BG60630B2 - Covalent modified polyanionic bacterial polysacccharides, their stable covalent conjugates with immunogenic proteins bound to hybride molecules, and methods for the preparation of the polysaccharides and conjugates and for proving the covalency - Google Patents

Abstract

The invention relates to covalently modified bacterial polysaccharides and proteins, their covalent conjugates linked by a hybrid engineered molecule that allows for proof of covalentness and facilitates the purification of the conjugated product with protein from an immunogenic bacterial membrane or other proteins. Conjugates are components useful in bacterial vaccines. The invention also relates to methods of preparing said polysaccharides, proteins and conjugates and to demonstrating covalent linkages between polysaccharides and proteins.

Description

(57) The invention relates to covalently modified bacterial polysaccharides and proteins, their covalent conjugates linked through a hybrid molecule that allows for the demonstration of covalence and facilitates the purification of the conjugated product with a protein from an immunogenic bacterial membrane or other proteins. Conjugates are components that are useful in bacterial vaccines. The invention also relates to methods for the preparation of said polysaccharides, proteins and conjugates and to demonstrate the covalency of the relationship between polysaccharides and proteins.

Claims (54) covalently modified polyanionic bacterial polysaccharides, the stable covalent conjugates with immunogenic proteins associated with hybrid molecules METHODS FOR MAKING THE REFERRED TO SUCH POLYSACCHARIDES AND CONJUGATES AND DEMONSTRATION OF covalently

The invention relates to covalently modified bacterial polysaccharides and immunogenic proteins, to their covalent conjugates linked by a hybrid molecule, which allows for the demonstration of covalency and facilitates purification by immunogenic bacterial membranes or other proteins that are conjugates to bacterial conjugates.

BACKGROUND OF THE INVENTION

Purified encapsulated bacterial polysaccharides have been used to create vaccines against related bacteria, but the resulting immune responses are often less satisfactory than desired, especially in very young children or individuals with imperfect or deficient immune systems. For example, the encapsulated polysaccharide of Haemophilus influenzae type b cannot provoke an immune response in children, rendering this polysaccharide ineffective in the occurrence of serious pediatric medical problems caused by H. influenzae type b. Enhancing the immunogenicity of these polysaccharides can often be accomplished by combining them with proteins, e.g. see. lit. source 1-3.

This mode of operation may be used in the selection of proteins to be combined with these polysaccharides, however, some proteins, such as pertussinogen, are nonspecific stimulators of the young child's immune system. These proteins may to some extent increase the immune response to polysaccharide antigens, but unfortunately, such nonspecific activation is at the root of undesirable biological effects (so-called reactogenicity). The most preferred specific enhancement of the immune responses of these polysaccharide antigens can be achieved in children by conjugating these polysaccharides with suitable proteins, as reported for the first time by W. F. Goebel (4).

The method of combining polysaccharides and proteins should also be carefully considered. The immunological increase is due to the molecular proximity of the polysaccharide determinants and the determinants of the protein carrier, these parts cannot be easily separated in the biological system. The non-covalent complexes resulting from the polyanionic nature of the polysaccharides and the polycationic nature of the protein carrier can stimulate the immune responses, but these complexes are chemically unstable and the resulting immune response manifests as T-cell independence. In contrast, covalent polysaccharide and protein conjugates would achieve greater chemical stability and may exhibit T cell-dependent immune responses.

Covalent polysaccharide-protein conjugates have been reported in the literature, but the exact nature of covalent binding has not been proven or quantified, since the only covalency assay is in vivo activity and methods disclosed in the literature cannot be reproduced. The polysaccharides of Haemophilus influenzae, type b, and Streptococcus pneumoniae, type 6A, are reacted with bromocyan, then with adipic acid dihydrazide, followed by tetanus toxoid or horseshoe hemocyanin proteins from cancer (2 and

5). Pneumococcal type 19F polysaccharide is attached to calf serum albumin directly by forming imines (Schiff bases) from the reducing ends of the polysaccharides and free amine groups (i.e. lysines) of the protein, then these amines are reduced with sodium cyanoborohydrochloride. 6).

Additionally, the polysaccharides bound to the diazotized aromatic amines are attached to protein tyrosines as described in lit. source 7, and the polysaccharides bound to the aromatic amines are converted to isothiocyanates, which then bind to the free amino groups of the lysine of the proteins in lit. source 8. In each case, however, the resulting conjugate is only characterized by its gel permeation chromatography behavior. In yet another example (9), the polysaccharide, pullulan, is activated with cyanuric chloride and then reacted with tetanus toxoid. In this case, the conjugates are characterized by electrophoresis, which only indicates that they are different from the starting material.

In none of these cases is a covalency other than that observed at aggregated molecular weight, which confuses the covalency with the interaction between polyanions and polycations in the molecular complexes, and these complexes also give an aggregated molecular weight.

It is an object of the invention to bind the polysaccharide determinants to the determinants of the protein carrier so that the molecular proximity of these moieties is maintained in biological systems. Another object of the invention is the covalent attachment of encapsulated polysaccharides to protein carriers and the creation of a method by which the covalent nature of this linkage can be demonstrated and quantified. An additional object of this invention is to provide chemically stable polysaccharide protein conjugates that exhibit T-cell dependence and which may be useful as components of vaccines for the extraction of a protective serum antibody against a particular bacterium, in particular a bacterium related thereto. from which the polysaccharide is derived.

It is an object of the invention to provide a method for solubilizing polysaccharides, in particular polyanionic polysaccharides, and covalently modifying these polysaccharides in the process of producing polysaccharide-protein conjugates.

It is an object of the invention to provide a method for purifying and concentrating covalently linked polysaccharide protein conjugates to remove unconjugated macromolecules and excess reagents.

It is a further object of the invention to provide a method of using these conjugates in immunologically effective vaccines for use against meningitis and otitis media.

SUMMARY OF THE INVENTION

The invention is directed to covalently modified bacterial polysaccharides and chemically stable conjugates of these polysaccharides with covalently modified immunogenic membrane proteins, viral protein subunits, synthetic polypeptides, bacterial toxins or other immunogenic immunogenic proteins, polymorphisms and polymers. The polysaccharide-protein conjugates of the invention are linked by hybrid-built molecules containing a covalent thioether group, in which the hybrid structures are atomic chains that bind macromolecules such as polysaccharides and proteins, parts of these hybrid structures derived from one modified molecule, e.g. some of them from another modified macromolecule (eg functionalized protein).

By the process of the invention, the polysaccharide is treated in one or more steps so as to be covalently functional for the preparation of a polysaccharide with free electrophilic centers or free thiol groups. Preferably, the polysaccharide is first solubilized in a non-hydroxyl organic solvent, then modified with a bifunctional activating agent before reacting with the bis-nucleophile thereafter. The polysaccharide with nucleophilic functional groups is then either reacted with an agent to produce free electrophilic sites or reacted with a reagent to produce free thiol groups. By suitable selection of the bis-nucleophile, i. to one that would react with an activated polysaccharide and result in a covalently-modified polysaccharide with free electrophilic sites or a thiol group or by selection of the appropriate nucleophile, further introducing functional groups into the nucleophilic functional polysaccharide can be avoided.

Regardless of the covalent modification of the polysaccharide, the appropriate bacterial protein carrier reacts with the reagents forming free thiol groups or free electrophilic centers in a one- or two-step process. Suitably covalently modified polysaccharides and proteins are then reacted to form covalent polysaccharide protein conjugates and purified to remove unconjugated macromolecules and excess reagents and to allow for the determination of an immunogenic dose on a covalently linked basis.

The covalent nature of the bond can be absolutely demonstrated and determined by cleavage (such as by hydrolysis) of the polysaccharide from its free electrophilic or thiol groups and cleavage (detachment) of the protein from its free electrophilic or thiol groups, then assayed for the detection of thioether vessels constructed molecules such that the determination of the concentration of the hybridly constructed molecules corresponds to a marker amino acid assay (lysine) to determine the covalency of the protein.

Immunogenic vaccines containing an immunologically effective amount of polysaccharide protein conjugates or derivatives thereof can then be prepared very readily based on general knowledge of the preparation of vaccines.

The conjugates of the invention may be any stable polysaccharide-protein conjugates linked through hybrid structures including a thioether group and a primary amine that forms hydrolytically-labile covalent bonds with the polysaccharide or protein.

Preferred conjugates of the invention, however, are those which can be represented by the formula Ps-AESB-Pro or Ps-A'-S-E'Β'-Pro, in which Ps is a polysaccharide; Pro is a bacterial protein and AESB and A'-S-E'-B ¹ represent hybrid structures that contain hydrolytically stable covalent thioether bonds and which form covalent bonds, for example hydrolytically labile ester or amide bonds with macromolecules, Pro and Ps. In the structure A-ES-B, S is sulfur, E is the conversion product of the thiophilic group, which product reacts with the thiol group and is represented by the formula

wherein R is H or CH ₃ and p is 1 to 3; And it is

W N

II I

- C - N / CH / Y / CH / - NH zm L n in which W is O or NH, m is O to 4, η is 0 to 3, and Υ is CH ₂ , O, S, NR ', or CHCO ₂ H, where R 'is H or C ₍ - or

C ₂ -alkyl, such that when Υ is CH, then m and η cannot be zero, and when Υ is O or S, then y is greater than 1 and η is greater than I; and B is

Ζ /

- (CH ₂ ) CH (CH ₂ ) _q D with q being 0 to 2, Z being NH ₂ ,

Oh

II

NHCR '

COOH or H, where R 'and p are as defined above and D is

Oh

II

C, NR ¹ or

In the fl fl

N - C / CH _2/2 C

Then in the structure, A'-S-E'-B ', S is sulfur; And it is

W

II

-CNH / CH / R "ζ" as a is from 1 to 4, and R "is CH ₂ , or

BUT Y '

I II /

NCCH / CH / ² p as Y 'is NH ₂ or NHCOR', and W, or R 'have the meanings defined as above, and E' is a thiophil group conversion product which is reacted with a thiol group and is presented by

R

I

- CH as R is as defined above and B 'is

Oh

II

- with or E 'is

O and B is ⁷

About II

- / CH, / c as pe 1 to 3. Further from the hybrid structural AESB and A-SE'-B ', ESB and A'-S'-E' components are determinable and quantifiable and this identification reflects the covalence of the conjugation linking the thioether sulfur conjugate derived from the covalently modified polysaccharide to that part of the structure derived from the functionalized protein. The polysaccharides of the invention can be any bacterial polysaccharides with acidic groups, but are not limited to certain more specific types. Examples of such bacterial polysaccharides include Streptococcus pneumoniae types 6A, 6B, 10A, 11A, 18C, 19A, 19F, 20, 22F, 23F polysaccharides; Group B streptococcal types la, lb, II and III; Heamophilus influenzae (H.flu) type b polysaccharide; Neisseria meningitidis groups A, B, C, X, B, W 135 and 29E polysaccharides; and E. coli KI, KI2, K13, K92 and KI00 polysaccharides. Particularly preferred polysaccharides are the encapsulated polysaccharides on the group consisting of H.flu type b polysaccharide, as described by Rosenberg et al. at 10 and 11; Streptococcus pneumoniae type 6B or type 6A polysaccharides, as described in (12), the pneumococcal type 19F polysaccharide, as described by C.J.Lee et al. in (13) and the pneumococcal type 23F polysaccharide as described in (14).

The proteins of the invention have proven safety and demonstrative immunogenicity, but are not limited to any particular type. Suitable proteins are bacterial membrane proteins, which are from different plant proteins, such as e.g. edestine or soybean trypsin inhibitor, viral protein subunits, such as hepatitis A or B, herpes gD or gC, EpsteinBarr or varicella zoster subunits; synthetic polypeptides; diphtheria or tetanus toxin, but are preferably N.meningitidis outer membrane proteins (meningococcal) in the serotype, which are cellular stimulators. Proteins of this serotype are described in lit. sources 15 and 16.

The Ps-A-E-S-B-Pro conjugates of the invention may contain structures whose components include derivatives of: carbon dioxide, 1,4-butanediamine and S-carboxymethyl-N-acetylhomocysteine;

1,5-pentanediamine, and carbon dioxide and 8-carboxymethyl-acetylhomocysteine; carbon dioxide, 3-oxa-1,5-pentanediamine and S-carboxymethyl-N-acetylhomocysteine; carbon dioxide, 1,4-butanediamine and Scarboxy methyl-N-acetylcysteine; carbon dioxide, 1,3-propandiamine and 3-carboxymethyl-1-benzoylhomocysteine; carbon dioxide, 3-aza6

1,5-pentanediamine and S-carboxymethyl-N-acetylcysteine; and carbon dioxide, 1,2-ethanediamine, glycine, and S (succin-2-yl) -1M-acetylhomocysteine. The Ps-A'-S-E'-B'-Pro conjugates of the invention may contain structures whose components include carbon dioxide derivatives and S-carboxymethylcysteamine; carbon dioxide and S- (acarboxyethyl) cysteamine; carbon dioxide, 8- (succin-2-yl) cysteamine and glycine and carbon dioxide and carboxymethylcysteine.

In the process according to the invention, the polysaccharide is covalently modified by a) solubilizing it in a non-hydroxyl organic solvent; b) then activating it with a bifunctional reagent, c) reacting this activated polysaccharide with a bis-nucleophile and finally, if necessary, d) further introducing functional groups into the modified polysaccharide or by reaction

1) with a reagent generating electrophilic (or thiophilic) centers or by 2) reacting with a reagent generating thiol groups. The protein, in turn, 1) either reacts with a thiol group generating reagent or 2) with a thiolphyl center generating reagent, then the covalently modified polysaccharide and the functionalized protein react together to form a covalently bound conjugate and the final mixture is purified to remove of unreacted proteins and polysaccharides.

The method according to the invention also includes the selection of a nucleophile or a bis-nucleophile that will react with the activated polysaccharide to produce a covalently modified polysaccharide with free electrophilic centers or free thiol groups, and therefore there is a need for further processing for functional purposes. the introduction of functional groups) of the bis-nucleophil-modified polysaccharide before reacting the covalently modified polysaccharide with a covalently modified protein. Also, the introduction of a functional group into any part of the protein can occur in more than one step, depending on the selection of the reagents in these steps.

A. Preparation of the polysaccharide. In the first step aimed at covalent modification of the polysaccharide, the solid polysaccharide should be solubilized.

To the extent that the nucleophilic alcohol hydroxyl groups of the polysaccharide cannot chemically compete with the hydroxyl groups of water in aqueous electrophilic reagents, the polysaccharide should be dissolved in an involuntary (non-hydroxyl) solvent. Suitable solvents are, for example, dimethylformamide, dimethylsulfoxide, dimethylacetamide, formamide, N, N 'dimethylimidazolidinone, or other similar polar, aprotic solvents, preferably dimethylformamide.

In addition to the use of these solvents according to the invention, it has been found that in the conversion of polysaccharides according to the invention (e.g. encapsulated H. influenzae type b polysaccharides, which are ribose ribitolphosphate polymers, and pneumococcal types 6B, 19F and 13F) having acidic hydrogen , as phosphoric acid mono- and diesters in the form of a suitable salt, these polysaccharides become readily soluble in the above solvents. The acidic hydrogens in these macromolecules can be replaced by large hydrophobic cations, such as tri- or tetra-N- to C ₅ / alkylammonium, 1 azabicyclo [2.2.2] octane, 1,8-diazabicyclo [5.4.0] undec- 7-ene or similar cations, in particular three or tetra- / C to C ₅ / alkylammonium, and the resulting three or tetraalkylammonium analog salt of the phosphorylated polysaccharides is readily dissolved in said solvents at about 17 to 50 ° C, and stirred from 1 min to 1 h.

The partially hydrolyzed H.influenzae type b polysaccharide is converted to the tetrabutylammonium salt, then dissolved in dimethyl sulfoxide (17), but this product is no longer antigenic and therefore unsuitable for the preparation of vaccines. In contrast, the present invention also solubilizes the non-hydrolyzed polysaccharide by passing it through a highly acidic cationic ion exchange resin in tetraalkylammonium form, or by carefully neutralizing the polysaccharide with a tetraalkylammonium hydroxide, such as the preferred one, polysaccharide for use in an immunogenic vaccine.

The following steps are aimed at overcoming other physicochemical constraints to create covalent bonds with polysaccharides, such as the lack of functional groups in polysaccharides other than hydroxyl groups that are sufficiently reactive with conventional or in particular used reagents to introduce functional groups which connection is desired. Activation of the polysaccharide to obtain activated polysaccharide. reaction with bis-nucleophiles to produce a nucleophilic functional polysaccharide or its functionalization with reagents creating either electrophilic centers or thiol groups, all aim at covalently modifying the polysaccharide and creating functional groups therein in preparation for conjugation.

In the next step, the solubilized polysaccharide is activated by reaction with a bifunctional agent at about 0 to 50 ° C and with stirring for 10 minutes to 1 hour. at a critical ratio of the activating agent and the polysaccharide in the range from 1: 5 to 1:12. In the known method, activation was effected by reacting the polysaccharide with bromocyan. However, bromocyanine-activated derivatives that tend to adjacent diols show transient stability during dialysis against phosphate buffer. Therefore, while bromocyanine activation is still possible according to the invention, this reagent is underutilized for the activation of polysaccharides and is not preferred. Instead, preferred bifunctional polysaccharide activation reagents include carboxylic acid derivatives

About II

R ² -C-R ³ such as R ² and R ³ may be independently azolyl, such as imidazolyl; halides; or phenyl esters such as p-nitrophenyl or polyhalophenyl.

The particularly preferred carbonyl diimidazole reagent is reacted with the hydroxyl groups to give the imidazolylurethanes of the polysaccharide and aryl chloroformates, for example nitrophenyl chloroformate, to give mixed polysaccharide carbonates. In any case, the resulting activated polysaccharide is very sensitive to nucleophilic reagents, such as amines and is therefore transformed into the corresponding urethanes.

In the next step, the activated polysaccharide is reacted with a nucleophilic reagent, for example an amine, in particular diamines, e.g.

N N

I I

HN / CH, / Y / CH7 - NH as m is 0 to 4, overrides CH ₂ , O, S, NR 1, CHCO ₂ H, such as R 1 is H or C 1 - or C ₂ -alkyl, such that when Y is CH ₂ , then both m and η cannot be equal to 0, and if Υ is O or S, then m is greater than 1 and η is greater than 1, with the reaction proceeding to a large excess of amine (e.g., 50 to 100 times the molar excess of amine with respect to the activating agent used). The reaction is carried out in an ice bath for 15 minutes to 1 hour at approx. 17 ° to 40 ° C.

Activated polysaccharide, when reacted with a diamine such as 1,4 butanediamine, gives a urethane form of a free amine polysaccharide into which functional groups can then be introduced by acylation. Mixed carbonates also readily react with diamines to give free amino groups.

Alternatively, the activated polysaccharide may be reacted with a nucleophile, such as diaminoalkane monohaloacetamide, for example 4-bromoacetamidobutylamine (18), for the preparation of covalently modified polysaccharide with free electrophilic sites. Or, the activated polysaccharide can be reacted with an aminothiol, such as cysteamine (aminoethanethiol) or cysteine, examples of derivatives well known in peptide synthesis to produce a polysaccharide with free thiol groups. In both cases, no further introduction of functional groups is required before the addition of the covalently modified polysaccharide to the modified bacterial protein carrier.

The final step of the polysaccharide preparation, the further introduction of functional groups, if necessary into the polysaccharide, can proceed either by reacting a nucleophil-functionalized polysaccharide with a reagent to obtain electrophilic (i.e., thiophilic) centers, or by reaction to produce thiol groups.

Suitable reagents for the preparation of electrophilic centers include, for example, those for the acylation of α-haloacetyl or α-halopropionyl derivatives, such as

O R II I x-c-c-n-x where R is H or CH ₃ ; X is Cl, Br or I; and X 'is nitrophenoxy, dinitrophenoxy, pentachlorophenoxy, pentafluorophenoxy, halide, O-N-hydroxysuccinimidyl) or azido, in particular chloroacetic acid or α-bromopropionic acid, the reaction being carried out at pH 8 to 11 (maintained at this interval) on the basis, if necessary) and at a temperature from about 0 to 35 ° C ^and for ten minutes to one hour. The amino derivative of the polysaccharide can be acylated with maleimidoamino acids (19) to give the following maleimido groups

wherein p is 1 to 3; with a 2-haloacetylating agent, for example p-nitrophenyl bromoacetate; or with an achaloketone-carboxylic acid derivative are carried out in a nitrogen atmosphere at about 0 to 35C and at a pH of 8 to 11 with the addition of a base if necessary to maintain the pH at the desired interval. For example, the amino derivative of the polysaccharide may react with

NHCOCH, _

//

HO ₂ C -C-CHCH ₂ Br (20) to form a polysaccharide with suitable thio-displacement-functional groups.

Reagents suitable for the formation (formation) of thiol groups are, for example, acylating reagents, such as thiolactones, e.g.

R ⁴ CNH

wherein R ⁴ is C, to C ₄ -alkyl or monoyl or bicycloaryl, such as C ₆ H ₅ or C ₁₀ H ₁₃ , and p is 1 to 3;

NHCOR ⁵

I

- O SSCH / CH, / CH - COX ', where w is 0 to 4, R ⁵ is C - to C ₄ -alkyl or C ₆ H ₅ and X' has the meanings given above, followed by treatment with HSCH ₂ CH ₂ OH, or

NHCOR ⁵

I

C, H ₄ - S - S - CH, / CH, / CHCHOX ¹ such as m, R ⁵ and X ¹ have the meanings defined above and then treatment with dithiothreitol. Such reactions produce a polysaccharide with suitable functional groups.

These steps then yield covalently modified polysaccharides of the type Ps-AE * - or Ps-A ¹ -SH-, with E * being -CCCH or

and A, X, R, X and p have the meanings defined above.

B. Protein production. Separate introduction of functional groups into the protein so that it can be attached to the polysaccharide includes the reaction of the protein with one or more reagents to produce a thiol group, or the reaction of the protein with one or more reagents to produce electrophilic, for example, thiophilen, center.

In the method of conjugation with an electrophilic-functionalized polysaccharide, the protein is reacted in one or two steps with one or more reagents to produce thiol groups, such as acylating reagents used to prepare thiol groups on the polysaccharide, as discussed above. Thiolated proteins can be obtained by amination of carboxy-activated proteins, such as those mentioned in (21), with aminothiols. A preferred embodiment of this step of the process involves the direct acylation of the free amino groups, for example lysyl groups, of the protein with N-acetylhomocysteineethiolactone at about 0 5 to 35 ° C and pH 8-11, for 5 min to 2 h, using equal weight amounts of reagents.

When E'B is

the conditions and method for preparing a functional group protein are the same as those described above for the preparation of the corresponding maleimidic acid-activated polysaccharide.

To conjugate a covalently modified bacterial polysaccharide with free thiol groups, the protein is acylated with a reagent having an electrophilic center, and such an acylating agent may be e.g.

О СН _Ч О

II I II

CHF ₂ C - X 'and CHF - CX' such as X and X 'have the meanings given above; and

About II n / ch ₂ /, c / co

- X 'as X is defined above. Suitable proteins with electrophilic centers also include, for example, those obtained by acylation of free lysyl amino groups with a reagent, 35 e.g.

'or by reacting a carboxy-activated protein with monohaloacetyl derivatives of diamines. In both reactions the temperature is from 0 to

35 ° C, duration from 5 min to 1 45 h and pH from 8 to 11.

C. Preparation of conjugate.

The formation of the conjugate is then merely a matter of reacting any covalently modified polysaccharide with free electrophilic centers with any of the proteins with free thiol groups at pH 7 to 9, in approximately equal weight amounts, in an atmosphere of nitrogen for 6 to 24 h 5 at a temperature of 17 to 40 ° C.

Examples of such reactions include it

II III

Ps - CNCH ₂ CH ₂ CH ₂ CH ₂ NHCCH ₂ Br +

NHCOCH,

I

HSCH ₂ CH ₂ CHCO - Pro>

OH O NHCOCH.

II I, 11I

PsCNCH ₂ CH ₂ CH ₂ CH ₂ NHCCH ₂ SCH ₂ CH ₂ CHCOPro wherein the activated polysaccharide that has reacted with N-acetylkoxy has reacted with 4-bromoacetamido-homocysteine thiolactone to butylamine, reacts with a protein, 20 conjugates, and

HSCH ₂ CH ₂ NHCCH ₂ CH ₂ CPro ----------->

Oh

II n.

CH ₂ CH ₂ NHCCH ₂ CH ₂ C Pro s ^z (wherein Y is a C ₂₈ alkyl radical), such as the amino derivative of the polysaccharide, which reacted with activated maleimidic acids, is reacted with a carboxyactivated protein which is aminated with aminothiol to give get the conjugate.

Similarly, any of the covalently modified free thiol group polysaccharides may be reacted with any of the pro45 theins having free electrophilic centers to form a covalent conjugate. An example of this12

what a reaction it is

0 II 0 II 0 II Η 1 PsCNiHCH ₂ CH ₂ SH + ProCCH ₂ CH ₂ C - N / CH _2/4 NHCOCH, Br OH OH Ο ο II 1 II 1 II II

PsC NCH.CH SCH C N / CH, / NHCCFLCH CPo

2 2 2 4 2 2 as an activated polysaccharide that reacted with aminothiol, reacted with a carboxy-activated protein that reacted with the monohaloacetyl derivatives of diamine to form a conjugate.

The electrophilic activity of excess haloacetyl groups should be eliminated. The interaction of a low molecular weight thiol conjugate such as n-acetylcysteamine accomplishes this goal. The use of this reagent, n-acetylcysteamine, also allows to confirm the determination of the haloacetyl groups used (see section D), as the resulting S-carboxymethylcysteamine can be determined by the method of Spackman, Moore and Stein .

These conjugates are then centrifuged at about 100,000 XG using a fixed angle rotor for about 2 hours at 1 to 20 ° C or subjected to any purification procedure involving gel permeation, chromatography, ion exclusion, stepwise centrifugation, or other separating adsorption chromatography, to remove non-covalently bound polysaccharides and proteins, using the covalency analysis of the hybrid structure (see below) as a method of chasing the desired biological activity.

Further separation of the reagents can be accomplished by column chromatography excluding a certain sample size or in the case of particularly long insoluble proteins, such as N-meningitidis B serotype from the outer membrane, this separation can be accomplished by ultracentrifugation.

D. Covalency Confirmation Analyzes.

Conjugation assay for confirmation of covalency and therefore stability of the conjugate is performed by hydrolysis (preferably with 6N HCl at 110 ° C for twenty hours) of the conjugate, then quantitative analysis of the amino acid from the hydrolytically stable structure containing the thioether linkage , and the protein-forming amino acids. The involvement of the amino acids of the protein can be removed, if necessary, by comparison with a standard amino acid suitable for the included protein, such as the residual value for the amino acid with the remaining amino acid from the interval reflecting on the covalency of the conjugate, or the amino acid being conjugate, to express the protein's amino acid standard in the assay. Covalency analysis is also useful for controlling purification, noting the increase in the concentration of biologically active components. In the above examples, the hydrolysis of

About NHCOCH.

II I

PsCNCH ₂ CH ₂ CH ₂ CH ₂ NHCCH ₂ SCH, CH ₂ CHCOPro results in the release of S-carboxymethylhomocysteine,

NH,

I

HO ₂ CCH ₂ SCH ₂ CH ₂ CHCO ₂ H hydrolysis of

Oh

II II.

CH ₂ CH ₂ CH ₂ NHCCH ₂ CPro * leads to the release of aminodikarboksilnata kiselina20 and hydrolysis of ho ₂ cch ₂ chsch ₂ ch ₂ nh ₂

NO ₂ P

OH OH oo

II | II AND IIII

PsCNCH ₂ CH SCH ₂ CN / CH / NHCCH CH ₂ CPro results in the release of S-carboxymethylcysteamine.

H ₂ NCH ₂ CH ₂ SCH ₂ CO ₂ H is released upon cleavage of the Ps-A-ES-B-Pro molecule by peptide bonds and other hydrolytically unstable bonds. Chromatographic methods such as Spackman, Moore, and Stein can be applied and the amount of amino acid components determined.

E. Applications.

One or more of the conjugates of the invention may be used in mammals for active or passive protection prophylactically or therapeutically against bacteremia caused by related organisms, for example in preferred embodiments of the invention, Haemophilus influenzae type b or Str. pneumoniae, type 6B, 19F or 23F. Active protection can be ensured by injecting an effective amount (an amount capable of producing measurable amounts of antibodies, for example 2 to 50 pg) of the polysaccharide in conjugated form from each of the conjugates administered in a dose, the entire antiserum derived from animals, pre-injected with conjugate or conjugates or with globulin or other antibody containing fractions of the present antiserum, with or without a pharmaceutically acceptable carrier, such as aseptic saline. Such globulin is obtained from complete 5 antisera by chromatography, salt or alcohol fractionation or electrophoresis. Passive protection may be provided by standard monoclonal antibody procedures or by immunization of suitable mammals as a host. The administration of an additive is also expected to be within the scope of the invention.

In a preferred embodiment of the invention the conjugate is used for active immunogenic vaccination of humans, in particular infants and children. For added stability, these conjugates can also be lyophilized in the presence of lactose (e.g. at 20 pg / ml of H.flu polysaccharide / 4 pg / ml lactose or 50 pg / ml of pneumococcal polysaccharide / 10 mg / ml lactose) before use .

The preferred dose is the amount of each of the conjugates or their derivatives to be administered corresponding to 25 µg of polysaccharide in conjugated form.

Haemophilus * inoculum medium for pneumococcal polysaccharide conjugates and 10 pg of polysaccharide in conjugated form for single dose H.flu conjugate form polysaccharide. If necessary, one or two more doses of the conjugate or its derivatives of H.flu type b polysaccharide are administered in an amount corresponding to 10 pg of the polysaccharide in conjugated form.

The invention is further explained by the following non-limiting examples.

Example 1. Preparation of a capsular polysaccharide of H. influenzae, type b (PRP) inoculum and its development.

Step A. A lyophilized tube of 20 ka with H. influenzae, type b (cultured from Ross 768, obtained from the State University of New York) was suspended in 1 ml of sterile Haemophilus inoculation medium (see below 25) and distributed in 19 chocolate agar plates (BBL). After incubation at 37 ° C for 20 hours, the culture of each plate was resuspended in 1-2 ml of Haemophilus medium and pooled.

Soy peptone 10g / l NaCl 5 g / l NaH PO 3.1 g / l NaHPO. 13.7 g / l K ₇ HP (In L 4 2.5 g / l distilled water to a certain volume

'The pH of the solution was adjusted to a certain value of 7.2 ± 0.1 (typically pH 7.23) and the solution was sterilized by autoclaving at 121 ° C for 25 min.

Stage B. Two liter non-sealed Erlenmeyer flasks.

One third of the resuspended bacteria referred to in Step A are used to inoculate three two-liter flasks, each containing about 1.01 from the Haemophilus crop and the production medium60630 to (see below). The flasks were then incubated at 37 ° C on a rotary vibrator at 200 rpm

Seeding of Haemophilus in about 5 hours. The typical value for OP ₆₆₀ at the end of the incubation period is 0.37.

production environment and

NaH ₂ PO ₄ 3.1 g / l At ₂ HPO ₄ 13.7 g / l Soy peptone 10 g / l Diafiltrate of extract from yeast (1) 10 mg / l K ₂ NRA ₄ 2.5 g / l NaCl 5.0 g / l Glucose 5.0 g / l Nicotinamidadine dinucleotide / IAD / (3) '2 mg / 1 Hemin (4) 5 mg / l

The salts and soy peptone are dissolved in small volumes of hot, pyrogen-free water and reduced to a definite final volume by the addition of hot pyrogen-free water. The fermenters or flasks are then sterilized for about 25 minutes at 121 ° C and after cooling, the diafiltrate of yeast extract 1), glucose 2), IAD 3) and hemin 4) are added aseptically to the fermenters or flasks before inoculation.

1) Yeast Diafiltrate: Dissolve 100 g of yeast extract (Amber) in 1 liter of distilled water and ultrafiltrate with Amicon DS-30 to remove molecules with a molecular weight of 50,000. The filtrate was collected and passed through a 0.22 µm membrane as a sterile product.

2) Glucose is prepared as a 25% solution in distilled water.

3) A 20 ml / ml IAD stock solution was sterilized by filtration through a Millipore filter (0.22µ) and added aseptically just before inoculation.

4) Hemin 3X stock solution was prepared by dissolving 200 mg in 10 ml of 0.1 M NaOH and the volume was reduced to 100 with distilled sterilized water. The solution was sterilized for 20 min at 121 ° C and added aseptically to the final medium before inoculation.

Stage B. 70-liter fermenter

Three liters of the product obtained from Step B were used to inoculate a 70 liter fermentor containing 41.4 l of production medium and seed of Haemophilus prepared as described above and 17 ml of UCON B625 antifoam. The initial pH was 7.4.

The fermentation takes place at 37 ° C and 100 rpm stirring, the process being monitored by optical density (OP) and pH values until the characteristic value is reached

0.39 for OP (after about 5.5 hours).

Stage G. 800 liter production fermenter

Approximately 40 l of the product from step B was used to inoculate a 45 liter 800 liter fermenter containing 570 l of the production medium prepared as described above and a UCON LB625 antifoam.

The fermentation is carried out at 37 ° C with agitation at 100 rpm, with the OP and pH checked every two hours, while the OP values remain the same for the two-hour period during which the fermentation ends (characteristic end value for the OP and 0.54 after 12 h.

Culture harvesting and inactivation.

Approximately 600 l of the batch was inactivated by collection in an inactivation tank containing 12 l of 1% thirosal.

Clarification

After 18 h inactivation at 4 ° C, the culture was centrifuged in Sharple centrifuges at a low speed ranging between 1.3 and 3.0 Ι / min and adjusted to keep the product clear. The supernatant obtained after centrifugation (15,000 rpm) was used to recover the product.

Isolation and concentration by ultrafiltration.

The supernatant from the two production fermentations was collected and concentrated at 2 to 8 ° C in a Romicon ultrafiltration apparatus (50,000 Daltons) with hollow fibrous cartridges (4.5 t ² membrane surface; 2.0 Ι / m air flow and 138 kN pressure / m ² ), followed by concentration so that after approx

Concentrate 1200 to 32.5 for 4.5 h. 1. The filtrate is removed.

Precipitation with 48% and 61% ethanol

To the 32.5 l of the retained ultrafiltrate was added 30 I 95% ethanol, dropwise over 1 h, with stirring and at 4 ° C to a final concentration of 48 vol% ethanol. The mixture was further stirred for 2 h at 4 ° C until complete precipitation and the supernatant was collected by a single, 10.2 cm Sharple centrifuge at 15000 rpm (flow rate = 0.27 l / min). The undissolved portion was removed and the purified liquid was brought to 61% ethanol by addition of 20.8 1 95% ethanol for 1 h. The mixture was stirred for 3 h to obtain complete precipitation.

Secondary sludge regeneration after centrifugation.

The resulting ethanol -48% soluble, ethanol-insoluble 61% precipitate was collected by centrifugation at 15,000 rpm in a 10.2 cm centrifuge and Sharple (flow rate = 0.62 l / min) and the 61% ethanol supernatant removed. . The crude product obtained is 0.377 Kg moist paste. Extraction with calcium chloride

377 g of the insoluble in 61% ethanol product was mixed in a Dimex dispersion vessel at 2 to 8 ° C with 6.5 l of cold distilled water. To this mixture was added 6.5 1 cold 2M CaCl ₂ .H ₂ O and the mixture (at final concentration = 1.0M CaCl ₂ ) was extracted at 4 ° C for 15 min. The vessel was then rinsed with 2 L of 1M CaCl, 2H ₂ O to give a 15 I final volume.

Sedimentation with 23% ethanol

1 of the CaCl ₂ extract was adjusted to 23% ethanol content by the addition of 4.48-195% ethanol dropwise, stirring at 4 ° C for 30 min. After further stirring for 17 h, the mixture was centrifuged on K ₂ Ultracentrifuge at 25,000 rpm (165 ml / min flow rate) for

6.5 h at 4 ° C. The supernatant was decanted through a cheesecloth to remove the surface-floating lipid-like particles and the insoluble mat sediment was removed after centrifugation.

Precipitation with 37% ethanol and collection of the crude paste product

The supernatant soluble in 23% ethanol was adjusted to 37% ethanol by the addition of 4.33 to 95% ethanol, dropwise with stirring for 30 min. The mixture was allowed to stand with stirring for 1 h, then without stirring for 14 h to achieve complete precipitation. The resulting mixture was then centrifuged on a 10.2 cm Sharple centrifuge at 15000 rpm (flow rate 0.2 l / min) to collect the crude polysaccharide precipitated after centrifugation.

Spraying

After centrifugation, the precipitate was transferred to a 3.8 IW mixer containing 1 liter of absolute alcohol and mixed with it for 30 s at high speed. Mixing is continued for 30 seconds on and off until a solid white powder is obtained. The powder was collected on a Buchner funnel with a Teflon filter disc and washed sequentially in citu with two one-liter portions of absolute ethanol and two 2-liter portions of acetone. The material was then dried in vacuo at 4 ° C for 24 h, resulting in 68 g (dry weight) of product.

Phenolic Extraction The resulting 68 g of dry material after the nebulization step was resuspended in 12 l of 0.488M sodium acetate at pH 6.9 using a Dimex dispersion vessel. The sodium acetate solution was immediately extracted with 4.48 l of fresh aqueous phenolic solution, obtained as follows: 900 ml of 0.488M sodium acetate, pH 6.9, was added to a 2.3 liter bottle of phenol (Mallinckrodt crystalline) in a 20 liter autoclave and mix until completely dissolved. Each phenolic extract was centrifuged for 2.5 h at 30000 rpm and 4C in a K ₂ Ultracentrifuge (nuclear electronics) to destroy the emulsion. The aqueous effluent was further extracted three times with 3.2 fresh aqueous phenolic solution in an analogous manner. The phenolic phases are removed.

Diafiltration

The aqueous phase from the phenol extractions above (17.6 1) was diluted with 300 1 of cold distilled water and diafiltered at 4 ° C on an Amicon DC-30 ultrafiltration apparatus using H ₁₀ ₽ ₁₀ cartridges. The ammonia apparatus was rinsed and the liquid added so that the final volume was 17.5. The ultrafiltrate was removed.

Precipitation with 67% ethanol

0.438 1 2.0 M CaCl _{2 was added} to

17.5 l dialysate from the previous step (final CaCl ₂ concentration was 0.05 M) and the solution was adjusted to 67% ethanol content by the addition of one hour dropwise and with vigorous stirring of 34.88 l 95% - ethanol. After stirring for 4 hours, the mixture was left standing for 12 hours at 4 ° C, the clear supernatant was poured and the precipitate collected by centrifugation in a 10.2 cm Sharply centrifuge (15000 rpm) at 4 ° C for 45 min. The resulting polysaccharide precipitate from the centrifugation is atomized into a 3.8 liter Wering mixer on and off for 30s with 2 1 ethanol (absolute), collected on a Buchner funnel with a Teflon filter disc and washed in citu with 4 liter portions absolute ethanol followed by two one-liter portions of acetone. The sample was then dried in vacuo at 4 ° C for 20 h. The resulting product is 39 g of dry powder.

Ultracentrifugation in 20% ethanol and collection of the final product

The product obtained after the above step in an amount of 39 g was dissolved in 15.21 l of distilled water, to which 0.39 1 0.05 M CaCl _{2 2} .2H ₂ O was added, bringing the solution to a concentration of 0.05 M CaCl ₂ and the total volume to 15.6 l (2.5 mg polysaccharide / ml), after which the mixture was brought to 24% ethanol by the addition of 4.93 l 95% ethanol dropwise over 30 min. The mixture was clarified immediately by centrifugation in a K ₂ Ultracentrifuge containing K ₃ titanium beaker and K _and Noril core (30000 rpm and 100 ml / min) for 3.5 h at 4 ° C. The precipitate was removed and the clear supernatant (volume 19.8 L) was adjusted to 37% en ethanol by addition of 4.23 1 95% ethanol for 30 min with stirring. After stirring for a further 30 min, the mixture was left at 4 ° C for 17 h and then collected by centrifugation at 5 15000 rpm in a 10.2 cm Sharple centrifuge (45 min required) ).

The resulting paste was transferred to a Waring mixer with a capacity of 4.5 l, absolute ethanol, and mixed at 10 highest speeds for 4 or 5 cycles of switching on and off for 30 s until solid white powder was obtained. This powder was collected on a Buchner funnel with a 15 Citex Teflon disk and washed sequentially in citu with two 0.5 liter fresh portions and one 1 liter portion of absolute ethanol and two one liter portions of acetone. The product was removed from the funnel and transferred to a tared vacuum drying vessel at 4 ° C for 25.5 h. The final product amounts to 34.7 g dry weight and its properties are described in the following table:

Table 1-1

HIb POLYZAHARID

DATA FROM CHEMICAL RESEARCH

Analysis of

Result, in% by weight

Humidity (TG) 13.5% Protein 0.0% Nucleic acid 1.3% Ribose (pentose) 35.1% Phosphorus 7,8% K _d (Sepharose 4B) .05.35 K _d (Sepharose 2B) .43.60

The following procedures were used to perform the analysis.

1. Humidity - Standard Thermogravimetry (TG) (weight loss up to

100 ° C) using a Perkin-Elmer 45 thermocouple TSG-1.

2. Protein - Lowry Method (22).

3. Nucleic acid - Ultra19 violet method (23)

4. Ribose - The Bial Method (24)

5. Phosphorus - Molybdenum Method (25)

6. K. - Determination of Sepharose

4B by refractive index

Table 1-2

Determination of pyrogenic substances (HIb Polysaccharide) Concentration / gg / ml / kg Max, temperature rise. * 0 ° C (3 rabbits) 0.1 (polysaccharide) 0.2, 0.2, 0.1

* 1.0 ml / kg dose

The polysaccharide is further identified by agar gel diffusion as follows. Double agar diffusion was performed using Heland Model D plates. The prepared antisera against H. influenzae Ross 768 strain was placed in the middle wells while the polysaccharide mass at 50, 25, 12.5, 6.2 and 3.1 gg / ml is inserted into the side wells. The plates were incubated at 20-25 ° C in a humidified chamber for 24 h. Precipitin bands were observed between the parent polysaccharide and the specific antiserum at polysaccharide concentrations of 50, 25 and 12.5 gg / ml.

Example 2. Preparation of a Membrane Protein from Neisseria meningitidis Bl 1, serotype 2.

A. Fermentation

1. Neisseria meningitidis, Group Bll A tube containing lyophilized Neisseria meningitidis culture obtained from WRAIR, Washington, was opened and Engonbroth (BBL) was opened and the culture was plated on chocolate agar and incubated at 37 ° C with CO ₂ for 36 h, during which time the culture was collected in a medium of 10% skim milk (Difco medium) diluted to 1 ml and frozen to -70 ° C. The organism is positively identified by agglutination with a specific antiserum provided by WRAIR and a typical antiserum provided by Difco.

The culture from the first passage was dashed on chocolate agar plates and incubated at 37 ° C with 50% CO ₂ for 18 h, during which time the culture was harvested in 10% skimmed milk, reduced to 1 ml and frozen to 70 ^(, C. The organism is again positively identified by agglutination with a specific antiserum provided by WRAIR and a typical serum provided by Difco.

The culture flask of the second passage was thawed and dashed on a Mueller-Hinton Columbia Sheep Blood agar plate (CBAB-BBL). The latter were incubated at 37 ° C with 5% CO ₂ for 18 h, after which the culture was collected in 100 ml medium of 10% skim milk, adjusted to 0.5 ml and frozen at -70 ° C. The organism is positively identified by agglutination with specific antisera, sugar fermentation and gram staining.

The culture vial of this passage is thawed, diluted with Muller-Hinton broth and seeded on 40 Muller-Hinton agar plates. The plates were incubated at 37 ° C with 6% CO ₂ for 18 h, after which the culture was collected in 17 ml of 10% skimmed milk medium, adjusted to 0.3 ml and frozen to -70 ° C. The organism is positively identified by staining per gram, agglutination with a specific antiserum and oxidase test.

2. Fermentation and collection of cell paste

a) Development of the inoculum. The inoculum develops from two 0.5 ml frozen ampoules of Neisseria meningitidis, group B, VI (passage 4). Four MüllerHinton agar bottles were inoculated, the culture was harvested approximately 18 hours later, and used as inoculum for 5 L of Gottley's dialysis medium at pH 7.29. The optical density (OP) was adjusted to 0.065 at 660 nm (Perkin Elmer). The organisms were grown in 5 liter Erlenmeyer flasks (each containing 1 L of medium, see below) at 37 ° C in a vibrator. The OP was controlled at 45-, 75-, and 120-min intervals. Approximately 4 l of fermentation culture with an optical density (OP 0.660) of 0.81 were obtained.

A sample of 3 ml was taken for Gram staining, bar strokes were isolated on SWAB agar plates, Müller-Hinton agar plates and yeast extract plates, and agglutination was checked.

All reactions are satisfactory.

b) 70 liter fermenter: Approximately 3600 ml of germ culture is used to inoculate a sterile 70 liter fermenter containing 42.6 l of complete production medium.

Conditioners for the 70 liter fermenter include 37 ° C, 185 rpm with 10 Ι / min airflow and continuous pH control at pH 7.0 for

5.5 h.

The culture was seeded on Müller-Hinton agar plates, yeast extract dextro, and rabbit blood (Merck) agar plates at 37 ° C and checked for agglutination with an antiserum for Neisseria meningitidis, group B. Growth was normal and the agglutination reaction is positive. For this sample, the final OP was 0.840 at 660μ after 5.5 h.

c) 800 liter production fermenter: approximately 46.2 l of the seed culture is used to inoculate the IU of a sterile 800 liter fermenter containing 568.2 l of the complete production medium. The batch was incubated at 37 ° C, 100 rpm with 60 Ι / min oxygen influx and constant pH control at 7.0.

Before the batch was inactivated, the culture was placed in Muller-Hinton agar plates, dextrose agar plates and rabbit blood agar plates at 37 ° C and tested for agglutination with antisera for Neisseria meningitidis, group B. Culture development in these cells normal and agglutination response - positive. For this batch, the final OP was 2.24 thirteen hours after inoculation.

3. Complete medium for nephelometric flasks and 70- and 800-liter fermenters

Fraction A

L-Glutamic acid

NaCl

At ₂ HPO _4. Anhydrous

NH Cl

KC1

L-cysteine hydrochloride

B. Gottschlich Dialysis Fraction

1280 g of Difco yeast extract was dissolved in 6.4 l of distilled water. The solution is dialyzed in 2 hollow fiber dialysers Amicon DS-30 with 3 cartridges H 10SM. The dialysate and 384 g of MgSO ₄ .7H ₂ O and 3200 g of dextrose were dissolved in the dialysis and the total volume was adjusted to 15 l with distilled water. The pH was adjusted to 7.4 with NaOH and sterilized by filtration through a Millipore filter (0.22 pk) and added to the fermenter containing fraction A.

For nephelometric flasks. 1 l of fraction A and 25 ml of fraction B were added and the pH was adjusted to 7.0 - 7.2 with NaOH.

For the 70 liter fermenter. 41.8 l of fraction A and 900 ml of fraction B were added, bringing the pH to 7.0 - 7.2 with NaOH.

For the 800 liter fermenter. 553 l of fraction A and 15,0 l of fraction B were added, the pH was adjusted to 7.0 - 7.2 with NaOH.

d) Collection and inactivation. After fermentation, phenol 10.5% v / v final concentration is added to a separate vessel, into which the cell broth is then transferred. The material was left at room temperature with gentle stirring until the viability of the culture was lost (about 24 h).

(e) Centrifugation. After about 24 h at 4 ° C temperature, 614.4 I of

1.5 g / l

6.0 g / l

2.5 gl

1.25 g / l

0.09 g / l

0.02 g / l of inactivated culture fluid was centrifuged in Sharple centrifuges. The weight of the cell paste after the addition of phenol is 3.875 kg.

B1- Isolation.

Step 1. Flushing of bacterial cells. For each isolation, 2000 g of the above phenol-inactivated paste was suspended in 800 ml of sterile distilled water and stirred with a magnetic stirrer to form a granular suspension. The suspended cells were centrifuged at 20,000 xg for 60 min at 5 ° C.

Step 2. Extraction. The washed cells were suspended in 2000 ml of 0.1 M Tris-0.01 MEDTA buffer, pH 8.5, with 0.5% sodium deoxycholate (TED Buffer) with Sorval omnix mixer 3 times for 60 s. Transfer the homogenized suspension into 16 500 ml Erlenmeyer flasks to extract at 56 ° C in a water bath for 15 min with shaking.

The extract was centrifuged at 20,000 xg for 60 min at 5 ° C. The viscous supernatant liquid was decanted (total volume 1980 ml) and left at 4 ° C.

The extracted cell pellet was resuspended in 2000 ml of TED Buffer as described above. The suspension was extracted for 15 min at 56 ° C and centrifuged as above. The supernatant was decanted (2100 ml volume) and left at 4 ° C.

Step 3. Concentration by ultrafiltration. The extracted supernatants from Step 2 were poured (4005 ml total volume). 2 1 of the casts are dosed into a 2 1 New Brunswick fermentation vessel connected to a Millipore Pellicon filter fitted with 2 0.45 μm foam membranes. The extracted supernatant was left at 25 ° C in the fermentation vessel for a concentration of 90 minutes. The sample was concentrated ten times at an average pressure across the membrane of 187 KN / m ² .

Step 4. Collection and washing of the serotype protein. The product stored after Step 3 (205 ml) was centrifuged to precipitate the protein from the particular serotype at 160000 xg for 2 h at 5 ° C. The supernatants are decanted and discarded.

The protein pellets were weighed (8.12 g) and then suspended in TED Buffer (190 ml buffer; 20 ml / g pellet) manually with a glass stirrer and a Dounce homogenizer. The suspension was extracted at 56 ° C for 15 min (at temperature) in 500 ml Erlenmeyer flasks with shaking. The suspension was centrifuged at 160,000 xg for 2 h at 5 ° C. The supernatant was decanted and discarded (190 ml volume).

Wash the precipitate a second time in 190 ml 5 TED Buffer as described above.

Step 5. Product Recovery. The washed protein sludge from step 4 was suspended in 100 ml of distilled water with a glass stirrer and a Dounce homogenizer until 10 more complete suspensions were obtained. Lowry protein was obtained from this suspension. amounting to 17.0 mg / ml. At this stage, 200 mg of the suspension is removed for experimental purposes. 15 Dilute the remainder of the suspension (91 ml) to 8.0 mg / ml with 102.4 ml of cold distilled water. The aqueous suspension was centrifuged at 12,000 xg for 15 min to purify 20 units.

The supernatant product is carefully withdrawn with a pipette to avoid soft aggregate particles. The product was recorded (182.5 ml volume) 25 and equal quantities were tested for sterility and pyrogens (sterile product; no pyrogens). The product was stored at 4 ° C as a sterile mass until used for conjugation30 when analytically characterized. The final product was 9.5 mg of Lowry protein / g from the original cell paste.

Table 2-1

Meningococcal serotype protein solution - 2

Chemical research data

Analysis for

Lowry protein

Nucleic acid *

The result

4.1 mg / ml

RNA 1.8%

DNA (diphenylamine) 0.6%

Neutral sugars * 1,05

Antron sialic acid 3.0%

Molecular Weight SDS-PAGE 40,000 d * Calculations are made as% of Lowry protein.

The following methodologies are used in the analyzes.

A protein as described in Example 1.

2. Nucleic acid - Color development is observed using the orcinol reaction (Bial), which corresponds to 1.8% of RNA calculated as% of protein concentration. Diphenylamine test for DNA indicated 0.6% _o DNA content as% of the protein in the bulk solution.

3. Neutral sugars - Neutral sugar content is calculated as% of protein using an anthron colorimetric test (26).

4. Sialic acid - the content of sialic acid is determined by the resorcinol-HCl method (27).

5. Molecular Weight - The molecular weight of mercaptoethanol denatured protein is found as determined by SDS-polyacrylamide gel electrophoresis (28).

Example 3. Preparation of a conjugate between a polysaccharide of H. influenzae type b and an outer membrane protein of N. meningitidis serotype B.

1. Preparation of Dowex 50x8 (200400 mesh) in tetra-p-butylamine form.

360 ml of fresh Dowex 50x8 (200-400 mesh) highly acidic cation exchange resin was loaded into a sterile chromatographic column and washed with 1500 ml of sterilized distilled water (c.g.) water and left overnight in 800 ml of the same water. The resin was then washed successively in 1 1 60:40 c.g. water: methanol, 1 1 40:60 c.g. water: methanol and 11 sterilized distilled (c.g.) water. The resin was then sterilized by immersion in 650 ml of 3N hydrochloric acid (200 ml HCl diluted to 800 ml with water). This acidic resin formulation was left to age for 19.5 hours and then washed with water to remove excess acid.

To this column was then added 700 ml of a 1: 1 mixture of water: 40% tetrabutylammonium hydroxide, which percolated through the resin until the flow was basic (pH about 10). The resin is then washed to remove excess base from approximately 2 L of water and then transferred to a sterile jar. The final flow is sterile and free from pyrogens.

II. Preparation of the tetra-p-butylammonium salt of H.influenzae type b polysaccharide (HIb)

A 250 ml round-bottom flask fitted with a magnetic stirrer is charged with 3.29 g of HIb and 84 ml of water. The mixture was stirred for 20 min and then an additional 15 ml of water was added. Stirring was continued for another 30 min until all HIb was incorporated into the solution. The HIb solution was then fed to Dowex 50x8 (200-400 mesh, tetrabutylammonium form) in a 45x270 mm column. Rinse with 10 ml of water. Cover the column with water and apply pressure using a hand pump (via Millex FG, 0.22 μ filter).

The 50 ml fractions were collected in sterile Nalgen centrifuge tubes and each was tested for organic matter by applying quantities of approximately 10 μ using sterile capillaries to determine, e.g., silica gel plates. . The plate was sprayed with CelV (SO _4/2) H ₂ SO ₄ solution (1% CeIV / SO _4/2 in 10% aqueous sulfuric acid), heated on a hot plate and the organics amounts were detected as black spots. A total of 190 ml of tubes 2, 3, 4 and 5 were collected in a 250 ml centrifuge tube, mixed and then distributed into 6 weighed 250 ml round-bottomed flasks, labeled A to F. One of the they were found to be sterile and free from pyrogens.

The contents of the six flasks were frozen in a mixture of dry ice and acetone and lyophilized. The dry sample has the same Kd as the original HIb.

III. Preparation of polysaccharide and butanediamine adduct (HIb-BuA ₂ )

Step A. Adjustment of 1,4-butanediamine solution. 1.46 g of 1,4-butanediamine dihydrochloride were charged to a 100 ml round-bottom flask and dissolved in 58 ml of water. 5.0 ml of 2.5 N NaOH was added, bringing the pH to 10.35. The solution was filtered through a 0.22 μm Sybron-Nalge filter.

Step B. Activation of HIb and reaction with 1,4-butanediamine. A magnetic stirrer and 17.5 ml of dimethylformamide were added to a flask A (of section II) containing 0.64 g of the tetra-n-butylammonium salt of PRP. The mixture was stirred at room temperature for 25 min, whereby almost all the material was incorporated into the solution.

mg of carbonyldiimidazole are weighed in 6 ml of sterile serum ampoule and then added at once to the dimethylformamide solution. The flask was sealed and stirred at room temperature for 35 min. During this time, 32 ml of the butanediamine solution prepared according to A was placed in a 100 ml round bottom flask equipped with a magnetic stirrer and stirred in an ice bath for about 5 min.

After stirring for 35 minutes, the dimethylformamide solution was added by pipette to a cold solution of

I, 4-Butanediamine. Stirring in an ice bath continued for 15 min, after which the ice was removed and stirring continued for 17 min.

Stage S. Dialysis and lyophilization. The solution was then transferred to an autoclaved Spectropor 2 dialysis tube (cylinder volume 0.21 ml / mm; ~ 43 cm) and dialyzed at 4 ° C. First, the solution was dialyzed in 8 L of 0.01M phosphate buffer at pH 7.0 for 5 h, then dialyzed twice in fresh 8 L phosphate buffer, for a further 5 h, then for 11 h. Finally, the solution was dialyzed with 18 l of water for 6 h.

The dialysis was dispensed into two 250 ml round-bottom flasks (125 ml each) after taking a test sample for sterility and pyrogens (as a result it was found to be sterile and free from pyrogens). The contents of these flasks are frozen in a mixture of dry ice and acetone and lyophilized by the method set out in section

II. As a result, a total of 480 mg was obtained. Fluorescence analysis showed 468 nanomoles of NH ₂ / mg.

IV. Preparation of polysaccharidebutanediamide-bromoacetamide (HlbBuA ₂ -BrAc).

Step A. Preparation of p-nitrophenyl bromoacetate. 6.39 g (45 mM) of bromoacetic acid and 6.25 g of 745 mM) p-nitrophenol were placed in a 250 ml round bottom flask and dissolved in 50 ml of methylene dichloride (CH ₂ Cl ₂ ). The solution was stirred in an ice bath for 10 min and then 10.3 g (50 mM) of dicyclohexylcarbodiimide dissolved in 10 ml of CH ₂ Cl ₂ was added thereto. The reaction mixture was then stirred at 4 ° C for 17.25 h.

The precipitated dicyclohexylurea then filtered and the filter concentrated to dryness in vacuo. The yellow residue was then added to 35 ml of 1-chlorobutane, recrystallized to give

6.5 g of the product with a melting point of 85-87 ° C and this product was added to 100 ml of cyclohexane, recrystallized to give 4.59 g of p-nitrophenyl bromoacetate, melting point 86-87 ° C.

Calculations for C _g H ₆ NO ₄ Br indicate for C: - 36.92, H - 2.30, N 5.38, Br - 30.77. Found: for C, 37.66; H, 2.48; N, 5.28; Br, 30.57.

Step B. Reaction of HIb-BuA ₂ . 380 mg of HIb-BuA ₂ prepared according to Section III above (from two flasks) was dissolved in 37 ml buffer pH 9.15 in a 250 ml volumetric flask and with a magnetic stirrer. To this solution was added 346 mgp-nitrophenyl bromoacetate (from step A above) dissolved in 9 ml of acetonitrile and the mixture was stirred at 4 ° C for 24 h, then transferred to a dialysis tube (spectropor 2, see section III ). This solution was then dialyzed against 18 L of water for 5.25 h and then against fresh 18 L of water for 17.25 h (in both cases at 4 ° C).

Dialysate 100 ml was successively filtered through a 0.45 x 20 μm pore size Sybron Nalge filter. It is then divided equally into six 100 ml round-bottom flasks, frozen and lyophilized as in Section II. A total of 0.28 g of HIb-BuA ₂ BrAc was obtained. The fluorescence sample shows 128 nanomoles NH ₂ / mg, yielding 340 nanomoles bromoacetyl groups / mg as a difference. The nephelometric analysis shows the same antigenicity as the parent polysaccharide.

V. Conjugation of HIb-BuA ₂ -BrAc with a membrane protein of N. meningitidis into which functional groups (NMP) are introduced.

Step A. Function introduction of NMP with N-acetylhomocysteine thiolactone. To 6 ml of serum ampoule containing 42 mg of ethylenediaminetetraacetic acid and 8 mg of dithiothreitol was added 5 ml of borate buffer pH 11.3. From this solution 3.8 ml were placed in a 50 ml round-bottom flask and added

11.5 ml of a solution of N. meningitidis membrane protein (NMP). The pH of the resulting mixture was adjusted to 11.39 with 40-50 μΙ of 2.5 N NaOH.

The flask was capped and the air replaced with nitrogen using a refractory valve. 53 mg of N-acetylhomocysteine thiolactone was added to a nitrogen box and the resulting solution was allowed to age at room temperature for 16.7 hours. This solution was then fed into a column containing 120 ml Sephadex C25. Elupranation was carried out with a phosphate buffer of pH 8 and 5 ml fractions were collected and tested with the Elman test for thiol groups. Separation of high molecular weight thiol groups from low molecular weight ones is carried out.

Stage V. Conjugation

High molecular weight fractions were collected and added to one of the 50 ml flasks containing 0.06 g of HIb-BuA ₂ -BrAc (section IV). This solution was placed ~ for aging in a nitrogen box at room temperature for 6 h, then subjected to sterile Spectropor dialysis at 4 ° C against 18 L of water for 15 h, then fresh fresh 18 L of water for 24 h.

Stage C. Centrifugation

The dialysate (approximately 32 ml) was pipetted into 25 and 7 ml fractions in two polycarbonate centrifuge tubes and centrifuged at 4 ° C for 2 h at 37,000 rpm (100,000 xg) in a Beckmann Ti 60 rotor. The supernatants were decanted and the precipitate was transferred to a Dounce homogenizer in about 8 ml of water, homogenized and returned to one of the centrifuge tubes. The latter was supplemented to 25 ml with water, which caused complete resuspension, and this tube was centrifuged at 37,000 rpm (100,000 xg) at 4 ° C. 2 h. The secondary supernatants were decanted and the precipitates were homogenized in 8 ml of water. Transfer the homogenate to a sterile 15 ml centrifuge tube and make up to 15 ml with water.

After aging at 4 ° C, a small amount of solid granules precipitated overnight, which was removed by brief (5 min) rotation in a clinical centrifuge at 2500 rpm.

The above activation, conjugation and centrifugation are repeated twice in the same manner. They are analyzed for protein content and polysaccharide for S-carboxymethylhomocysteine (S CMCS), lysine ratio, sterility and pyrogenicity. All samples are sterile and pyrogen free. The remaining results are presented in the table below.

Sample Polysaccharide gg / ml Protein gg / ml Ratio S SMNS / Lysine 1. 105 1210 .09 .011 2. 154 1700 .09 .019 3. 166 1800 .09 .027

The constant ratio of the polysaccharide to the protein that characterizes the conjugate confirms the repeatability of the process, and the ratio of S-carboxymethylhomocysteine to lysine is an indication of reaction efficiency, with a result above 0 proving the covalency of the bond between covalently modified proteases40.

The solutions were collected for clinical use and lyophilized in the presence of lactose (20 gg / ml polysaccharide 4 gg / ml lactose).

Example 4. Preparation of a conjugate between a polysaccharide of H influenzae type B and an outer membrane protein of N. meningitidis, serotype B, closed with N-acetylcysteamine

The preparation of the polysaccharide with introduced functional groups, HlbΒιιΑ, -BrAc, was carried out as described in Example 3 (sections I to IV). The preparation of NMPs with functional groups introduced is the same as described in Example 8, Section IVA.

To a flask containing 4 ml of thiolated protein (5.6 μΜ SH according to the Elman analysis) was added 59 mg of HIb-BuA ₂ -BrAc (300 ημ bromoacetyl). The flask was sealed for sterility, degassed, replacing the air with Ν, and allowing the solution to age 18.5 hours. 6 ml of water was added and the solution was transferred to a 10 ml polycarbonate centrifuge tube and centrifuged for 2 h at 43,000 rpm in a Beckman 75 Ti rotor at 4 ° C. The supernatant was removed and the pellet was resuspended with a Dounce homogenizer in 10 ml of 0.1M pH 8 buffer containing 106 mg of N-acetylcysteineamine, the solution was degassed and left at room temperature for 19.5 h.

It was then centrifuged as described above (43,000 rpm,

4 ° C. 2 h, 75 Ti rotor). The precipitate was suspended (without homogenization) in 9.7 ml of water and centrifuged relative as described above. The resulting precipitate was resuspended by homogenization in 25 ml of water and then diluted with 30 ml of water to form an aqueous suspension of a product having a structure. Analysis of the co10 nugget shows the following.

Concentration of:

polysaccharide-188 gg / ml, protein-1200 pg / and HIB / Protein concentration 15 SCMHC / lysine = 0.096

S-carboxymethylcysteamine / lysine = 0.20

Example 5. Testing Animals for the Reaction of an Antibody with an Influenzae Type B Polysaccharide conjugate Fa 20 Type B and an N.meningitidis Outer Membrane Protein, Serotype B

The conjugate obtained according to the procedure described in Example 3 was subjected to an immunogenic response (reaction) test in ICR / Hamic and Rhesus monkeys at different ages and the results were systematized in Tables 2 and 3.

Conjugation analysis shows the following.

Polysaccharide concentration

Protein concentration

P _s / Protein ratio

Yield P

S

276 pg / ml

2.38 gg / ml

0.12

6.1%

Table 5-1

Reaction against serum antibody in ICR / Ha mice of different ages immunized with H. influenzae type b polysaccharide conjugates and protein

Sample

RIA Titer (CMT) ng antibody / ml mouse age, days *.

21

35

H.influenzae polysaccharide protein 282 ** 211 1 8645 15860 conjugates 201 ** * age at first injection; mice injected with 2 pg /

0.1 ml on day 0 and day 14; bleeding on day 21 ** separately; 7-8 mice in all groups

The activity of the conjugate between H. influenzae polysaccharide type b and protein was tested in mice and the results are shown in Table 2.

The conjugate is shown to be highly immunogenic.

___________________________________________________ Table 5-2 Thymic Dependence Test of Nude Mice Immunized with H. Influenzae Hyp b Polysaccharide - Protein Conjugate

Sample Polysaccharide dose Ria titer (GMT) ng antibody / ml Nu / Nu Mouse * Nu / + Mouse * conjugates of H.flu 1 2.5 1,802 th most common 11,362 th most common polysaccharide- protein - salt 1 50 control 2 55

+ mice injected subcutaneously at 0 and 14 days; bleeding at 21 days

The response in hlu / Nu mice is reversed, indicating that the conjugate is about 15% of the response in Nu / + thymus - dependent antigen

Table 5-3

Serum antibody response in rhesus monkeys immunized with H.influenzae type b polysaccharide protein conjugates

Rhesus Monkeys Dose Age * Polysaccharide

RIA Titer (GMT) ng antibody / ml days

14 28 42

2-3 months 20gg 50 116 195 3036 4 months 20 pg 50 414 437 1548 18 months 20 pg 124 5858 4785 7447

* three monkeys in a group

As shown in Table 2, the polysaccharide conjugate of H.influenzae type b showed high immunogenicity in Rhesus monkeys of different ages.

Example 6. Conjugation of Pneumococcal Type 19 F Polysaccharide and N.meningitidis Outer Membrane Protein

I. Preparation of the tetra-n-butylammonium salt of polysaccharide pneumococcal type 19 F. A 25 mm round bottom flask equipped with a magnetic stirrer was charged with 50 mg of polysaccharide type 19F (Merck) and 5 ml of water and the mixture was stirred for 20 min. this solution is passed through a 3 ml Dowex 50x8 column (200-400 mesh, tetraammonium form), eluted with water and collected in a 25 ml Erlenmeyer flask.

The solution is tested for the content of polysaccharides on silica gel plates sprayed with CelV (SO ₄ ) / H ₂ SO _{4 solution} , which are then heated on a hot plate to give the amount of polysaccharide that can be detected like black spots. The solution containing 19 F polysaccharide was freeze-dried and 52 mg was recovered.

II. Reaction of the tetrabutylammonium salt of pneumococcal type 19 F with carbonyldiimidazole, followed by reaction with 1,4-butanediamine (19 FBiA ₂ ).

Step A. Preparation of 1,4-butanediamine solution. Dissolve 40 mg of 1,4-butanediamine dihydrochloride in 1.0 ml of water and bring the pH to 9.15 with 2.5 NaOH.

Step B. Activation of 19 F polysaccharide and reaction with 1,4-butanediamine.

In a 25 ml round-bottom flask containing 20 mg polysaccharide 19

F in tetrabutylammonium form was added a magnetic stirrer and 4 ml of dimethyl sulfoxide (DMSO). The mixture was stirred for 20 min at room temperature, then the mixture was dissolved. 5 mg of carbonyldiimidazole is added and the reaction mixture is stirred for 30 minutes at room temperature. During this time, the 1,4-butanediamine solution obtained in step A was transferred to a 25 ml round-bottom flask equipped with a magnetic stirrer and stirred in an ice bath for about 5 min. After 30 min, a solution of DMSO with pipette was added to the cold solution of 1,4-butanediamine. Stirring in an ice bath is continued for 15 minutes, after which the ice bath is removed, but stirring is continued for another 15 minutes at room temperature.

Stage B. Dialysis and lyophilization.

After performing the processes described above, the solution was transferred to a Spectropor 2 dialysis tube and dialyzed at 4 ° C with stirring. The solution was dialyzed against 4 l 0.01 M phosphate buffer at pH 7.0 8 h, then dialyzed against 4 l 0.01 M phosphate buffer at pH 7.0 8 h. Finally, the solution was dialyzed against 4 l of water for 6 h. The solution was then lyophilized and 19 mg of the butanediamine derivative type 19F polysaccharide (19P-ViA ₂ ) was recovered. Fluorescence analysis showed 100 nm NH ₂ / mg substance.

III. Reaction of 19 F-BuA ₂ with p-nitrophenyl bromoacetate

Stage A. Reaction with

IQF-BuAj. 15 mg of 19 F-BuA ₂ prepared as indicated in II above was suspended in 2 ml pH buffer

9.15 and 25 ml round bottom flask fitted with a stirrer (magnetic) and stirred for 10 min until all material has dissolved. To this solution was added 15 mg of p-nitrophenyl bromoacetate dissolved in 0.2 ml of acetonitrile. The mixture was stirred for 24 h at 4 ° C and transferred to a Specropor 2 dialysis tube. Dialyzed twice against 4 l of water. The sample was freeze-dried to give 9 mg of N-bromoacetylated derivatives of 19 F-BuA ₂ (19 F-BuA ₂ BrAc).

Fluorescence analysis showed 57 nM NH ₂ / mg resulting in the presence of 43 nM bromoacetyl groups / mg. ^-

IV. Conjugation of 19F-BuA ₂ -BrAc with outer membrane protein of N.meningitidis (NMP) with inserted functional groups.

Step A. Function introduction of NMP with N-acetylhomocysteine thiolactone. 43 mg of ethylenediaminetetraacetic acid and 8 mg of dithiothreitol were dissolved in 5 ml of saturated borate buffer, pH 11.30. Transfer 0.4 ml of the above solution into a 15 ml centrifuge tube and add 1 ml / 13.7 mg of the N.meningitidis outer membrane protein (NMP) solution. The solution was degassed and placed in a nitrogen atmosphere at room temperature for 16 h. The solution was then diluted to a total volume of 2.5 ml by the addition of 1.1 ml of phosphate buffer at pH 8.0. Then this solution was applied to a PD _J0 column (Sephadex G25M), which was pre-equilibrated under N phosphate buffer at pH 8,0. The sample is eluted with

3.5 ml phosphate buffer pH 8.0. The thiol content was determined by Elman analysis and amounted to 1.89 μΜ / sample.

2.5 ml of sample was applied to a second PD10 column also pre-calibrated with phosphate buffer pH 8.0. It was eluted with 3.5 ml of phosphate buffer at pH 8.0. The Elman thiol content is 0.44 μΜ / sample.

Stage B. Conjugation. To a 15 ml pyrex centrifuge tube containing the protein solution, add 9 mg 19 F-BuA ₂ -BrAc from step III above. The solution was allowed to age at room temperature for 6 h. A dialysis tube of Spectropor 2 was then transferred and dialyzed at 4 ° C against 4 l of water for 8 h and then again against 4 l of water for 8 h. A certain amount is freeze dried for amino acid analysis.

Found: lysine 0.141 μΜ / mg S CMCS. 0.0062 µΜ / mg, with a value greater than 0 that the covalency of the bond is proved.

Stage C. Centrifugation. The dialysate (approximately 10 ml) was pipetted into a polycarbonate centrifuge tube and centrifuged at 4 ° C for 2 h at 37,000 rpm (100,000 g) in a Beckman Ti60 rotor. The supernatants were poured and the precipitates were transferred to a homogenizer with about 2 ml of water, where they were homogenized and returned to one of the centrifuge tubes. Make up to 10 ml with H ₂ O and centrifuge again at 37,000 rpm (100,000 g) at 4 ° C for 2 h. The second supernatant was poured and the precipitate homogenized in 8 ml of water. The homogenate is stored in a 15 ml plastic centrifuge tube and tested for immunogenicity.

Table 6-1

Reaction against serum antibody in ICR / Mice immunized with polysaccharide conjugate of pneumococcal 19 F-protein from outer membrane of meningococcal B serotype Sample Dose, mg porlisaccharide RIA Titre (GMT) ng / ml * Ps 19F-Pro conjugate 0.5 17,338 th most common

* mice were injected intraperitoneally on day 0, day 7, day 28. Bleeding on day 35.

As Table 6-1 shows, the conjugate is highly immunogenic.

Example 7. Pneumococcal 19 F Polysaccharide Binding and Hemp Seed Protein Triplet (Edestin)

I. Preparation of the tetra-n-butylammonium salt of a 19 F pneumococcal polysaccharide

Fill a 50 ml round-bottom flask with a magnetic stirrer with 105 mg polysaccharide type 19 F (Merck) and 6 ml water. The mixture was stirred for 20 min and the solution was passed through a 6 ml Dowex 50x8 column (200-400 mesh, tetra-n-butylammonium form). The column was eluted with water and the eluant was collected in a 50 ml Erlenmeyer flask and then analyzed for polysaccharide content on silica gel plates sprayed with CelV (SO ₄ ) ₂ / H ₂ SO _{4 solution} heated on hot plates. The corresponding polysaccharide content is detected as dark spots. The solution containing 19 F polysaccharide was freeze-dried and 112 mg of the tetra-n-butylammonium salt at 19 F was recovered.

II. Interaction of the tetra-n-butylammonium salt of pneumococcal type 19 F with carbonyldiimidazole, after a reaction with 1,4-butanediamine. Step A. Preparation of the 1,4-butanediamine solution.

175 mg of 1,4-butanediamine dihydrochloride were dissolved in 7 ml of water20 and the pH was adjusted to 9.5 with 2.5N NaOH.

Step B. Activation of the type 19 F polysaccharide and reaction with 1,4-butanediamine

In a 50 ml round-bottom flask containing 112 mg of polysaccharide 19F in tetrabutylammonium form was added a magnetic stirrer and 5 ml of dimethyl sulfoxide (DMSO). The mixture was stirred at room temperature for 10 min, after which the material was dissolved. 13 mg of carbonyl diimidazole was added and the reaction continued for 35 min with stirring. During this time, the 1,4-butanediamine solution obtained in step A above was transferred to a 50 ml magnetic stirrer flask and placed in an ice bath, stirring for about 5 min. Next

35 min stirring The DMSO solution was pipetted into the cold solution of 1,4-butanediamine solution. Stirring was continued for 15 min, during which time the ice bath was removed and returned for an additional 15 min with a solution at room temperature.

Stage B. Dialysis and lyophilization

The solution was dialyzed in a Spectropor 2 dialysis tube at 4 ° C. The solution was initially dialyzed against 4 l 0.01 M phosphate buffer at pH 7.0 for 8 h, then dialyzed against 4 l 0.01 M phosphate buffer at pH 7.0 for 8 h. Finally, the solution was dialyzed against 4 l of water for 4 h. The solution was then lyophilized and 80 mg of the butanediamine derivative of type 19 F polysaccharide (19F-BuA ₂ ) was recovered. The fluorescence sample shows 77 NH ₂ / mg. -

III. Reaction of 19F ViA ₂ with p-nitrophenyl bromoacetate

Step A. Reaction of 19F ViA ₂ mg 19F BuA ₂ obtained in section II was dissolved in 4 ml pH buffer

9.15 in a 25 ml round-bottom magnetic flask flask and stir for 10 min until all material has dissolved. To this solution was added 50 mg of p-nitrophenyl bromoacetate dissolved in 0.5 ml of acetonitrile. The mixture was stirred at 4 ° C for 24 h and then transferred to a Spectropor 2 dialysis tube. It was dialyzed twice against 4 l of water. The sample was then lyophilized to give 44 mg of the N-bromoacetyl derivative of 19F BuA ₂ (19F BuA ₂ BrAc). A fluorescence sample showed 7.5 nm NH ₂ / mg resulting in the presence of 69.5 nm bromoacetyl rpyna / mg.

IV. Conjugation of 19 F-BuA ₂ BrAc with functionalized and purified hemp seed (edestin) globulin.

Step A. Purification of edestine by high performance liquid chromatography (HPIC)

240 mg of twice crystallized hemp seed edema (Sigma) was dissolved in 4 ml of 3M guanidine at pH 7.0. The sample was shaken vigorously and 0.1 ml of mercaptoethanol was added. A large amount of foam forms during shaking.

The sample was left at room temperature for 1 hour and centrifuged to remove the foam, filtered through a Mileks-GV 0.22 micron filter (Millipore). Half of the sample (2 ml, 120 mg) was injected on a TSK 3000 column - molecular sieve with the following parameters: flow rate: 1 ml / min; λ max: 280 nm; solvent: 3B guanidine; UV range: 2.0; speed: 0.25 cm / min.

The corresponding fractions as determined by Y B analysis were collected and dialyzed on Spectropor 2 against 30 I of water for 16 h. Replacing 3M guanidine with water during dialysis causes precipitation of purified edestine.

The whole sample was transferred from the dialyzer to a centrifuge tube and centrifuged for 5 min. The precipitate containing the purified edestone was collected and dried in vacuo in the presence of P ₂ O ₅ . The remaining half of the starting sample (2 ml. 120mg) was then injected and passed through identical steps. Isolate is 110 mg of purified edestine. The purified edestine is then dissolved in 2.0 ml of 3M guanidine, centrifuged, filtered and chromatographed two more times using the above procedure. After three refinements, a total of 18 mg was isolated, representing a single peak in the analytical B TSK 3000 column.

Step B. Introduction of functional groups into triple-purified edestine.

mg of ethylenediaminetetraacetic acid and 5 I mercaptoethanol were placed in 1 ml of 3M guanidine. To this solution was added 14 mg of the three times purified edestine obtained according to step A above. The pH of the solution was adjusted to 9.5 with 20 ml of 2.5 M NaOH and the solution was degassed and placed under an atmosphere of N ₂ .13 mg N-acetylhomocysteine thiolactone was added to a nitrogen chamber and the resulting solution was allowed to age in a nitrogen atmosphere at room temperature for 16 h.

The solution was then diluted to a final volume of 2.5 ml by the addition of 1.4 ml of 3M guanidine and applied to a PD10 column (Sephadex G 25 M Pharmacia), which was pre-calibrated in the presence of N ₂ with 3M guanidine. The sample was eluted with 3.5 ml of 3M guanidine. The thiol content was determined by an Elman sample and found to be approximately 4.38 μΜ / sample. 2.5 ml of sample is applied to a second column of PD10, pre-calibrated with 3M guanidine and then eluted with 3.5 ml 3M guanidine. The thiol content determined by Elman is 3.24 μΜ / sample.

Stage V. Conjugation.

7 mg bromoacetylated polysaccharide 19 F type (19F-BuA ₂ -BrAc) section III was added to a centrifuge tube containing the edestine solution. This solution was allowed to age in a nitrogen chamber at room temperature for 6 h. It was then transferred to a Spectropor 2 dialysis tube and dialyzed repeatedly for 4 l of water for 8 h. The whole sample was freeze-dried and a small portion was sent for amino acid analysis.

Found: leasing 0.105 μΜ / πίβΛ CMC, 0.003 μΜ / mg, demonstrating the covalency of the link between modified polysaccharides and proteins.

Example 8. Preparation of a Conjugate of Streptococcus agaloctiae (STREP B-TURE III) Polysaccharide and N. Meningitidis Protein in the Outer Serotype

I. Preparation of Tetra-n-Butylammonium Salt from Step B (III).

100 mg of the polysaccharide obtained in step B III (prepared according to the method described in US patent No. 4.413057) was dissolved in 4 ml of water and passed through a 7 ml Dowex 50x8 column (200-400 mesh), cation exchange. resin, tetrabutylammonium form. The column was eluted with water and the fractions (3 ml) were checked for organic matter by CelV (SO ₄ ) ₂ / H ₂ SO ₄ method. The corresponding fractions were lyophilized to give the 100 mg tetrabutylammonium salt of Strep B (III) polysaccharide.

II. Preparation of the polysaccharidebutanediamine product Strep B / III / Vi ₂ ) mg of the tetrabutylammonium salt of Strep B (III) was dissolved in

2.5 ml of dry dimethylformamide and stirred for 10 minutes until completely dissolved. 5 mg of 1,1-carbonyldiimidazole is added in one go and the solution is left at room temperature for 35 minutes. This solution was then added to 3 ml of a solution containing 80 mg of 1,4-butanediamine 2CH1 whose pH was adjusted to 10.3 with 2.5 N NaOH and which was cooled in an ice bath. The resulting mixture was left in an ice bath for 15 min then at room temperature for 15 min.

The mixture was then dialyzed against 4 l of 0.1 M phosphate buffer (pH

7) three times, respectively for 5 hours, 17 hours and 7 hours. The last dialysis against 4 L of water for 18 h was followed by lyophilization, resulting in 32 g of butanediamine product from Step B (III), namely Strep B (III) -BuA. The fluorescence sample showed 212 nM NH ₂ / mg.

III. Preparation of polysaccharidebutanediamine bromoacetamide (Strep B / W / -VIA ₂ -BrAc)

26.8 mg (Strep B / 1N / -VIA ₂ ) was dissolved in 2.5 ml of borate buffer with pH 9 and 28 mg of p-nitrophenyl bromoacetate in 0.4 ml of acetonitrile was added to the solution. The mixture was stirred at 4 ° C for 23.5 h and then _TO was dialyzed at 4 ° C for 30 l of water for 17 h and then against 4 l of water for 6 h. Lyophilization gave 27 mg _{τ of the} product. The fluorescence sample showed 35 nM NH ₂ / mg, resulting in 177 μΜ / mg bromoacetyl as a difference. The nephelometric sample shows complete antigenicity.

IV. Conjugate of (Strep B / III / ViA ₂ ~ BrAc and N.meningitidis-type membrane protein (NMP) with functional groups

A. Functional group introduction to NMP: 10 ml of the NMP solution (5 mg / ml) were loaded into a polycarbonate tube at 43,000 rpm. 2 h at 4 ° C in a Beckmann Ti rotor. The supernatant was removed and the pellet was resuspended in 4 ml of borate buffer pH 11.3. containing 33.6 mg disodium hydrochloric ethyldiaminetetraacetic acid, 6.4 mg dithiothreitol. Suspension is performed with a Downes homogenizer. The mixture was transferred to a centrifuge tube, covered with serum, degassed, nitrided and 55 mg of N-acetylhomocysteine thiolactone added. The resulting mixture was allowed to age under nitrogen for 18 h at room temperature. The pH was then adjusted to 7.25 with 2.6 ml of 1M KH ₂ PO ₄ and 2.6 ml of 0.1 M phosphate buffer, and the mixture was transferred to a centrifuge tube (under nitrogen). Centrifuge as above (43000 rpm. 2 h, 4 ^U C, Ti 75 rotor). After removal of the supernatant, the precipitate was resuspended in 10 ml, pH 8.0, 0.1 M phosphate buffer (with Downs homogenizer). Re-centrifugation of this suspension, followed by resuspension of the precipitate in 4 ml of buffer with pH 8.0, resulted in a solution whose thiol titre showed a total of 5.6 μΜ SH. The control sample indicated that this was due solely to protein and small molecules such as hydrolyzed thiolactone.

B. Conjugation and purification. To the 4 ml of resuspended precipitate was added 24 ml (under nitrogen) of (Strep B / III / -BuA ₂ -BRAc and the mixture was allowed to age 18.75 h at room temperature. The mixture was transferred to 10 ml polycarbonate. centrifuge the tube and cover with water After centrifugation the pellet is resuspended in 10 ml of water and centrifuged again The final pellet is resuspended in 15 ml of water as above and the suspension has a protein content of 2.7 mg / ml and polysaccharide content 0,263 mg / ml.

The ratio of S CMCS / lysine is 0.044.

Example 9. Preparation of E. colli K1 encapsulated polysaccharide

Inoculation and development of the embryo

A lyophilized sample of E. colli K1 (obtained from Dr. John Robbins, BOB) was melted and diluted with approximately 1 ml of Trypticase-hysoy glucose (THG) medium. A Tryptocase-soy inclined arap was inoculated for 1 day before fermentation and incubated overnight at 37 ° C, during which time the developing culture was removed and suspended in 1 L THG medium. The THG medium was obtained by autoclaving 9.5 l of solution A at 121 ° C for 90 min, cooling and adding 500 ml of solution B. which was autoclaved separately at 121 ° C for 30 min.

Solution A

a. Trypticase-soy broth 300 g

b. Hysoy (Sheffield) 100 g

c. Phenol row 90mg

g. UCON LB-625 Antifoam (Union Carbide) 10 ml

e. Distilled water sufficient to give a solution 9,5 1

Solution C

a. Dextrose (anhydrous) 50 g

b. Distilled water, sufficient to give a solution of 500 ml

Fermentation

One liter of THG medium inoculated with agar stock culture was cultured in a 2 liter Erlenmeyer flask at 37 ° C with stirring at 200 rpm for 6 h when cell germination was observed. This one liter is then seeded (inoculated) in 10 l THG medium in a 14 liter fermenter in which air flow is 2 Ι / min and stirring is 200 rpm, pH is adjusted to 6.8-7.4 d 10% NaOH for 6 h when two identical values of OP (optical density) were observed.

Collection and clarification

The final fermentation culture of the examples described was added to a 22 liter plastic bottle containing hexadecyltrimethylammonium bromide (final concentration 0.3% w / v). The bottle was allowed to stand for 4 h at 4 ° C, during which time the polysaccharide precipitated with hexadecyltrimethylammonium bromide was allowed to precipitate, the sample was tested for inactivation and centrifuged in a Sharpies laboratory centrifuge at approximately 30000 rpm for 20 min, followed by the supernatant for 20 min. is removed. The cell pellet (approx. 66 g) was maintained for isolation purposes.

Suspension and extraction

Three precipitates from three fermentations were suspended separately in 400 ml 25 1.0 m CaCl ₂ and these suspensions were extracted into an Omni-mixer, immersed in an ice-cold water bath for 30 min at position 2 and collected.

Sedimentation with 25% ethanol to remove impurities

373 ml of absolute ethanol was added dropwise (up to 25% ethanol concentration), with stirring, to 1130 ml of a CaCl ₂ suspension from the previous step and the mixture was left overnight at 4 ° C. The resulting precipitate was removed by centrifugation in a Beckmann J-21, B centrifuge at · 11,000 xG for 30 min at 4 ° C and refluxed.

Sedimentation with 75% ethanol to collect crude polysaccharide

2560 ml of absolute ethanol was added dropwise (to a final concentration of 75%) by stirring to 1280 ml of clear supernatant liquid from the previous precipitation step and the mixture was left overnight at 4 ° C to ensure complete precipitation of the crude polysaccharide.

Recovery of crude polysaccharide

The insoluble polysaccharide is recovered by centrifugation with a Beckman-21 B centrifuge at 11000 xG for 30 min at 4 ° C and washed once with about 200 ml absolute ethanol and once with about 200 ml acetone, discarding the washing liquids, drying the insoluble product in a vacuum at 4 ° C over anhydrous CaCl ₂ (yield 4.6 g).

Phenolic extraction and dialysis

4.6 g of the crude polysaccharide is suspended in 400 ml of 0.488 M sodium acetate, pH 6.9, at 11.5 mg / ml by a Daunce homogenizer and this solution is extracted three times with 200 ml of an aqueous solution of phenol, obtained by the addition of 180 ml 0.488 M sodium acetate, pH 6.9 in a half-liter bottle of Mallincrodt. Each phenolic solution was then centrifuged at 11,000 xG for 30 min, at 4 ° C to break down the emulsion, and the aqueous phases were aspirated, combined and extracted with the phenolic phases removed.

The combined aqueous phases were dialyzed at 4 ° C for 24 h by varying the distilled water so that the final dialysis number was greater than 1: 100,000.

Sedimentation with 75% ethanol to collect polysaccharide

7.6 ml of 2M SaS1 ₂ were added to 305 ml of dialysate from the previous step to a final concentration of 0.05 M SaS1 ₂ and 938 ml of absolute ethanol was added dropwise (to a concentration of 75% ethanol). After standing overnight at 4 ° C, the resulting precipitate was collected by Beckman centrifuge centrifugation for 30 min at

11000 xG and 4 ° C, then washed once with about 200 ml of absolute ethanol, once with about 200 ml of acetone and dried in vacuo over anhydrous CaCl ₂ at 4 ° C (yield 1.7 g).

Ultracentrifugation

1.7 g of the polysaccharide were resuspended in 170 ml of 0.05 M CaCl ₂ , 18.9 ml of absolute ethanol was added dropwise with stirring, and the solution was centrifuged at 100,000 xG at 4 ° C for 2 h.

Product Collection

The resulting clear supernatant was separated by decantation and 468 ml of ethanol was added dropwise (to a concentration of 75% ethanol) with stirring to precipitate the polysaccharide. The mixture was left overnight to allow complete precipitation and the product was collected by centrifugation at 11000 xG for 30 min at 4 ° C, washed once with 200 ml absolute ethanol, once with 200 ml acetone and dried in vacuo over anhydrous. CaCl ₂ at 4 ° C (yield 1.46 g).

Ultracentrifugation 1.46 g of the polysaccharide was resuspended in 150 ml of 0.05 M CaCl ₂ , 50 m absolute ethanol was added. dropwise (up to 25% concentration) and the solution is ultracentrifuged at 100,000 xG at 4 ° C for 2 h.

Collection of the final product

The resulting 190 ml clear supernatant was removed; _{of the} precipitate by decantation and add 190 ml of ethanol dropwise (to a concentration of 50%) with stirring. The mixture was left for two days at 4 ° C until complete precipitation and the final product was collected by centrifugation on a Beckman J-21 B centrifuge at 11000 xG for 30 min at 4 ° C. Finally, the product was washed once with about 200 ml of ethanol, once with about 200 ml of acetone and dried in vacuo over anhydrous CaCl ₂ at 4 ° C (yield 1.2 g).

Example 10. Preparation of a conjugate between E. Colli K1 encapsulated polysaccharide and an outer membrane protein of N.meningitidis serotype B

I. Preparation of the tetra-n-butylammonium salt of E. colli K1 polysaccharide

103 mg of E. colli K1 polysaccharide prepared by the method of Example 9 was dissolved in 2 ml of water and the solution was passed through a 4 ml column of Dowex 50x8 (200-400 mesh, tetra-n-butylammonium form). The column was eluted with water and the fractions (3 ml) were tested for organic matter by the Ce / 1V / SO ₄ / H ₂ SO ₄ method. The appropriate fractions were lyophilized and the product was dried in a desiccator over P ₂ O ₅ to give 134 mg of tetrabutylammonium salt.

II Preparation of polysaccharidebutanediamine adduct (E. colli K1 ViA ₂ )

134 mg of the salt obtained in section I was dissolved in 3 ml of dry dimethylformamide and stirred for 12 min.

12.7 mg of 1,1-carbonyldiimidazole was then added at once and the solution was stirred for 30 min. This solution was then added to 6 ml of ice-cold aqueous solution containing 145 mg of 1,4-butanediamine 2CH1 whose pH was adjusted to 10.35 with 2.5 N NaOH. This solution was stirred for 15 min in an ice bath and 20 min at room temperature. It was then dialyzed three times against 4 l of 0.1 M phosphate buffer (pH 7) for 5.5 h, 16 h and 3.75 h. The last dialysis against 4 1 H ₂ O ran for 4.5 h. Lyophilization gave 73 mg of E. colli KI-ViA ₂ . The fluorescence sample indicated 180 nM NH ₂ / mg.

III Preparation of polysaccharidebutamine diaminobromacetamide (E. colli Ki - BuA ₂ -BrAc) mg E. colli K1 - ViA ₂ was dissolved in 6.7 ml of borate buffer with pH 9 and 70 mg of p-nitrophenyl bromoacetate in 1.5 ml of acetonitrile was added . The mixture was left for 19 h at 4 ° C and then dialyzed against 32 I H ₂ O and against 4 l of water for 8 h and 13 h, respectively. The solution was lyophilized to 74 mg of E. colli K1 BuA ₂ -BrAc. The fluorescence sample gave 50 nM NH ₂ / mg, resulting in a difference of 20 nM bromoacetyl / mg.

IV Conjugation ^- E. colli K1 BuA ₂ -BrAc with N.meningitidis membrane protein with introduced functional groups (NMP).

Obtaining NMPs with functional groups is the same as in Example 8, section IV-A. To a centrifuge tube containing 4 ml of thiolated NMP protein (9 μΜ SH by Elman sample) was added 25 mg of E. colli KI - BuA ₂ -BrAc (120 µm bromoacetyl / mg). The tube was covered with serum, degassed, treated with nitrogen and allowed to age for 18.5 h. This was diluted with 6 ml of pH 8 buffer and centrifuged for 2 h at 43,000 rpm at 4 ° C in a Ti 75 rotor. The supernatant was removed and the pellet was resuspended in 10 ml of pH 8 buffer with a Dounce homogenizer and then centrifuged again as above. The second centrifugation precipitate was suspended in 10 ml of water, centrifuged as above for the third time. The precipitate was then resuspended in 20 ml of water.

Conjugation analysis shows the following.

Polysaccharide content

336 pg / ml

S CMCH / lysine is 0.015.

Protein content 1000 pg / ml

Ps / protein ratio

0.34

Claims (28)

Patent Claims 1. Stable, covalently-linked polysaccharide-protein conjugates, including polyanionic bacterial polysaccharides with acid groups and immunogenic proteins, linked by hybridly constructed molecules containing thioether bonds, which can be represented by the formula AE-SB in which E represents a R represents H or II I O R II I or —CH — C N / CH7 Y / CH, / NH 1 m 2 η with m being 0 to 4, η 0 to 3, 25 CH ₂ , then m and η are not equal to nuW is 0 or ΝΗ and Υ is CH ₂ , O, S, NR ', la, and when Υ is 0 or S, then m is either CHCO ₂ H, with R' being H or C, 2,3 or 4 and η is 2 or 3 and B is or C ₂ -alkyl such that when Υ is Ζ I - (CH ₂ ) _p CH (CH ₂ ) qD as p has a value of 1 to 3, q is 0 to 2, Z is NH ₂ , NHCR ' II CO ₂ H or H, and D is О Н О О II I II II C, NR ', NC / CH, / ₂ C, where R' has the meanings given above. 45 2. Stable, covalently-coupled according to claim 1, wherein the hybrid-constructed molecules have the following formula of our polysaccharide-protein conjugate39 OH O NHCOCH, II I II I cnch ₂ ch ₂ ch ₂ ch ₂ nhcch ₂ sch ₂ ch ₂ chco The polysaccharide-protein conjugates of claim 1, wherein the bacterial encapsulated polysaccharide with acid groups is selected from the group of polysaccharides Haemophilus influenzae type b polysaccharide, and Streprococcus pneumoniae type 6B, 19F and 23 F polysaccharides. The polysaccharide-protein conjugates of claim 1, wherein the immunogenic protein is a meningococcal B serotype protein from the outer membrane or edestin protein. 5. A stable, covalently-linked 10 polysaccharide-protein conjugate consisting of Haemophilus influenzae type b polysaccharide linked by a structure of formula OH BUT NHCOCH II II HI I cnch ₂ ch ₂ ch ₂ ch ₂ n cch sch ch chco c Meningococcal protein B serotype from the outer membrane. 6. The polysaccharide-protein conjugates of claim 1, wherein the bacterial encapsulated polysaccharide containing acid groups is a polysaccharide of pneumococcal type 6B, the immunogenic protein is a mening20 coccyx B serotype of the outer membrane, and a hybrid molecule may be formed OH O NHCOCH II I II I CNCH CH CH CH NHCCH SCH CH CH 7. The polysaccharide-protein conjugates of claim 1, wherein the bacterial encapsulated polysaccharide having acid groups is a pneumococcal type 19 F polysaccharide, the immunogenic protein is a meningococcal B serotype of the outer membrane and a molecularly formed molecule can be represented. HE About NHCOCH, II I cnch ₂ ch ₂ ch ₂ ch ₂ nhcch ₂ sch ₂ CH ₂ CHCO 8. The polysaccharide-protein conjugates of claim 1, wherein the bacterial encapsulated polysaccharide having acid groups is of pneumococcal type 23 F, the immunogenic protein is of meningococcal B serotype of the outer membrane, and the hybridly constructed molecule can be 40-formed molecule. OH O NHCOCH, II I \\ I CNCH ₂ CH ₂ CH ₂ CH ₂ NHCCH ₂ SCH ₂ CH ₂ CHSO A composition comprising an immunologically active amount for both active and passive protection of mammals from bacteraemia caused by certain organisms from stable, covalently linked polysaccharide protein conjugates according to claim 1, an antiserum derived from these conjugates or gamma globulin, or other fractions of this antiserum, comprising an antibody and a pharmaceutically acceptable carrier. The composition of claim 9, further comprising an adjuvant. A composition according to claim 9 or 10, wherein the polysaccharide-protein conjugates contain one or more members of the group consisting of a polysaccharide of Haemophilus influenzae type b linked by a hybrid molecule of the formula OH O NHCOCH, II I II I CNCH CH CH CH NHCCH SCH CH ₂ CHCO with meningococcal B serotype protein from outer membrane; pneumococcal polysaccharide type 6B, conjunction | 1 I cnch ₂ ch ₂ ch ₂ with serogroup meningococcal protein B from outer membrane, pneumococcal type 19 F polysaccharide, bonded OH II I cnch ₂ ch ₂ ch ₂ with meningococcal B protein in the outer membrane and pneumococcal type 23 F polysaccharide, conjunction II I CNCH CH CH is detected by a hybrid molecule of formula About NHCOCH II I: h ₂ nhcch ₂ sch ₂ CH ₂ CHCO by hybrid constructed molecule of formula About NHCOCH II I: h ₂ nhcch ₂ sch ₂ CH ₂ CHCO Zahn by a hybrid constructed molecule of formula About NHCOCH, II I ₂ nhcch sch ₂ sn ₂ snso with meningococcal protein B serotype from outer membrane. The composition of claim 11, wherein the immunologically effective amount is an amount of each of the conjugates in the composition such that each conjugate contains from 2-50 mg of the polysaccharide in conjugated form. A composition according to claim 9 for active and passive protection of humans. The composition of claim 12, wherein the immunologically effective amount is an amount of each conjugate in the composition such that each conjugate contains 25 mg of polysaccharide in conjugated form for co-conjugates of pneumococcal polysaccharides and 10 μg of polysaccharide in conjugated form for conjugates of polysaccharides of Haemophilus influenzae type b. 15. A method of treating mammals against bacteraemia caused by related organisms, comprising administering to said mammals an immunologically effective amount of a composition comprising one or more types of polysaccharide-protein conjugates containing bacterial encapsulated polysaccharides having acidic groups bound via hybridly constructed thioether linkage molecules to immunogenic proteins, as well as a pharmaceutically acceptable carrier or adjuvant, or both. The method of treating mammals according to claim 15, wherein the polysaccharide-protein conjugates include one or more members of the group consisting of Haemophilus influenzae type b polysaccharide linked through a hybrid molecule of formula HE ON NHCOCH II I II I CNCH CH CH CH NHCCH SCH is a Meningococcal type B protein from an external polysaccharide linked via a hybrid membrane, a pneumococcal type 6B molecule of formula HE ON NHCOCH, II I II I CNCH CH CH CH NHCCH SCH taken with an outer membrane Meningococcal serotype B protein, a pneumococcal type 19 F polysaccharide linked by a hybrid molecule of formula HE ON NHCOCH, II I II I CNCH CH CH CH CH NHCCH SCH CH with a Meningococcal serotype protein detected by a hybrid outer molecule molecule and polysaccharide formulated with pneumococcal type 23 F harbor, OHO NHCOCH. II I II I CNCH CH CH CH NHCCHSCH CH cnso with an outer membrane meningococcal serotype B protein. The method of treating mammals according to claim 15, wherein the subject of treatment is infants and children and the effective amount of the composition in a single dose is an amount corresponding to 25 pg of polysaccharide in conjugated form for conjugates of pneumococcal polysaccharides and 10 pg of polysaccharide in conjugated form 45 for polysaccharide conjugates of Haemophilus influenzae type b and protein. The method of treating mammals according to claim 17, wherein one or two immunization compositions are added to the amount of polysaccharide-protein conjugate containing Haemophilus influenzae type b polysaccharide linked by a hybrid molecule of formula HE ON NHCOCH II I II I CNCH CH CH CH NHCCH SCH CHCH with meningococcal type B protein from the outer membrane corresponding to 10 µg polysaccharide in conjugated form for use in the treatment of infants and children. 19. Polysaccharide protein conjugates according to claim 1, in which HE II I 20. 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