have previously been sensitized to the expensive carrier protein used for the immunization secondary education. Accordingly, the ability of a mouse to produce a secondary antibody response to a PRP-silver conjugate is dependent on the presence of T lymphocytes inoculated with specificity for the carrier protein. Demonstration of the ability of MIEP to provide a vehicle inoculating anti-PRP antibody responses was done in mice that were inoculated with F.RP covalently attached to a heterologous vehicle, diphtheria loxoid &lt; DT &gt;. Was

adoptive transfer in order to determine whether a. administration of lymphocytes inoculated with MIEP alone was sufficient to generate helper T cell activity effective for the formation of anti-PRP antibody in response to PEP-Î ± 1βPC. Similar secondary snti-PRP antibody responses were induced by PRP-OMPC when lymphocytes inoculated with MIEP or OMPC were transferred, indicating reconhecimento cell recognition of OMPC residues in the FlIEP portion,

The PRP-npEP conjugates were tested for immunogeneicity in mice as well as in newborn Rhesus monkeys. The immune response in both animal models shares, with newborn humans, a deficiency in their ability to. generate antibody responses against independent T-antigens such as histamine polysaccharides. These animals are normally used as templates for determining the immune response of newborn humans to various antigens.

)

One or more of the conjugate vaccines of this invention may be used in mammalian species either for active or passive protection prophylactically or therapeutically against infectious agents including bacteria, rickettsia, parasites, and viruses, or. against toxins or toxins, allergens, and mammalian cells or other eukaryotic cells. The active preparation can be carried out by injection of an effective amount capable of producing measurable amounts of antibodies (for example, 2 to 50 pg) of an antigen (for example PRP) in the form of MIEP-conjugate of each of the conjugates to be administered The use of an adjuvant (eg, alum) is also intended to be within the scope of this invention. Passive delivery may be carried out by injection of the total antiserum obtained from animals ρrecipiently dosed with MIEP-conjugated or conjugated, or globulin or other antibody-containing fractions of

said antisera, with or. without a pharmaceutically acceptable carrier, such as sterile saline. Such globulin is obtained from the total antiserum by chromatography, fractionating in alcohol or salt or electrophoresis. Passive protection may be performed by standard monoclonal antibody procedures or t by immunization of suitable mammalian hosts)

In a preferred embodiment of this invention, the conjugate is used for active immunogenic vaccination of humans, preferably newborn infants, immunocompromised individuals (such as asplannic individuals to foot-chemo patients), and the elderly. For additional stability, these conjugates may also be lyophilized in the presence of lactose (for example, 20æg / ml PRF / 4mg / ml lactose) prior to use,

A preferred dosage level is an amount of each of the conjugated IL-1β, or its derivative to be administered, corresponding to between about 2 to 2Âμg of PEP in the form of the MEP-conjugate for conjugates of H, influsnzae, serotype B polysaccharide in a single administration. If necessary, one or two additional doses of the polypeptide, or derivative thereof, may also be administered from the H. influenzae polysaccharide serotype B in an amount corresponding to from about 2 to 2% by weight of PRP in the form of the conjugate, the invention is further defined by reference to the following examples, which are intended to be illustrative to non-limiting,

Fig.

Preparation of OHPc from Neissaris boyitidis Blí serotioc H. 1 Neisseria meningitirfis group B1 i &gt;

A tube containing the lyophilized culture of meninitidis (obtained from Dr, SI, Artenstein, Walter Reed, Institute of Research, 1985). Washington, DC; was opened 'and Eugonbroth &lt; BBL) was added. The culture was scored on Musllsr Hinton agar slopes and incubated at 37 ° C with 5% CDâ,, .delta. for 36 hours, at which time the growth was collected in skim milk (50% Difco), and the fruits were frozen at -70 ° C. The identity of the organism was confirmed by specific antiserum agglutination provided by WRAIR®, and standard serum supplied by Difco®

One vial of the second pass culture was thawed and scratched onto 10 plates of Columbia Sheep Blood agar (CBBAB8BL). Plates were incubated at 37øC with 5% CD durante for 1 hr at which time the growth was harvested at ! € ·? €> ml of 100% skim milk medium were removed in 0.5 ml amounts and frozen at -70 ° C, the organism was positively identified by specific antiserum agglutination, sugar fermentation and coloration,

One vial of this, passageway was thawed. diluted with Hu.el.-Hin ton Broth and scratched on 40 plates of Huei-Hexagonal plates. The plates were incubated at 37 ° C with 20% CQ-S for 18 hours, after which time the growth was collected in 17 mL of

of skim milk at 10%, pliers were withdrawn in amounts of 0.3 ml and frozen at -78 ° C. The organism was positively identified in Sram staining, specific antiserum agglutination &amp; test of rust © »2« Fermentation the collection of the pastei of the cells

The. Development of the Inoculum The inoculum was grown in a frozen flask of Maisseria meninqitldls from Group B, B-1 above (passage 4). Ten slopes of Mulex-Hinton agar were inoculated and six were harvested for approximately 1 hour later, and used as inoculum for 3 vials of 250 ml. of Botschilch yeast medium dialysate at pH 6.35. THE

Du, was adjusted to Θ 16, incubated to 0 ° C. to be between 1

1.8 ml. From this culture was used to inoculate each 5 μL. The Erlenmeyer flasks (each containing 1 liter of medium, see below) and incubated at 37 ° C on a shaker at 2 ° rpm. A O.D. was monitored at one hour intervals following inoculation,

4 liters of culture broth were obtained in a solution of 1.2B.

7 Liter Seed Fermenter Approximately 4 liters of seed culture were used to inoculate a sterile 7 liter fermenter containing about 4 liters of complete production medium (see below). The conditions for the fermentation of 7 Â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ .. final was 0.732 after 2 hours »

Brewer's Production Brewer

Approximately 40 liters of seed culture was used to inoculate a &gt; liters containing 568.2 liters of complete production medium (vsr abai 7 ·). This batch was incubated at 37øC, 100 rpm with air spray of 6Θ L / min and constant pH control at pH For this portion, the final D 0 0 was 5, 58 hours after inoculation. And L-glutamic acid MaCl (II), and the compounds of formula (I)

Ma, J-iPO ", anhydrous ν 'SJ. ', * NHgCl 1.25 α / Iitro κι .; i 0.09 q / liter L-cysteine HCl 0-0-2 o / l-ln

Fraction B CDialised from Sotschlich Yeast

The Difference Yeast EKtract was dissolved in 6.4 liters of distilled water. The solution was dialyzed in 2 days of NaOH-3a concave fiber with B108M cangas. 3B4 g of Na2 SO4 and 320M q '4 ji. and the total volume brought to 15 liters of water was then adjusted to pH 4 with Na 2 SO 4, sterilized by passage through a filter of &lt; 3.22æ, and transferred to. the a, the termer

Er1enroeyer: The traces of A? Cc? The reaction mixture was stirred at &lt; RTI ID = 0.0 &gt; F &lt; / RTI &gt; 1 fractions of Fraction A and 90 ml of Fraction B and pH 7.10-7.7 with NaOH. For the fermenter, 80 liters of water were added. Fraction B and pH ~ 780 ° C with NaOH. Cohen et al. i. After the fermentation, it was completed in a separate vessel. for the q i 1 / jj) 1 the broth of the dried fruit? yielded a concentration of ϋ ~ fi fi fi fiololol # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # temperature. until the culture is no longer good. . .W . The title compound was prepared in the presence of about 10% by weight of the title compound (about 24.0% by weight)

Centrifugation

After about 24 hours at 4Â ° C, the 14 Âμl of inactivated culture fluid was centrifuged through continuous centrifuged centrifugals of the cell paste after treatment with phenol was 3Â ° C 87Â ° B Isolation of QMPC Step 1h Concentration and diafiltration The phenol inactivated culture was concentrated to

about 3 litros liters and diafiltered in sterile distilled water using concave fiber filters, 2 æEMKA).

Pass an equal volume of 2X TED buffer. EDIA 5,500, pH 8.5, 10% sodium deoxyleolate was concentrated in vacuo. IZt «·" SUSPS; ieratu f &quot; ** Referred to &gt; The present invention relates to a process for the preparation of a compound of formula (I) 1 μl of 4 ° / 0 SD at + 4 ° C. The samples were extracted with continuous flow centrifugation and reextracted in TED buffer as described above. The supernatants were pooled and stored at 4 ° C.

Step 3. Concentration by Uitrafiltration The pooled sample was transferred to a temperature-controlled vessel connected to polysulfone Θ micron AB-Tech. The extract temperature was maintained at 25 ° C in the vessel throughout the concentration process. The sample was concentrated :: times at a mean transmembrane pressure between 75Â ° C - and i65s36 kPa.

Step 4. Picking and Washing the OliPC The retentate from Step 3 was centrifuged at about ΙθθΘ & h g \ rpm) at about c / k? in a continuous flow centrifuge at a flow velocity of from 3 to 50 ml / min and the excess diene is discarded.

The OMPC pellet was suspended in TED buffer (19 μl tamplap 20 mL / g pellet) and Step 2 and Step 4 were repeated twice (omitting Step 3)

Step 5. Product Recovery OMPC

The washed pellets from Step 4 were suspended in 10 ml of distilled water with a glass rod and a Dounce homogenate to ensure complete suspension. The aqueous OMPC suspension was then filter sterilized by passing through a filter of Θ, 22 μl, and the TED buffer was replaced with water by diafiltration against sterile distilled water using a concave fiber filter of æ51 μM EKIS: LD2

Preparation of H. influenzae type b capsular polysaccharide (PRP)

Development of Inoculum and Seed

V

A freeze-dried tube of Haemophilus influenzae type fa? (cultivated from Ross 768, received from the State University of Nen York) was suspended in 1 ml of sterile Haemochyl Intracellular Medium (see below) and this suspension was spread on 9 slopes of Chocolate Agar (BBL), pH of medium the inoculum was adjusted to 7.2 Â ± 0.1 Cum Cum value was pH 7.23) and the medium solution was sterilized prior to use in autoclaves. After 20 hours of instillation at 37Â ° C in a candle pot, growth of each plate was suspended in 1-2 ml of Hasfflaohilus inoculum medium and pairs of slopes were collected.

Hagmophi Inoculum Medium icss g / Liter 1Θ 2Θ SQjâ

NaCl Nâ,

In "HPO. έ. 4] &lt; * 4-ipn volume-ΔCJ-S. li B X.

The resuspended cells from each pair of slopes were inoculated in vacuo containing 2-500 ml of 5% FCI The following steps were performed for 250 m: L for B m B B 37 ° C for about 2 hours. (B) (b), and (b) (b), and (b). OB (below): - (-) - - - - - - - - - - - - - - - - - - - - - »

Stage 2 Erlenroeyer Bottles Not Stressed (Baffled) of 2 liters - 5 mls were used. of the culture of Phase A (I) above to inoculate each of the five two-liter vials, each containing about 1.9 liters of production medium and whole Haemophilus seeds (below). The flasks were incubated at 37øC on a rotary shaker at about 60øC; 3 hours »A value of DQ; at the end of Q &amp; Q was about 10%.

HaeniQphilus Comolsto and Haemophilia)

I- * D Γ * 1. CF CF CF CF CF Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia Dia (1) 10 (1), (1) and (2). 5 w / L NaCl 5.0 g / L Glucose (2) g / L Nicotinamide adenine nucleotide (NAD) <3) mg / Hemin (4) 5 ml / L

The soya salts and psptona were dissolved in small volumes of pyrogen-free water and heated to the correct final volume with additional hot pinhole free water. or flasks were then autoclaved for about 25 minutes at 121 ° C, and after cooling, yeast extract diafiltrate (1), glucose C 2) s NAD C 3), and hematin (4) were added. ) to the vials or fermenters prior to inoculation, (1) Yeast extract diafilirario; were dissolved in 10 ml of yeast extract of the brewer CAmber) in 1 liter of distilled water and ultra-sterilized using an incase Amxcon uu a 3 * ® with 50 kd cartridges. The filtrate was collected. (i.e., from 1 to 4). X L. s U of 0, -X X fi n Already x Cffcj with a cut

(2) The glucose was sterile and distilled water.

(3) A stored solution of the SMAD containing 2 &amp; mg / mL was sterilized by passage through a (0.22 μ filter) and added acceptably just prior to inoculation. (4) A concentrated solution of 3X Hemina was prepared by dissolving 200 mg in ΊΘ ml NaOH Θ. The solution was sterilized for 20 minutes at 120Â ° C and added skeptically to the final medium prior to inoculation.

Phase C Seed Fermenter 7-liter-Tris liters of the Phase B product were used to inoculate a fermenter containing about 4Θ liters of Complete Production and Seed Haemep-hi1 us (prepared as described above) and 17 ml of UCON B62S antifoam agent. The pH at inoculation was 7.4 =

Production Fermenter Phase of 80 ± 1 - Approximately 40 liters of the product of &quot; Phase C &quot; was added to inoculate a 100æl fermenter containing 57Θ liters of Production Medium and Haemophilus Seed (prepared as described above, measured to the required volume), and 72 ml of UCON LB625 anti-spp. The reaction mixture was stirred overnight at room temperature for 1 hour and then cooled to room temperature overnight. After 10 minutes, the reaction mixture was allowed to warm to room temperature. The reaction mixture is then allowed to stand for 1 to 5 minutes, then the mixture is allowed to stabilize for a period of 1 to 2 hours after fermentation. Approximately 6 liters of lotus per harvest in a "north" tank containing 12 liters of timber-

¢ 51.¾ I

CLARIFICATION

For 18 hours at 4 ° C. the io to tox csntri * · &quot; (1) at a flow rate adjusted to maintain the clarity of the product (varying between 1.3 and 3 liters per minute), D supernatant obtained after centrifugation (15ΘΘ rpm) was used to recover the product, the supernatant of two production fermentations was pooled and concentrated at 2 to 8Â ° C in an ultrafiltration unit

Ronticon XM-5® with 20 cc of concave fi bres with a color (c: u.tof f) of 5 (kd) (area of medium) Lpm 137.8 kPa). concentration was such that after approx. hours, about 19Âμl had been concentrated to 57.4 liters, the filtrate was discarded, 48% E-VAN ETHANOL PRECIPITATION

They were added, __ .1. "" "" "" "And" 5% by weight of ultrafiltration of 53 liters of 95% stanol for 1 hour with stirring at 4 ° C to a final concentration of 487% ethanol, The mixture was stirred for additional hours at 4 ° C. s dante was collected at. to follow passage through

pellet was discarded and the clarified fluid was brought to 82% ethanol with addition of 40.7 liters of 95% atanol over a one hour period. The mixture was stirred for an additional three hours to ensure complete precipitation.

(A) (b) (c) (c) (c) (X) (a) Continuous flux-traverse 8-harples of 16 cm at 1 ° C. ; Q ΰ O about 0.4 liters per minute. The resulting 48% ethanol-insoluble precipitate soluble in 48% ethanol was collected by centrifugation in a Sharples flow-through centrifuge from θθ, ΐώ cm to δ5ββ rpm with a flow rate of about 24 liters per minute and was The crude product yield was about 1.4 kg of 82% wet melt in 24.5 ml of the mixture, 24.3 ml of water, and evaporated to dryness. 4 * 0 Γ o 2 liters of CHLORIDE OF CHLORIDE 0 1.4 kq of the ethanol-insoluble material at 82% liters of CaCl2 = 2H2O2, cold; for 15 minutes. The vessel was then washed with 0.1 M CaCl2 Â · 2HCl, resulting in about 500 liters of final volume. Ac-SO WITH 23% ETHAMOL

The 5 €? 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 12, 13, 16, 16, for 30 minutes. c. In the case of ethanol, the amount of ethanol in the reaction mixture is approximately 1%. The supernatant was collected and the pellet was discarded.

PREPARATION WITH ETHANOL AT -58 ° C AND COLLECTED PRODUCT PASTE The 25% ethanol-soluble supernatant was brought to 38% ethanol by the dropwise addition of 13.9 liters of 95% et-anal with stirring, over a period of 38 minutes. The mixture was then allowed to stand under stirring for one hour, then without stirring for 14 hours, to ensure precipitation. tion. The mixture was evaporated to dryness. The crude product was then centrifuged for 10 minutes at a flow rate of 0.2-1 A. per cent (20% by weight) per liter of powder. from H. inf lu censae br rpm

The centrifuge pellet was transferred to a 4.545 liter Waring blender containing 2 to 3 liters of absolute ethanol and mixed for 30 seconds at higher speed. The mixture was continued for 30 seconds (on &gt;, and 30 seconds off Cof) until a hard white powder resulted. The powder was collected on a Buchner funnel with a Teflon filter dial and washed sequentially in situ with two 1 liter portions of absolute ethanol and 2 liter portions of acetone. The material was pre-dried in vacuo at 4 ° C for 24 hours, resulting in about 337 g (dry weight) of the product.

EXTRACOKD WITH FENDL pa · SSQ of rO · · on 12 one-way was QS f! sodium ene

About 168 grams of the dry crushed material (about half of the total) were resuspended in 12 liters of 0.488 M sodium acetate, pH 6.9, with the Daymax dispersion aid. The acetate solution was extracted with 4.48 liters of a fresh aqueous solution followed by 590 ml of sodium acetate,

5 483 (1), pH 9> were added to each of the eight bottles (50 kg OB) in a pressure vessel of 20 liters and mixed in the suspension. Each phenol extract was centrifuged for 2 hours at 30 DEG C. The aqueous effluent was extracted three additional times with fresh aqueous phenol solution as described above. The phases of? phenol were rejected "

The aqueous phase of the above first phenol extract (12.2 liters) was diluted with 3θ θ liters of fresh distilled water Amicon S EJC-30, Η 10ΡΘ para, to which was washed to 31.3

Lithocyclohexylamine at a concentration of 3: 1: 1. The reaction mixture was cooled to 0 DEG C. The reaction mixture was cooled to 0 DEG C. The reaction mixture was cooled to 0 DEG C. The reaction mixture was cooled to 0 DEG C., anol completely, and the filtrate was dried in vacuo using 3 parts of wash added to the residue so that the volume of the ultrafiltrate was discarded. ETHANOL PRECIPITATION At 87%

The volume of the dialysate of the step at the final pH was 0.15 and the solution was added dropwise. w q to C | x ti to ÇS; 48.5 5% ethanol 95% ethanol. After serving for 12 hours at 4 cc. The suspension was cooled to 0 ° C for 5 minutes. After stirring for 2 hours, the mixture was shaken for 2 minutes and the mixture was shaken for 3 minutes. in a funnel of Buchner et al. , and washed. in situ, with .j. ^ ^ &amp; of all the extracts were dried over magnesium sulfate, dried over magnesium sulfate, filtered and evaporated to dryness, followed by two 1-liter portions of acetone. The sample was then dried on a heavy plate, in vacuo, at 40 ° C for 24 hours. The yield was about 120 grams of dry powder. The surrender

ULTRACEWTRIFUBATION IN ETHANOL 29% AND COLLECTION OF FINAL PRODUCT

The 212.6 grams of the above dried powder were dissolved in 82.9 liters of distilled water, to which 2.13 liters of CaCl2 = 2H2O, 0.05 M CaCl2), 2.5 mg of polysaccharide / mL), &amp; 3. my you. The reaction mixture was cooled to -78 ° C. From 29, 86 liters from 1 to 4 years, and from 1 January to 1 January. The composition is then cleaved by a diluent in a diluent in the presence of a distilled water. (30000 rpm) of 15 ml per flask volume (min) of 4 KC. The reaction mixture was filtered and the filtrate was evaporated to dryness. The filtrate was evaporated to dryness, and the residue was evaporated to dryness. Λ ____ LlSij. to x, After ii ti ·? In addition, the present invention relates to a process for the preparation of a compound of formula (I). The reaction mixture was stirred for 4 hours at room temperature for 17 hours. The precipitate was dried over magnesium sulfate and evaporated to dryness. The precipitate was evaporated to dryness, The resulting slurry was charged to a mixed Waring of 4, 54o in the tent with 2 liters of absolute ethanol and mixed at the highest viscosity, or 5 cycles from: 30 seconds to 5 seconds off until a hard white powder is formed. This powder was collected on a Buchner funnel equipped with a Zifcex teflon disc and sequentially washed in situ with two 0.5-liter portions, fresh and a 1-liter portion of absolute ethanol, ε with two portions of ice . The product was removed from the funnel and transferred to a heavy

drying in vacuo at 4Â ° C (for 25 hours), the final yield of the product was 79Â ± 1 grams dry weight. gXEMPL0 "3

Cloning of the genomic DNFT encoding the MIEP

I

About 1æg of the phenol-inactivated N. maningitidis cells Cver Example 1) were placed in a fresh tube. Phenol-inactivated cells were resuspended in 567æl of 1 mM TE ETR1S-HC1 TE buffer, 1 mM EDTA, pH 8, 3 3. Resuspended cells were added 30 μg of 10% SDS, and 3 μl. of 2 mg / KiL proteinase! (Sigma) cells. The cells were mixed and incubated at 37 ° C for about 1 hour, whereupon 1 μg of NaCl was added and vigorously stirred. 8æl cetyltrimethylammonium bromide (0.8% CTAB) in NaCl, 7H were then added, mixed vigorously, and incubated at 65 ° C for 1 minute. An equal volume (about 0.7 to The aqueous phase (upper) was transferred to a stirred solution of chloroform / isoquinoline alcohols (at a rate of 24Âμl respectively), mixed vigorously and centrifuged at about 5Â ° C. a fresh organic phase was discarded. Was it added to the aqueous phase? an equal volume of phenol / chloroform / isoamyl alcohol in a 25: 24 ratio, respectively), mixed vigorously, and centrifuged at 40.degree. The aqueous (upper) phase was transferred to a new tube and 6 volumes (about 42 μl) of isopropyl alcohol were mixed vigorously and the precipitated DMA was centrifuged at 1 ° C. The supernatant was discarded, and the pellet was washed with 70% ethanol. The DMA pellet was dried and resuspended in 10æg TE buffer, s represents the N genomic DNA, meningitidis.

Two DMA oligoreclotides that correspond to the 5 'end of the MIEP gene and to the 3' end of the HIEF gene were synthesized. CMurakamij E.C., et al. &Lt; 1909) &quot; Infection and Immunity, 57. pg. 2318-233. The specific DMA Dligonucleotide sequence for the 5 'end of the MIEP gene was 5'-ACTASTTQChsAAAAAATCCCTh-e to the 3' end of the MIEP fois 5 * BAATTCASATTA8BAATTTSTT-3 gene. These DNA oligonucleotides were used as primers for the amplification of CPCR polymerase chain reaction) of the MIEP gene using i. nanograms of N. meningitidis genomic DNA. The FCR amplification step was performed according to procedures provided by the manufacturer (Perkin Elmer). The amplified MIEP DMA was then digested with the EcoRI restriction endonucleases. The 1.3 kilobase DNA fragment &lt; kb 5, containing the complete coding region of MIEP, was isolated by electrophoresis on a 1.5% agarose gel, by gel electrophoresis, , &lt; 1987), Ausubsl, R Η = E s, Hoo re, D.D., Smith, d .A., Sei · * = 0 2. Ç>; ρΗ '3 · .; 8reene Publishing Assoe = -.] =, The vector plasmid pUC-i9 was digested with Spel and EcoRI. The gel-purified Soel-EcoRI DNA was ligated into the vector pUC-19-Sodium-EcoRI and was used as a template for transfection of the MIEP DNA. JP kpb were obtained from a restriction endonuclease map, and the MIEP DNA was sequenced to assure the distribution of the vector pcl / 1 .Gai 10p (B) FsB'1; ____s. The Sal i ύ ΐαχ promoter isolated from plasmid Q. CBroachet et al., (1983), "Experimental Manipulation of Expression, InouePl (Ed.), Et al.

The gel purification gene of the 5 kilogram base pair fragment (kpb) obtained after cleavage of the 5 'Hind 511 HindIII terminator was isolated from the vector pBAP =, tHDH2 CKniskern. et al, &lt; 1986), the extracts were made with a gel of the 1.1 V C fragment with the Hind III Ϊ So So So So Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os Os. pUCISdeltaHind 1 (QS x Hind III III was deleted by digestion of the pUC18 Hind III fragment with cohesive termination with the fragment

Kienow from DMA polymerase and E. coli, and DMA binding to T4 and gt; which had been digested with BamHI and Sohl to create the vector of the pGal1Θ-tADH1 origin. This has a unique HindIII cloning site at the junction Sal ΙΦρ ,, ΑΟΗί

The SalIθρ single-strand Hind III clonagogal site was changed to a single BamHI cloning site by digestion of Sal 10p-tADH1 with Hind III. gel purification of the cut DMA, and ligation, using DMA ligase and T4, to the following ligand Hinç [IΣ ΐ - BamH I s

5? ASCTCGGATCCtó-3 3 &quot; -BCCTAS8CTCG plasmid? The resulting HindIII site generated the HindIII site generated a unique BamHI site.

the transformers were unique from the HindIII line of the vector, pIII.8alli with Hind III and BaMHI., fragments Gelato * tADHi fax isolated from pB &amp; CBBC) tHDHi by digestion with SmaI and SphI, terminated co- DMA polymerase T4, and purified by gel, the yeast virus vector pCl / 1 EBrake et al., &Lt; 1984), Proc. Nat'l. Acad, Sci., U.S.A., 61, pg, 4642-46463 was digested with SphI «terminated cohesively with unpurified T4 DWA polymerase. This fragment was ligated into the vector with DWA ligase. The ligation reaction mixture was then used to transform cells from E. ccsi111 to ampicillin-resistant cells, and hybridized to a line 3.

The new construct BamHI was confirmed by digestion

EXAMPLE

Construction of an Evprc Vector Sequences of DMA primers are 11 æl of Yeast with π · t%. IQtóv}

A complete DMA fragment of MIEP was generated by Spel and EcoRI, purification in parallel with polymeric DMAs containing the coding region by digestion of pUC19â "¢ MIEP # 7 with MIEP DMA gel, terminated and T4. The inner yeast pressure vector pC1 / 2, Ga11P- <B) AuH1 was digested with the polymerase gut alkaline. BamHI, dephosphorylated with foil, and terminated cohesively purified on gel to remove the sphatase with a vector, the fragment, terminated cohesively, 1.1 kbpd

nXEP 1iq pCl / i, BalΙΦρίEOAdHl t! finished

cohesively, ε the reaction reaction mixture was used to transform competent E. coli DH5 cells into ampicillin-resistant cells. The transformers were analyzed by hybridization to a DNA oligonucleotide labeled &quot; P ';

DNA preparations have been made from the formation of positive transformants and digested with KpnI and SaIl for the preparation of a peptide having the amino acid sequence set forth in SEQ ID NO: 1. verify that the MIEP fragment was in the correct orientation for expression of the Bali promoter. Further confirmation of the DNA construct was obtained by dideoxy-sequencing of the Î ± 1Î ± promoter in the MIEP coding region, Î ±, the βpression of the MIEP by the transformants was detested by Western blot analysis. Recombinant MIEP produced in the transformants co-migrated on polyacrylamide gels with purified MIEP from OMV vesicles, and was immunologically reactive with MIEP-specific antibodies. EXAMPLE 6

Construction of a Yeast MIEP Expression Vector with a 5 'J Modified MIEP DNA Sequence ---------------------------- ---------------------------------------

A DMA oligonucleotide containing a HindIII site, a conserved yeast 5 'untranslated (leader), a methionine initiation codon (ATB), the first 89 codons of the mature MIEP (starting with Asp at the + 2 posição position ) and a Kpnl site <at position +89) was generated using polymer chain reaction (PCR) technique = PCR was performed as specified by the manufacturer (Perkin Elmer Cetus) using the plasmid

CDCIHD pUC19Mi.EP42 # 7 coroo the matrix ε the following DNA precursors? b'CTAAGCTTAACAAAATSSACGTTACCTTGTACGSTftCAATT3 'b * ACSGTACC8AASCCBCCTTCAAC3 ..

To remove the 5 'region of the ΜΊΕ cl clone, plasmid pUC 19β11EP42 # 7 was digested with ΚρηI to HindIII and the kpb vector fragment was purified on agar gel. The 280 ph PCR fragment was digested with KonI and HindIII , purified on agarose gel, and ligated with the 3.4 kbp vector fragment. The transformants of E. coli HB101 CBRL) were analyzed by hybridization of DMA oligonucleotides and the DNFT of positive transformers was analyzed by restriction enzyme digestion. * To ensure that mutations were not introduced during the PCR step, the generated DMA by PCR of 280 bp of the positive transformants was sequenced. The resulting plasmid contains a HindIII-EcoRI insert consisting of yeast NTL, AT8 codon, and the complete open reading frame (CORF) of MIEP starting at the Asp amino acid codon +20).

Yeast MiEP expression constructs were constructed as follows. The pSali0 / pcl / i and p-SAP / pCi / i vectors CVlasuk, B.P., et al., (1989) 264 * pg * 12106-121123 were digested with BamHI. non-cohesively terminated (flash) with the Klencw fragment of DNA polymerase I, and dephosphorylated with calf intestinal alkaline phosphatase. These linear vectors were ligated with the treated Klenow and the gel purified HindIII? coRI fragment described above, which contains the NTL of yeast, ATB and MIEP ORF and forming pBal / pI / MIEP and pSAP / pBAP / pC1 / EP. The strain Bacoharomvces cerevisiae U9 Î "lgÎ ± pgal4-) was transformed with the plasmid pSali0 / p / pct / MIEP. The clones

Recombinant isolates were examined for the expression of MIEP. The clans were made to grow. 37 ° C with shaking in sinicric medium (leu -) containing 2% glucose &lt; w / v) to a DMF of about 10%. 2% (w / v) galactose was then added to induce MIEP expression from the Sal10 promoter. The cells were grown for an additional 45 hours as follows. The cells were then harvested by centrifugation. The cell pellet was washed with distilled water and frozen. The cell pellet was washed with distilled water and frozen.

Western Blot for ΜΪΕΡ Spare part

To determine if the yeast was expressing MIEP, Western blot analysis was performed. Novek Laemmli gels were used from 1 to 15 wells, at twelve percent. Yeast cells were plated in H₂O using glass beads (sodium dodecyl sulphate (SDS &gt; 2% can be used during the breaking process). Cell debris was removed by centrifugation for 1 minute at &gt; the supernatants were mixed with sample development buffer , as described for the purification in qsl of polyacrylamide of MIEP. Samples were grown at 35 mA using OMPC as a reference control until the bromophenol dye label exited the gel for nitrocellulose paper. of nitrocellulose was 5% in saline, which was added anti-rabbit MIEP on gel using

Proteins were transferred to 0.45 μm pore size using NOVEX. After transferring the blocked albumen with phosphate buffered bovine serum albumin for 1 hour, after 15 ml of a 1% dilution. (generated by immunization with purified HIEP

standard procedures) After incubation overnight at room temperature 15 ml of a 1% dilution of goat anti-rabbit IgG conjugated to alkaline phosphate were added. After 2 hours incubation the stain was developed using FAST RED TR SALT CS (q) and Naphthol-AS-MX phosphate (Sigma), EXAMPLE 7

Expression of Recombinant HXEP The pUC19-MIEP plasmid containing the Î »3 β-base pair ΜΪΕ inser gene insert was digested with restriction endonuclease S and EcoRI. The 1.1 kbp fragment was isolated and purified on an agarose gel using standard techniques known in the art. Plasmid pTACSD, containing the two-channel TftC promoter to a single ECORI site, was digested with ECORI. Cohesive films were formed both in the 1.3 kbp MIEP DMA and in the pTACSD vector, using DMA palimerass T4 (Boehringer Mannheim) according to the manufacturer's directives, the cohesively terminated 1.3 kbp MIEP DMA was bound to the cohesively terminated vector using DMA T4 ligase (Boehringer Mannheim) according to the manufacturer's instructions, the bound DMA was used to transform E. coli strain BH5aIQMAX (BRL) from those were plated on Sr-plates containing 25æg canamicide / ml. and € 5 &gt; μg of penicillin / ml and incubated for about 15 hours at 37 ° C. A DMA oligonucleotide with a MIEP homologue sequence was labeled with P and used to analyze nitrocellulose filters containing denatured colonies lysed from plaques transformants using standard DMA hybridization techniques. Colonies that were positive by hybridization were mapped using restriction endonucleases to determine the orientation of the MIEP gene. The expression of the MIEP by the transformants was detected by Western blot analysis. Combinylated MIEP would produce on the meridian transformants on psol iacri gels licked with purified MIEP from OMPC vesicles, and was immunologically reactive with the MIEP-specific antibodies.

EXAMPLE S

Preparation of Purified MIEP from OMPC liposomes or from Combinating Cells by Electrophoresis in Polyacrylamide Synthesis 1.0 D. gel,

The acrylamide / BIS gels (37.5), 18 κ 14 cm, 3 mm thick were used. The agglomeration gel was 4% polyacrylamide and the gel separation was 12% polyacrylamide. Approximately 5æg OMPC protein or cell protein, recombinant host by gel. To 1 mL of OMPC (50 mL) was added 4% of the sample buffer, 4% glycerol, DTT 3.0%, TRIS 10000, 0.001% bromophenol blue, pH 7.0). The mixture was heated at 105 ° C for 2 hours. minutes and allowed to cool to room temperature before being loaded onto the gel. The gel was developed at 20Â ° C for 1 hour with cooling to the bottom of the gel. It was cut from 1 to 2 cm (1%) of the strips. was discolored until Kd) became VX sivel ... A faix

The band was then placed in its original gel position and the MIEP area was excised from the rest of the gel using a scalpel. The excised area was cut into cubes (5 mm thick) and alloyed with HDwS buffer The elution was evaluated for purity by SDS-PASE. The eluate is combined with a common eluent assembly and dialysed at 60 mSi. i. can be dialyzed Ϊ! 1 "! Syllable% -v 4. a. ww νΛ protein equilibrated up to è. dryness The material was further purified by passage through a PD10-size column Pismataway, Piscataway, NJ), and stored at room temperature. EXAMPLE 9

Vehicle Activity of MXEP in a PRP-OMPC Covalent Conjugate

Male C3H / HeN mice (Charles River, Wilmington MA) were immunized intraperitoneally (IP) with PRP covalently bound to OMPC CPRP-uMPC? comprising 2.5 pg of PRP and 17 μα of OMPC) or PRP coupled to iPEP-DTn DT containing 2? 5-5? 7? PRP and 1.8-5? 4 DT?) CConnaught Laboratories , Willowdale, ONT), suspended in θ, 5 ml of phosphate buffered saline (PBS), were added. A second group of male C3H-HeN mice received either 17 μα of MIEP, 17 μg of OMPC, either 17 pg of OMPC-1AA (OMPC derived with N-acyl-homocysteine, thiolactone, closed) with iodoacetamide). Cell donors for adoptive transfer experiments were immunized twice IP, 21 days apart, and spleen cells were harvested 10 days after the second immunization. The foster transfer recipients were male C3H / HeN mice giving a. gamma irradiation in the total body of 5μg from a source of Cs and reconstituted immediately by intravenous injection of 8 κ10 spleen cells from each of two syngeneic donors separately inactivated with PEP-DT, and OMPC, MIEP , or OMPC-ΣAh.

Control mice received x1 S4 U sfi spleen cells

-S &quot; mice inoculated with PRP-QMPC and an equal number of dactyl cells from an uninoculated donor mouse. ISA were prepared in accordance with the procedure described in Marburg, et al., U.S. Pat. No. 4,882,317. , This PRP-derivatized chromate-filtered fax Sephadex 8-25 in 0.1 M sodium bicarbonate buffer, pH 8.4. N-Hydro-Kissuccinimidobiotin (Pierce Chemical, Rockfard, IL) in dimethylsulfoxide was added to the eluate at a final concentration of 0.3 mg / ml and reacted in the dark for 4 hours at room temperature (about 05-28 ° C). The unreacted β-hydroxysuccinimidobiotin was removed by filtration and evaporation, , G "in FBS. Pleated (Cambridge, MA) dye polyvinyl chloride ELISA plates were coated with 5æg / well of avidin (Pierce Chemical) at 1æg / ml in sodium bicarbonate buffer Μ, pH 9, b during night at room temperature and moisture of XW%. The plates were washed 3 times with 0.05 M TRIS-buffered saline, pH 8.5, containing ≥5% Tween-20 (TBS-T), and blocked with TBS-T plus gelatin a (blocking buffer) at room temperature and 10% moisture for 1 hour. Plates were stained without lavage. 50æg / well of ρρ.ρ "κ | α ^^ η5 was added in pgs at 15-40 ° C pg / ml, and plates were incubated for 1 hour. The plates were washed 3 times with TBS-T before sample addition. Samples were serially added two-fold in blocking buffer, and incubated for 2 hours at room temperature and 100% humidity. The plates were then washed 3 times with TBS-T, and appropriate alkaline phosphatase conjugated anti-immunoglobulins diluted in blocking buffer were added. The antibodies used were goat anti-mouse IgH (Jackson Immunoresearch, West IgB (Fc5 (jackson Immunoresearch), IgBl (gamma) CBRL, Baithersburg,,

IgG2a (gamma) (IgRL), Ig82b (gamma) (Southern Biotechnology Associates, Birmingham, ALK IgS3 (gamma) (Southern Biotechnology Associates), and goat anti-cosme IgG (Jackson Immunoresearch) plates were incubated for 2 hours at (%), washed with blocking buffer, and the development of the substrate was performed using p-n-triphenylphosphate (1 mg / mL in 1 M Alkanolamine, Kirkegaard and Ferry, Gaithersburg, MD). dilutions were considered positive if the diphosphorance of the sample exceeded the mean absorbance by more than 3 times the standard deviation of white reagents if the difference in absorbance between successive titers was greater than, were defined as the reciprocal of the highest dilution which gave a positive reaction in the ELISA as described above. The logarithms of the reciprocal titers were compared between the treatment groups by two-way analysis of the variance CLindeman, R.H., L., et al., Introduction to Bivariate and Fluctuariate Analysis, Scott Forssman &lt; puh.), Nev. York3.

B1Ê .to ..... guantifice. anti-PRF antibody

Experimental serum samples to be tested for the amount of anti-PRP antibodies were diluted to β2, β5 and β 20 using fetal calf serum as the leader. 25 Âμl of each diluted serum sample was transferred in duplicate into 0.5 mL RIA vials (Sarstedt). A PRI-labeled solution of JI was diluted to give 300-8 ° C counts per minute (cpm) for 5 & pL using phosphate buffered saline as the diluent, 50 Âμl of diluted β-I-PRP were transferred to each RIA flask, mixed vigorously and incubated for about 15 hours at 4 ° C "75 μl" of a saturated ammonium sulfate solution at 4 ° C were added to each RIA flask, vigorously mixed and incubated at 4 ° C for 1 hour. The RIA flasks were then centrifuged for 10 minutes at 0 ° C , α sohrenatestin was rejected and the cpm in the pellet was mediated by the g-sm-a counter.

A standard curve consisting of series of two dilutions of an antiserum containing a known amount of anti-PRP antibodies was prepared as described above and were assayed concurrently with the experimental serum samples. The amount of anti-PRP antibodies in the standard curve is between 14 pg / mL as the highest amount of antibodies and &amp; 5 &amp; The mean cpm of the duplicate samples was compared to the standard curve for calculating the amount of anti-PRP antibodies present in the serum samples exoerimals'

Antibody responses from adoptive transfer recipients Spleen cell containers inoculated separately with PRP-DT5 and either MIEP or GMPC or 1AA-QMPC, responded to PRP-OMPC immunization by producing equivalent amounts of anti-PRP IgG1 antibody and serum IgGa at 4 days &lt; see Figure 15. Irradiated mice reconstituted with spleen cells that were inoculated with either MIEP or OMPC or I-AA-OMPC vehicle had significantly higher anti-PRP IgG1 antibody titers &lt; p &lt; , Î »max, and Ig82λ 4) after immunization with PRP-OMPC than the control mice, giving spleen cells inoculated with PRP-DT but not with OMPC-inoculated spleen cells. Serum antibody responses è. immunization with PRP-OMPC in mice given spleen cells inoculated separately with PRP-DT and either MIP or OMPC or IAA-OMPC were comparable to those in mice given PRP-OMPC inoculated spleen cells (ρ &gt; β , 12 for IgB1 antibody on days 13, and ρ> 5 for IgS2a antibody on days 9-13). No response was observed

antibody when irradiated mice reconstituted with PRP-DT spleen cells inoculated with and inoculated spleen cells or with MIEP or. with OHPC were immunized with PRP without a carrier of protein. Statistical analysis was done by two-way analysis of variance (AMOVA &gt; CLindeman, R.H. et.

Introduction to Etl variate and Multiple Analysis, (1983), Scott Foresman (pub. &Gt;, New York)

These results demonstrate that MIEP worked in mice as well as OHPC to induce a vehicle helper T cell response to generate anti-PRP IgB antibodies. EXAMPLE 10

Mitotic activity of MIEP The MIEP purified from N OMPC, girl itid was assayed for mitoenaic activity. in a lymphocyte proliferation assay. Splenic lymphocytes from murine were obtained at. from mice C3H / HsN, C3H / FsJ, C3H / Heu, or Balb / Cn. The mice were either naturally or had been previously vaccinated with PRP-OMPC. The spleen cells were passed through a sterile fine mesh screen to remove stray debris and suspended in K 2 O 16 'medium (BIBCD) 10% fetal calf serum (Armor), 6mLthamine 2mH (BIECO) , 10 mM Hepes (BIECO), 10 μg / ml penicillin / 100 μg / ml streptomycin (IGCI)) and 50 μM (3-mercaptoethanol) (Eioracl) 3 Following pipetting to break cell groups, the suspension was centrifuged at 3Θ6

and the pellet was resuspended in a Utt-1c cap of 1 æl and the guinea fowl were placed at 90% (Fisher). TRIS-HCl 0.7 M a 10% (sigma), pH 7.23 at room temperature. Î'1 ml of cells / ml L of buffer for two minutes. BS cells were stratified from calf fetus and SsTiL from

COfB cells centrifuged at 4ΘΘΘ κ g for half K. Two cells were resuspended. These cells were implanted μl / well) together with 1? and then washed in K medium at 5 cells / ml. 96-well plates (1 μg of protein or sample of

A solution of the title compound was prepared as a white powder, dried (MgSO4) and evaporated to dryness. PR: p-QTPC and i. X-ray diffraction (X-ray diffraction) of the X-ray diffraction (X-ray diffraction) of the X- J α α α α α α α α α α α α α α * * * * * * * * * * * * * * * * * * * * * metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade metade ^ 3 each type of 1-in-base cavities in three stages suspended from the preparation only in c1u1ar

as described above include DMPC itself, and the stras were diluted 265 52, 105, and 130 above so that their concentrations were also incubated to determine the 3, 5, or 7 culture, the wells were 1æM H-thymidine (Amersham) containing 1 mCi / 25 s were collected 16-18 hours later in one, and the counts per minute &lt; CPH &gt; were liquid scintillation measurements * The network change in cpm was

We were pulsed with 25 μl of the Skatron collector cavity in a &gt; calculated by subtracting the cavity from per cells only in such &lt;. The stimulation index, mean number of cpm taken by K, of the mean of the experimental cpm was determined by dividing the average mean cpm by the mean of pm of the control wells.

As shown in the F vaccine the OMPC and PRP-OHPC populations of vaccinated mice did not appear to be due to HIEP being free of LF's pyrogenicity in rabbit, and Figure 2, the HEP vaccine as well as resulted in lymphocyte proliferation. ·-previously. This &lt; LPS) mitogenic-lipopolysaccharide activity since detestable, as measured by assays of the proliferative effect was greater than

than that which might have been caused by. The results of the present invention are summarized in the following table. EXAMPLE 11

Conjugation of the polysaccharide i -RP d N '

The ionic compositions were conducted in accordance with the teachings of US Pat.

1Θ mg of Μ¹ΈΡ eu were mixed? 3 mL of 0.1 M borate buffer, pH 3, pH 7.4, and pH 7.4. ) and 4 mg of dithiothreitol (Sigm to Chemical). The solution of the particles was vigorously flushed with H2. 125 mg of N-acetyl-homo-cysteinathiolactone &lt; Aldrich Chemicals) was added to the solution of EPl, and the mixture was incubated at room temperature for 16 hours. It was then double-dialed under N2 against 2 L of borate buffer pH 9.5 containing 4000 EDTA for 24 hours at room temperature. The thiolaria protein was then assayed for the content of uncle! by Ellman's reagent (Sigma Chemicals) and the protein concentration was determined by Bradford reagent (Pierce (Chemicals). For the conjugation of i.e., to PRP, a 1.5 fold (w / w) excess of PRP of H. influenzae serotype b bromoecyclic to the MIEF solution and the pH was adjusted to 9-9.5 with 1N NaOH. The mixture was allowed to incubate for 6 to 5 hours at room temperature. At the end of the reaction time, 25 μl of N-acetylcysteamine (Chemicals Dynamics) was added to the mixture, and was allowed to stand for 13 hours under N ° at room temperature. The conjugate solution was acidified to between pH 3 and

4 with 1N HCl, and centrifuged a. 1 ΘΘΘβ κ g for -10 minutes. 1 ml of the supernatant was applied directly to a Superose 6B FPLC (1 6 κ 5% cm cm-1 Pharmac. Ia) column. eluted with PBS. The empty volume peak containing the polysaccharide-protein conjugate (PRP-MIEP) was pooled. The conjugate solution was then filtered through a Θ 22 μ filter for sterile ileum. EXAMPLE 12 *

Demonstration of Immunogenicity of PRP-MIEP Conjugates

Immunizations Male Balb / c mice (Charles River, Illilmington, HA) were immunized ΊΡ with PRP covalently conjugated to MIEP as described in Example 1i, using 2.5 μα of PRP at Θ55 mt. of preformed alum. Control mice were immunized with equivalent amounts of PRP given as PRP-CRII EAnderson, M. et al., (1985), J. Med. pg. 346-3513 &lt; 2.5 pg of PRF / 6.25 μα of CRM; 1/4 of the human dose), PRP-DT (2.5 pg PRP / 1.8 pg DTξ æ / ΊΘ human dose such that constant amounts of PRP were used), and PRP-OMPC (2.5 μg of PRP / 35 pg of OMPCs and 1/4 of the human dose)

The mice were immunized with each of the two vaccines. Each monkey received immunizations for immunization on days, blood, all of the two mammals, oestradiol, xanthine, PRP conjugates -IMP adsorbent in 2 ml of conjugate at two different sites - a total dose of 0.5 ml. The monkeys were 28, and 56, and samples were taken for four weeks.

The antibody responses were made using the ELISA described in Example 9, which assigned the subclasses of the immunoglobulin RIA response that quantitates the total anti-PRP antibody (see Example 9) was also used to evaluate the monkey's response. The antibody responses of PRP-MIEP conjugate containers are shown in Figure 3.

The results show that PRP-MIEP conjugates are capable of generating an immune response in mice consisting of anti-PRP IgS antibody and a memory response. This is in contrast to PRP-CRn and PRP-DT which do not induce anti- -PRP measurable. Thus, PI1I works as a

It will produce an anti-PRP antibody response when covalently conjugated to the PRP antigen. The purified MIEP is therefore an effective immunological vehicle protein replacing the heterologan QliPC in the construction of bacterial polysaccharide conjugate vaccines. E XEMPlU 13

Induction of Interleukin

following MIEP exposure,

American Type Culture Collectors designated ATCC TIB 214) [BilliSg S, 5 b Smith, K- (1977) Mature 268, pg. 154-1561 were maintained in RPMI 164® <GIBCQ> 5 supplemented with Defected Bovine Fetal Serum (HyClone), 2 μM L-glutamine (8IBC0), im μ / mL penicillin, Streptomycin ΪΘΘ μπ (GIBCQ) , 2-β-mercaptoethanol 2ÂμM BiBrad), Rat T-Cell Foliation (%), Labeling Research, Inc.). CTLL cells were seeded every 2 to 3 days to 1-2 cells / ml. Supernatants were tested for peripheral blood lymphocytes at 4 x 5æg / ml, 5æg / ml PRP-OMPC,

&lt; / RTI &gt; with MlhF

Serum of Bovinc X.-.begin) at 50 μm / ml 5 or Sa (Ts% çXQ in a plac-d-6 · cavity <C oSta tr) 3 having volum are equal to i- η Η λ W A supernatant is used for in vitro fertilization for 2 to 3 or 4 days in culturing the embryos. The T-cell assay was prepared as in SCR. Gilli et al., J. Immunol., 120 (120). 2027 &lt; (97%) for 5 to 10 minutes after dilution of 100 μl. .............. The cells were incubated for 1 hour in the above medium without rat-T cell polyhelion. All samples were prepared in triplicate in a 96-well plate ( The plates were incubated overnight at 37 ° C, 50% CO2. Each well was pulsed with 1 ÂμCi of β-thymidine (Amersham) for 10 hours, collected on an immediate BetaPlate CPharmacia filter A standard curve of IL-2 was determined simultaneously using recombinant human IL-2 (Cellular Products, Inc.). Quantitative determinations of IL-2 were calculated using the methodology de Oillis et ah, J "

Immunol. pg. 2027-2032 (1978) The &quot; Probit &quot; using Recombinant IL-2 defined 1 U / ml IL-2 as 50% of maximal H-thymidine incorporation. The test supernatants were also expressed as a percentage of the maximal H-thymidine incorporation from which the U / mL of γ-γ-mâ,,â,, could be determined. Figure 4 shows the result of this experience 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 time period '

Claims (6)

A method for increasing the immune response to antigens characterized by comprising administering a protein in substantially pure form, purified from the outer membrane of a Gram-negative bacterium, in a range. A method according to claim 1, characterized in that it comprises administration of the Class proteína protein of the outer membrane of Meisseria meninitidis. serogroup B, in the purely purely pure form. The method of claim 2, further comprising administration of an antigen or antigens, . A method as in which the antigens are derived from mammalian cells, fungi, rictecia, allergens, toxins or poisons, synthetic peptides, and polypeptide fragments. A method for increasing the immune response to antigens characterized by comprising administering a recombinant outer membrane protein of a Gram-negative bacterium, produced in a recombinant host cell, in a dosage range of at least 2 pg. A method according to claim 1, comprising administering a recombinant Class II protein from the outer membrane of Meisseria meningitidis serogroup B, produced in a recombinant host cell, also comprising administering an antigen or The method of claim 1 wherein the antigens are derived from bacteria, viruses, mammalian cells, fungi, fungi, allergens, toxins or poisons, synthetic peptides and fragments of polypeptides, Method for increasing the level of cykines in a mammal characterized as comprising administering a protein in substantially pure form purified from the outer membrane of a Bram-negative bacterium in a dosage range of at least 2 μ A method according to claim 9, which comprises administering the Class II protein of the outer membrane of Mecoseria msninqitidis, serogroup B, in substantially pure form, A method according to claim 9, which comprises administering a recombinant Class II protein of the outer membrane of Nsisseria meningitidis, serogroup B, produced in a recombinant host cell. A method for increasing the level of interleukin-2 in a mammal which comprises administering a protein in substantially pure form, purified from the outer membrane of a Branv-negative bacterium, in a dosage range of at least 2 pg. A method according to claim 12, characterized in that it comprises the administration of a membrane-precoating protein of a Gram-negative bacterium, produced in a recombinant host cell, A method for increasing the level of administration of Neisseria boyi, in a range of a mammal, comprising Class II protein of the outer membrane of the tetrad serogroup B, in substantially the form of at least 2 μg / ml The invention also relates to the use of a pharmaceutically acceptable carrier according to any of the preceding claims, in which the pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier is a pharmaceutically acceptable carrier. , Class II protein is expressed as a percentage of the total protein, Official Agent of Industrial Property RUA VtCTOR CORDON, 10-A 3. · 1200 LISBOA