JP2002511241A - peptide - Google Patents

Abstract

  (57) [Summary] Polypeptides up to 50 amino acids in length adapted for use as vaccines against streptococcal infections have been described. The polypeptide is (a) amino acids 150-168 of S. pyogenes protein H having the sequence QKQQQLETEKQISEASRKS; (b) the amino acid sequence for the outer membrane protein of a streptococcal strain corresponding to sequence (a); (C) fragments of sequence (a) or (b) having 6 or more amino acids; (d) sequences (a), (b) or (c) modified by deletion, insertion, substitution, rearrangement Are arranged.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to peptides and their use in vaccines against streptococcal infection.

BACKGROUND OF THE INVENTION There are a number of streptococcal species that cause disease states in humans and other animals.

[0003] Diseases caused by streptococci range from uncomplicated purulent diseases such as pharyngitis and skin infections to serious diseases such as sepsis and toxic shock syndrome.
Rheumatic fever and nephritis are non-suppurative sequelae due to acute infection with Streptococcus pyogenes. Other diseases caused by streptococci include scarlet fever, Tobihi, erysipelas, myositis, necrotic vascular contractions, septic arthritis, fragmone, colonization and destruction of heart valves (endocarditis), neonatal infections, conjunctivitis , Sinusitis, fly disease, umbilicus, meningitis, detachment and villous glanditis, postpartum sepsis, upper respiratory tract disease in humans, pneumonia, otitis media, wound infections, abscess, pyometra, mastitis, urinary tract There are infections, osteomyelitis, infectious fever of horses, dental pathogens in humans, and kidney infections in humans.

[0004] Many bacterial infections, such as acute pharyngitis, can be treated with antibiotics. However, in some parts of the world, resistance to antibiotics, especially erythromycin, is very common. Previously, the incidence of acute rheumatic fever associated with streptococcal pharyngitis has increased sharply. Acute rheumatic fever is associated with at least six different types of M-type Streptococcus pyogenes. Moreover, the number of serious streptococcal infections is increasing, with cases leading to bacteremia and sepsis, necrotic glandular vasoconstriction and myositis, puerperal sepsis, streptococcal toxic shock syndrome (STSS), and others. More.

[0005] For severe streptococcal infections, rapid and aggressive treatment is effective. Currently such treatments include surgical debridement, antibiotic treatment, intravenous infusion, respiratory support with oxygen and ventilation, dialysis, vasoconstriction to increase blood pressure, steroids and antithrombotic drugs, and the like. Significant streptococcal infections can lead to death.

[0006] Current vaccine strategies against Streptococcus pyogenes target outer membrane M-proteins. This protein is considered to be a key bacteriotoxic agent in that it may confer resistance to phagocytosis on bacteria. Furthermore, this protein elicits the potential to stimulate a deleterious host immune response through its superantigenic properties to generate a cross-reactive antibody response in humans. However, the type and number of M-proteins, and their potential to elicit a cross-reactive antibody response, are obstacles to preparing an effective vaccine against various serotypes of Streptococcus pyogenes.

[0007] New vaccines that can be used to develop additional treatment strategies for serious streptococcal infections and to overcome not only purulent streptococcal infections but also infections caused by other streptococcal species The need to create prescriptions continues.

SUMMARY OF THE INVENTION The present inventors have established a relationship between the virulence of S. pyogenes and the ability of S. pyogenes to aggregate with clinical infections. The ability to aggregate is associated with the immunogenic region of the H-protein of Streptococcus pyogenes. Amino acid sequences consistent with the amino acid sequence of this region are found not only in a wide range of Streptococcus pyogenes but also in the outer membrane proteins of other streptococcal strains.

Accordingly, the present invention provides a polypeptide of up to 50 amino acids in length suitable for use as a vaccine against streptococcal infections comprising: (a) replacing the sequence of QKQQQLETEKQISEASRKS with Suppurative streptococcal protein H
(B) an amino acid sequence of an outer membrane protein of a streptococcal strain corresponding to sequence (a); (c) a fragment of sequence (a) or (b) consisting of 6 or more amino acids Or (d) a sequence (a) that has been denatured by deletion, insertion, substitution, or rearrangement;
A sequence consisting of (b) or (c).

In the present invention, the first polypeptide has the sequence of (a), (b), (c), or (d) above, and the second polypeptide is naturally adjacent to the polypeptide first in nature. A chimeric protein that is not present is also provided.

From a further perspective, the present invention also provides a polynucleotide encoding the polypeptide of the present invention.

[0012] The present invention also relates to a regulatory sequence which constitutes the polynucleotide of the present invention and which can bind to a host cell transformed by the polynucleotide and the expression vector of the polypeptide encoded by the polynucleotide. Is also related.

A pharmaceutical composition comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier,
The present invention also provides a polypeptide of the present invention, an adjuvant in a pharmaceutically acceptable carrier or a vaccine composition comprising a polynucleotide of the present invention and a pharmaceutically acceptable polynucleotide carrier. are doing.

[0014] The present invention also relates to antibodies against the polypeptides of the present invention.

In a further broad aspect, the present invention provides a polypeptide having a length of up to 50 amino acids having the following structure: (a) Streptococcus pyogenes protein H having a sequence of QKQQQLETEKQISEASRKS (B) an amino acid sequence of an outer membrane protein of a streptococcal strain corresponding to sequence (a), wherein the sequence has a function of inhibiting aggregation or adhesion of the streptococcal strain; (C) a fragment of sequence (a) or (b) consisting of 6 or more amino acids, the fragment having a function of inhibiting streptococcal aggregation or adhesion; and (d) deletion, insertion, substitution Or a sequence (a) denatured by rearrangement,
A sequence consisting of (b) or (c), which has a function of inhibiting aggregation or adhesion of streptococci.

DETAILED DESCRIPTION OF THE INVENTION The polypeptides of the present invention are up to 50 amino acids in length adapted for use as a vaccine against streptococcal infection. The polypeptide has (a) the sequence QKQQ
It consists essentially of amino acids 150-168 of Streptococcus pyogenes protein H having QLETEKQISEASRKS. The polypeptide of the present invention also comprises (b) a sequence (
Amino acid sequence of outer membrane protein of streptococcal strain corresponding to a), (c) sequence (a
) Or (b) constitutes an amino acid fragment having a length of 6 or more, (d) a sequence (a), (b) or (c) modified by deletion, insertion, substitution or rearrangement It may be any of the arrays.

[0017] Streptococci can be classified into several groups by the Lancefield classification. This classification is based on group-specific antibodies present in cell wall polysaccharides. The key to Lansfield's classification is Group A, composed of Streptococcus Pyogenes, Group B, composed of Streptococcus agalactiae, Streptococcus equi and Streptococcus equi. Streptococcus equisimi
lis), Group C containing fecal enterococci (Enterococcus faecalis), Viridian containing mutans streptococci and sanguis streptococci.
Group. Strains that do not belong to the group include Streptococcus pneumoniae (Stre
ptococcus pneumoniae).

[0018] Within each group, bacteria are further classified by their serotype. In the case of group A streptococci, serotyping is performed by additional surface antibodies, M proteins.
This protein divides group A streptococci into more than 80 serotypes. Some of the M serotypes have been recruited for strong toxicity and pathogenicity. In particular, M1, 2, 3
, 4, 5, 6, 7, 8, 10, 12, 14, 16, 17, 18, 19, 22, 2,
The serotypes 4, 49, 55, 57 and 60 are associated with severe human infections.

S. pyogenes may express other outer membrane proteins, including immunoglobulin binding proteins. Protein H is an example of an outer membrane protein expressed by the M1 serotype of Streptococcus pyogenes. Protein H, along with other IgG proteins expressed by Streptococcus pyogenes, is now considered to be part of the same family as it is structurally related to the M protein capable of binding IgG. Nielson et al. Described the structure of the proteins H and M1 proteins of the AP1 strain of Streptococcus pyogenes (Biochemistry; 1995 Vol. 34 No. 41 13688-13698) and disclosed the entire sequence and domain structure of this protein. I have.

Although some strains of streptococci are known to form clumps, those that are prone to clumps are strongly related to the high toxicity of the bacteria. The examples given below show the role of protein H in the form of aggregates by protein H-protein H interaction. This cohesiveness is between 150 and 168 of protein H.
It is mapped to the above-mentioned sequence (a) constituting residue No. and is named APP. This is considered to be suitable for use as a vaccine against streptococci in view of the fact that APP plays a role in aggregation with bacterial toxicity. From a similar viewpoint, 145 to 145 of protein H adjacent to sequence (a)
Also of interest is residue 177, the sequence YQEQLQKQQQLETEKQISEASRKSLSRDLEASR, which is a 33-mer.

A comparative study of outer membrane proteins of a wide range of serotypes of Streptococcus pyogenes has revealed that similar sequences are present in many other strains of Streptococcus pyogenes . The same sequence was found in streptococci not belonging to group A. Accordingly, the present invention relates to the sequence (b) constituting the sequence corresponding to the outer membrane protein of the streptococcal strain. This includes the A, B, C, D, G groups and those proteins expressed on the surface of Viridian streptococci. In a preferred embodiment of the invention, sequence (b) is derived from the outer membrane proteins of group A streptococci, Streptococcus pyogenes. In another embodiment of the present invention, the amino acid sequence is a more virulent serotype of Streptococcus pyogenes, such as M1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 17, 18,
19, 22, 24, 49, 55, 57 and 60. The outer membrane protein may, for example, constitute one of the group A streptococcal M proteins. Examples of other proteins also include the proteins Arp and Sir22 expressed by certain streptococcal strains that are closely related to the binding of serum proteins. Relevant outer membrane proteins and sequences can be defined using APP or the 33-mer to establish the most appropriate arrangement when looking at the sequence of the streptococcal outer membrane proteins.

Examples of such arrangements are given in FIGS. 7 and 11. Examples of such sequences are shown below:

As can be seen from the example shown in FIG. 7, some of the highly virulent streptococcal species listed above contain a sequence corresponding to sequence (a), which emphasizes that the sequence is suitable as a vaccine. Some have. Other such sequences include those in the C2 and C3 repeat domains of protein H and have a high degree of similarity to APP.

The polypeptide of sequence (b) or (d) of the present invention is preferably at least 40% homologous to sequence (a) or the 33-mer described above with respect to its full length. At least 50% of the sequence (b) or (d) of the present invention is sequence (a)
It is even more desirable if they are homologous to at least 60%, 70% and 80%. The sequence (b) or (d) may comprise at least 90%, more preferably at least 95%, 97%, 98% of the sequence (a) or a sequence homologous to the 33-mer. More desirable.

As another method, a corresponding sequence can be obtained by hybridization using the nucleotide sequence shown in FIG. 10, which will be described later.

The present invention also relates to fragments of sequence (a) or (b) having an amino acid length of 6 or more, preferably 8 or more, more preferably 10 or more. . Such fragments can be up to 10 amino acids in length, but are preferably up to 12, 16, or 18 amino acids in length.

The modification by amino acid substitution, deletion or rearrangement is performed by (a), (b), or (c).
), For example, at amino acids 1, 2, 3, 4, 5 to 9 of sequence (a), preferably at amino acids 1, 2, or 3. For example, replacement can be performed in a conventional manner as shown in the table below. The same blocks in the second column, preferably in the same row in the third column, are interchangeable.

[0028] Other desirable substitutions can be made, for example, in comparison to the sequences described above. Preferably, substitutions are made in one or more of the sequences described above.

Except where otherwise stated, the sequences of the polypeptides described herein are represented by the conventional one-letter code and are the N-terminal to C-terminal arrangement. The amino acid polypeptides of the present invention can be modified to include amino acids that do not exist in nature or to increase stability in vivo. If the compound is made by synthetic methods, such amino acids are introduced during the manufacturing process. It is also possible to carry out denaturation by synthetic or recombination techniques.

The polypeptide of the present invention can also be made synthetically using D-amino acids. In this case, the amino acids are linked at the opposite positions C to N. This method is a conventional method for producing such a peptide.

A method for performing side chain modification on amino acids is also known, and side chain modification can be performed using the polypeptide of the present invention. Such denaturation methods include, for example, a reductive alkylation method of reacting with an aldehyde and then reducing with NaBH ₄ , and an amidine treatment for acylation with acetic anhydride. The carboxy terminal or carboxy side chain is an ester group,
For example, it can be blocked in the form of a C _1-6 alkyl ester.

The amino acid modifications listed above are not exhaustive. Those skilled in the art can modify amino acid side chains at desired positions using known chemical methods.

[0033] The polypeptide of the present invention can consist essentially of sequence (a), (b), (c) or (d).

In general, the polypeptides of the present invention will be selected and modified so as to maintain stability as a vaccine composition. As will be readily understood by those skilled in the art, polypeptides adapted for use as a vaccine composition in the present invention are those polypeptides capable of exhibiting a protective immune response against streptococcal infection. is there. The polypeptides of the present invention are selected primarily for their ability to inhibit bacterial aggregation or adhesion to epithelial surfaces. In terms of agglomeration formation and attachment, the arrays have been used as anti-aggregating agents. The corresponding sequence (b) is used to inhibit the aggregation of the streptococcal strain from which the sequence was derived. In other words, the sequence is capable of generally inhibiting streptococcal aggregation and / or bacterial attachment to epithelial cells. In this respect, bacterial agglutination means streptococcal agglutination as described in Example 1, which can be measured by an absorption test. Desirably, the peptide reduces aggregation by 30% or more, more preferably at least 50%. Additionally or in other words, the polypeptide can also inhibit bacterial attachment to epithelial cells, as shown in Example 4.

The fragments of sequences (a) and (b) can be selected on the basis of their ability to inhibit bacterial aggregation or adhesion. The modifications, substitutions, deletions, and rearrangements outlined above can also be chosen to maintain the ability of the polypeptide to inhibit bacterial aggregation.

Thus, according to a different aspect of the invention, the polypeptides we provide up to 50 amino acids have the following structure: (a) Streptococcus pyogenes protein H having the sequence QKQQQLETEKQISEASRKS of protein H
(B) a sequence corresponding to the sequence (a) of the outer membrane protein of a streptococcal strain, which has a function of inhibiting aggregation or adhesion of the streptococcal strain; c) a fragment consisting of six or more amino acids of sequence (a) or (b), which has the function of inhibiting streptococcal aggregation or adhesion; and (d) deletion, insertion, substitution, rearrangement Consisting of the sequence (a), (b) or (c) denatured by the above, which has a function of inhibiting the aggregation or adhesion of streptococci.

Since the polypeptide applied in this manner has been proposed to be used for inhibiting bacterial aggregation and adhesion to epithelial cells, it can also be used for reducing bacterial toxicity.

It is desirable that the polypeptide of the present invention be in an isolated form as much as possible. Although the polypeptide may be used in conjunction with a carrier or diluent that does not interfere with the intended use of the polypeptide, it is still believed that the isolated form is desirable. The polypeptide of the present invention is purified as much as possible. In this case, the polypeptide has a weight fraction of 9% in the preparation prepared through the separation and purification steps.
Generally, it occupies 0%, but sometimes it occupies 95% or more, and sometimes 99% by weight or more.

The polypeptides of the present invention can be produced synthetically or by recombination using techniques widely known to those skilled in the art. Synthetic manufacturing methods generally include the step of sequentially adding individual amino acid residues in a reaction vessel in which a polypeptide of the desired sequence is made. Examples of recombination techniques are described below.

The polypeptides of the present invention are up to 50 amino acids in length. More preferably, the polypeptide is no more than 40 to 45 amino acids in length, most preferably up to 30, 20, or 15 amino acids in length.

The present invention also relates to a chimeric protein composed of a first polypeptide of the present invention and a second polypeptide that is not adjacent to such a first polypeptide in nature. I have. In such a case, the polypeptide of the present invention has the sequence (a),
It is also possible to repeat (b), (c) or (d) or a combination thereof. The polypeptide may be cyclic. In some cases, the polypeptide is composed of repeating units linked through a linker sequence, such as a polylysine bridge. Peptides, especially keyhole limpet hemacyanin (limp
et hemacyanin), diphtheria toxoid, and tetanus toxoid, when used as a vaccine together with a carrier, it can also be formulated as a molten protein with the carrier.

The polypeptide of the present invention can be formulated into a pharmaceutical composition. The composition comprises the polypeptide and a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents include those used in formulations adapted for oral, topical, parenteral (eg, intramuscular, intravenous) administration. The formulations are usually prepared in a unit dosage form and prepared by methods known in the pharmaceutical arts.

For example, formulations suitable for parenteral administration include aqueous and non-aqueous systems containing antioxidants, buffers, bacteriostats, and solutes to achieve isotonicity with blood at the intended administration site. Includes sterile injectable solutions, aqueous and non-aqueous sterile suspensions containing suspending agents and thickening agents, liposomes or other particulate systems designed to deliver polypeptides into the body.

The polypeptides of the present invention can also be used to raise an immune response to prevent post-infection by streptococci.

Means for expressing the peptide immunogen include, but are not limited to, the following: keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, tetanus toxin and diphtheria toxin In the form of a peptide conjugated to a suitable carrier protein, such as a non-activated bacterial toxin, bacterial or mammalian heat shock protein; one containing the selected peptide and a T- or B-cell epitope (antigenic determinant). A recombinant bacterium or virus composed of one or more peptides or proteins, or a fused or chimeric protein expressed by a relative binding domain to a molecule present on the surface of lymphocytes; a live bacterial or viral vector Peptide or molten protein expressed or hidden on the surface by Free peptide in the form of protein.

Whether the peptide is trapped inside or expressed on the surface of lymphocytes; (
D, L) Quil A, incorporated in biodegradable microbeads prepared from lactic acid-glycolic acid copolymer or other polymers or suitable as diluent
Can be expressed on the surface of micelles prepared from saponins, cholesterol, phospholipids and the like.

To enhance the immune response, for example, 2 to 20 copies of the peptide vaccine
And / or multiple copies of the peptide can be administered, such as a synthetic polypeptide containing one or more copies of an additional T- or B-cell epitope. On the other hand, 2 to 20 copies of the peptide vaccine source and / or one or more copies of the additional T- or B-cell epitope may be chemically formed around the central matrix of lysine or other amino acids. It is expressed in the form of a synthesized branched oligomer.

[0048] The proteins used in the vaccines can be chemically denatured to facilitate serving on protein carriers and / or to enhance immunogenicity. Suitable modifications include, but are not limited to, adding a cysteine residue to each terminal that allows for polymerization via formation of a disulfide bond. Peptides can also be modified to enhance immunogenicity by conjugating a lipid such as α-aminohexadecanoic acid to the N-terminus.

The preparation of a vaccine containing an immunogenic polypeptide as an active ingredient is known to those skilled in the art. For example, such vaccines may be formulated into injectable preparations in the form of solutions or suspensions, into solid preparations adapted to be solutions or suspensions, or into liquids prepared prior to injection. The preparation may be emulsified or encapsulated in liposomes. The active immunogenic ingredient is often mixed with pharmaceutically acceptable additives that are miscible with the active ingredient. Suitable additives include, for example, water, saline, dextrose, glycerol, ethanol, or a combination thereof.

Furthermore, if necessary, the vaccine may contain various ingredients such as a small amount of a wetting agent or emulsifier, a pH buffering agent and / or an adjuvant for enhancing the effect of the vaccine. Useful adjuvants include alum or other aluminum salts, calcium salts, water-in-oil emulsions containing mineral oil, squalene or squalane, water-in-oil emulsions of squalene, Tween 85 or Span 85, etc. , But not limited to, squalene or oil in combination with a surfactant. Such emulsions may include N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), synthetic sulfolipopolysaccharide, nonionic block copolymer, quill A and / or monophosphoryl lipid A (MPL). Containing saponins, mannan or β-1-3
Carbohydrate polymers such as glucose, natural or recombinant bacterial toxins such as cholera toxin and Escherichia coli labile toxin, human interleukin-1 (IL-1), IL
-2, natural or recombinant cytokines such as IL-4, IL-5
Components such as s) can also be included. The effectiveness of an adjuvant can be determined by measuring the amount of antibody directed to the immunogenic peptide obtained from administration of a vaccine composed of various adjuvants.

The vaccine is administered by conventional parenteral routes, such as injection, for example, subcutaneously or intramuscularly. Other dosage forms include suppositories and other forms suitable for administration, oral administration,
In some cases topical administration to the nasal, rectal or vaginal mucosa, inhalation of liquid or powder preparations,
And so on. In the case of suppositories, traditional binders or carriers such as, for example, polyalkylene glycol triglycerides are added. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such preparations are prepared into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, powders and the like. If the vaccine composition has been lyophilized, the lyophilized product is liquefied, eg, a suspension, prior to administration.

Capsules, tablets and pills to be orally administered to patients are Eudragit
It may be enteric coated with "S", Euderagit "L", cellulose acetate, cellulose acetate phthalate, hydroxypropylmethyl cellulose and the like.

The polypeptides of the present invention are formulated into a vaccine in free or salt form. Pharmaceutically acceptable salts include salts with an acid (formed between the free amino group of the peptide), in which case inorganic acids such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid And an organic acid such as maleic acid. The form in which the free carboxyl group forms a salt is derived from inorganic bases such as sodium, potassium, ammonium, calcium and iron hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine. I will

The peptide vaccine stock may be administered in a single dose or in multiple doses, with a single dose of 2 to 500 micrograms and preferably a dose of 10 to 1000 micrograms of peptide. Desirably, two or more repeated administrations are performed.

In preparing the vaccine composition, other antigenic substances obtained from streptococcal proteins can be used in combination. For example, streptococcal extracellular cysteine proteinase (SCP) or an antigenic fragment thereof is also used in a vaccine formulation. SCP
Is thought to play an important role in the virulence of Streptococcus pyogenes. Active S
It is known that Streptococcus pyogenes deficient in CP productivity has substantially no toxicity. Synthetic peptides, such as those disclosed in WO 96/085692, exhibiting highly immunogenic epitope characteristics maintained in the SCP can be shared in a suitable vaccine composition.

Another streptococcal protein useful in vaccine formulation is a streptococcal inhibitor of complement-induced lysis and is termed SIC. This protein also plays a role in streptococcal virulence and toxicity and is disclosed in WO 97/137862.

As outlined above, the peptides of the present invention may be made by recombinant techniques. The present invention also provides a polypeptide of the present invention encoding a nucleic acid. Particularly preferred are polynucleotides whose 33-mer sequence is shown in FIG. 10 or fragments thereof encoding the 19-mer APP sequence (a).

The polynucleotide of the invention may comprise a coding sequence of sequence (a), for example the sequence shown in FIG. 10 for the 33-mer or a fragment thereof encoding the 19-mer APP sequence (a), or the coding sequence. Can be selectively hybridized to a sequence that is complementary to The polynucleotide of the present invention is a variant of the sequence shown in FIG. 10 that encodes the amino acid sequence of APP or 33-mer, the sequence (b)
Or a variant of the sequence encoding the polypeptide having (d). Briefly, a polynucleotide of the invention is a flanking sequence of nucleotides capable of selectively hybridizing to the 33 or 19-mer polynucleotide sequence set forth in FIG. 10 or the complement of such a sequence. .

The polynucleotide of the present invention, which hybridizes the sequence of FIG. 10 encoding the 19-mer APP sequence (a), can hybridize at a level higher than the background. Background hybridization is performed, for example, in DN
It can be caused by other DNA present in the A library. The level of signal elicited by the interaction between the polynucleotide of the present invention and the sequence of the polynucleotide of FIG. 10 encoding the 19-mer sequence (a) encoded the sequence (a) with other nucleotides. Generally at least 10-fold, preferably at least 100-fold, more than the interaction with the polynucleotide sequence of FIG. The strength of the interaction can be measured, for example, by radioactive labeling with a probe such as ³² P. Selective hybridization is performed in a medium of high stringency, for example, 0.
This can be achieved by using conditions of about 50 ° C. to about 60 ° C. in 03M sodium chloride and 0.03M sodium citrate.

The sequence of nucleotides that can selectively hybridize to the DNA coding sequence of sequence (a) or to a sequence that is complementary to that coding sequence generally has at least 50%, preferably at least 70 or 80%, Desirably at least 90 or 95% of the polynucleotide of FIG. 10 or at least 30, for the 33-mer over the entire length of the polynucleotide sequence listed in FIG.
Desirably it is homologous to the complement at least in the region of 40, for example 60, 80, 90 adjacent nucleotides. Methods for measuring the homology of polynucleotides are known. The UWGCG package providing the BESTFIT program can be used for homology calculations, eg, with its default settings (Deveraux et al, Nucl.
Acids. Res. 12 .387-395, 1984 ).

Any combination of homology and minimum size listed above, as well as more stringent combinations, ie, those with a high degree of homology over a longer range, may be used in the present invention. Can be used to define a polynucleotide.

The polynucleotide of the present invention can participate in a recombinant replication vector. Vectors are used to replicate nucleic acids in compatible host cells. In yet another example,
The present invention is directed to methods of introducing the polynucleotides of the present invention into replicable vectors, methods of introducing the vectors into compatible host cells, and methods of growing host cells under conditions that cause vector replication. Methods for making the polynucleotides of the invention are provided. The vector is recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines, and other eukaryotic cell lines, eg, insect S cells.
F9 cells are included.

The polynucleotide of the present invention, when present in the vector, desirably has an operable linkage to a normal sequence capable of providing a representation of the coding sequence of the host cell. The meaning of the term operable connection is parallel to the relationship in which the components in question are capable of performing their functions in the desired manner. A normal system capable of operable linkage with a coding sequence is achieved under conditions compatible with the reference sequence.

Such a vector can be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process comprises culturing host cells transformed with the expression vector described above under conditions that provide for expression of the coding sequence encoding the polypeptide by the vector, and then collecting the expressed polypeptide. .

The vector may be, for example, a plasmid or virus that provides a prototype for replication, but is preferably a promoter for expression of the polynucleotide, and is preferably a regulator of the promoter. The vector may contain one or more selectable marker genes, for example, an ampicillin resistance gene in the case of a bacterial plasmid, or a neomycin resistance gene in the case of a mammalian vector. Vectors are used in vitro, for example, to make RNA or to transfect or transform host cells.

The promoter / enhancer and other expression control signals are chosen to be compatible with the host cell for which the expression vector is designed. For example, yeast promoters include the yeast (S. serevisiae) GAL4 and ADH promoters. The virus promoter is SV40 large T antigen promoter, retrovirus LTR
Promoter, contains adenovirus promoter. These promoters can be easily obtained by those skilled in the art.

[0067] The nucleotide sequences and expression vectors of the invention can also be used in the preparation of a vaccine, as outlined above. Vaccines are composed of naked nucleotide sequences or combinations with cationic lipids, polymers and targeting systems.

The immunizing polypeptide prepared as described above is used for the production of polyclonal antibodies, monoclonal antibodies, and both antibodies. When a polyclonal antibody is required, a selected mammal (eg, mouse, rabbit, goat, horse, etc.) is immunized with the immunizing polypeptide of the present invention. The serum obtained from the immunized animal is collected and processed in a known manner. When the serum containing the polyclonal antibody against the polypeptide contains an antibody against another antigen, it can be purified by immunoaffinity chromatography. Methods for producing and processing polyclonal antibody sera are known to those of skill in the art.

[0069] Monoclonal antibodies to streptococcal epitopes in the polypeptides of the present invention can also be produced by methods known to those skilled in the art. General methods for producing monoclonal antibodies in hybridomas are known. Immortalized antibody-producing cell lines are generated by cell fusion or by methods such as direct transformation of B lymphocytes with tumor DNA, transformation with the Epstein-Barr virus, and the like. Panels of monoclonal antibodies produced against the polypeptides of the present invention are selected using properties such as isotype and epitope affinity.

Monoclonal and polyclonal antibodies directed against the polypeptides of the present invention are particularly useful in diagnosis, and neutralized antibodies are useful for passive immunotherapy. In particular, monoclonal antibodies can be used to raise anti-idiotype antibodies. Anti-idiotypic antibodies are immunoglobulins that provide an "internal view" of the antigen against the source of infection that requires protection.

Methods for raising anti-idiotype antibodies are known to those skilled in the art. Such anti-idiotype antibodies are useful for treating streptococci and for elucidating the immune region of the polypeptides of the present invention.

It is also possible to use fragments of the above-mentioned antibodies, for example, Fab fragments.

Examples Example 1 Surface Protein Promoting Aggregation of AP1 Bacteria WHO Streptococcal Information Research Collaborating Center for Referral
AP1 strain (40/58) of Streptococcus, obtained from ence and Research on Streptococci, Prague, Czech Republic, was obtained from Todd Hew.
When grown overnight at 37 ° C. in itt broth) (TH) (Difco, Detroit, MI), it was found to form aggregates. This visible aggregate quickly settles to the bottom of the test tube. When the optical density at 620 nm is measured over time, the degree of aggregation can be measured. Human plasma (10% solution) or human IgG (1.4 mg / ml) (Sigma Chem
ical Co., St. Louis, MO), no aggregation was observed during growth and sedimentation of the culture was much slower (FIG. 1). When the culture was examined microscopically, large aggregates of bacteria were observed except for those containing plasma or IgG, but in the case of plasma or IgG, the chains were short and no aggregates were observed (not shown).

The role of these molecules in the purification of aggregates is elucidated because proteins H and M1 proteins respond to IgG binding at the bacterial surface.

The papain and cysteine degrading enzymes produced by S. pyogenes are AP1
It has the function of removing protein H and M1 protein from the surface of bacteria. AP1 bacteria were added to 0.
Suspended at 1% (v / v) in 01 M Tris-HCl, pH 8.0 (2 ×
10 ⁹ cells / ml). Bacteria were cultured in papain (Sigma) and L-cysteine solution (containing 100 μg of papain and 28 μL of 1 M L-cysteine solution in 1 ml of cell solution) at 37 ° C. for 1 hour. Collect bacteria by centrifugation at 3000 xg,
S was washed twice and subjected to sedimentation analysis. Binding of ¹²⁵ I-labeled IgG to cells was also performed. To digest streptococci with cysteine-degrading enzymes, AP1 0.5 m
1 (2 × ^{10 10} cells / mL PBS) was incubated with 5 μg of the activating enzyme at 37 ° C. for 3 hours. The enzyme was neutralized by adding iodoacetic acid to 6 mM, and the cells were
After washing twice with S, sedimentation and binding of ¹²⁵ I-labeled IgG were measured.

Bacterial suspensions that have been slowly sedimented after treatment with this enzyme or cyanogen bromide (CNBr) no longer show IgG binding activity (FIGS. 2A, B). The peptide solubilized with CNBr was separated by SDS-PAGE, and bands were formed at apparent molecular weights of 54, 49, and 44 kDa, respectively. These bands, designated I, II and III in FIG. 2C, were assigned to the NH ₂ -terminal amino acid sequence. The sequences of bands I and II were identified as Asn-Gly-Asp-Gly-Asn and Glu-Val-Ala-Gly-Arg, and the sequences began at positions 42 and 82 of the M1 protein, respectively, while those of band III NH ₂ - terminal sequence (Glu-Gly-Ala-Lys -Ile) corresponds to a fragment of the protein H starting with 42 positions. The size of the fragment generated by CNBr is in good agreement with the position of the methionine residue in the M1 protein and protein H sequences. Data obtained from observations of IgG binding and aggregate formation indicate that proteins H and / or M1 contribute to cell-cell interactions caused by the formation of bacterial macroaggregates.

Example 2 Protein H binding to itself Protein H or M1 protein radioactively labeled with ¹²⁵ I using the Bolton and Hunter reagent (Amersham, UK) together with AP1 bacteria as before. Was cultured in TH. Protein H was found bound to the bacterial cells (FIG. 3A). The M6 serotype AP6 strain is a strain that does not express protein H, and neither protein H nor M1 protein shows affinity for this strain. Listed as a positive control in FIG. 3A is the binding curve obtained with ¹²⁵ I labeled IgG. Binding of protein H to AP1 bacteria is effectively inhibited by protein H (see below, FIG. 4B). Protein H and M1 proteins were applied from the cut end of the PVDC membrane, and the proteins H and M1 proteins labeled with ¹²⁵ I were used as probes. Although interactions were observed between Protein H molecules, the M1 protein did not bind to Protein H or itself (FIG. 3B). Furthermore, Sepharose-passivated Protein H was found to interact specifically with ¹²⁵ I-labeled Protein H (see below). This observation suggests that protein H contributes to AP1 aggregation by homosexual interaction.

Example 3 Identification of Self-interacting Regions of Protein H Using various fragments of protein H, the protein H molecule IgG, albumin,
The binding position to the FNIII domain was measured. Binding position for IgG and FNIII is NH ₂ - is located respectively in the terminal AB and A regions, binding to albumin residue is happening in C repeat protein H. FIG. 4C illustrates protein H, but also describes the fragments used in the experiments described below.

[0079] Fragment AB, except for radiolabeled protein H and fragment A, shows affinity for AP1 bacteria (FIG. 4A). Binding of radiolabeled protein H to AP1 bacteria is also a fragment AB
, But not in fragment A, suggesting that the junction is located in the B region (FIG. 4B). Nevertheless, a large portion of AB is required for inhibition as compared to protein H. Therefore, to obtain 50% inhibition, 300 times or more of the AB fragment is required. Furthermore, although fibronectin and albumin cannot block the binding of protein H to AP1 (data not shown), B suggests that it exhibits a self-aggregating region.

The IgG binding to protein H is located in the region covering the NH ₂ -terminal portion of B and the COOH-terminal portion of A. The fact that IgG inhibits bacterial aggregation indicates that the self-aggregating region may overlap with the IgG binding site. However, when radiolabeled protein H is applied to a protein H-Sepharose column, bound radioactivity cannot be eluted with excess unlabeled IgG (FIG. 5). In contrast, radioactivity elutes easily with 3M KSCN. The binding of the protein H fragment to the protein H-Sepharose was tested again and only the AB fragment had affinity (not shown).
This fragment NH ₂ from C1 repeated - has included terminal amino acid residues (Fig. 4
C), this sequence and / or the COOH-terminal portion of B raises the possibility of exhibiting self-aggregated regions. A peptide covering this region is synthesized (FIG. 4C).
) APP: aggregating protein H peptide (a ggregative p rotein H p eptide 150-
168).

On the surface of bacteria, proteins H and other members of the M protein family form α-helical coiled dimers. The homobifunctional cross-linker disuccinimidyl substrate (DSS) from Pierce in Me ₂ SO ₄ was added to the protein in PBS at 30 ° C. over 30 minutes to a final concentration of 1 mM. 1M Tris-H
The reaction was quenched by the addition of Cl pH 7.5 and 1 M NaCl. DSS was found to dimerize protein H (FIG. 6, stained, lanes 1 and 2). AP
To determine whether P interacts with the dimer, protein H is cultured with and without 21000-fold excess of unlabeled APP together with ¹²⁵ I-labeled APP, then DSS and SDS- Crosslinked with PAGE. One of the gels was stained (FIG. 6, colored) and one was autoradiographed dry.
(Figure 6, autodiagram). APP labeled with ¹²⁵ I preferentially
(Fig. 6, autoradiogram, lane 3).
Inhibited by PP (FIG. 6, autoradiogram, lane 4). The incomplete dimerization of protein H due to the presence of excess APP (FIG. 6, coloration, lane 4) may be due to the binding of the peptide to the monomer of protein H, thereby inhibiting dimerization and subsequent covalent bond formation. Have been. Experiments with APP show that there is a physical interaction with protein H, but irrelevant peptides do not crosslink protein H (FIG. 6, autoradiograph, lane 5). This peptide corresponds to one of the Ig light chain-binding domains of protein L, a different type of bacterial surface protein. This result indicates that the APP sequence is part of the self-aggregation region of protein H, and APP protein interacts in this region.

Example 4 APP Sequence Is Associated with Bacterial Aggregation, Attachment, and Resistance to Phagocytosis APP peptide was added to AP1 media to determine if the APP sequence also affected bacterial aggregation. APP, proteins H and M1 reduced the sedimentation rate (Table below), but peptides obtained from the COOH-terminal portion of protein H and peptides led to protein L had no effect. Table 1 Inhibition of AP1 aggregation (% reduction in OD after 1 hour) ¹ protein / peptide nmol / mL inhibition% protein H 1.7 99.5 M1 protein 1.3 98.6 APP 82.5 51.5 protein H peptide 351-376 64.6 0 protein L, B1- B4 5.0 0 protein L peptide 1 150.8 0 protein L peptide 2 82.4 0 ¹ AP1 were grown overnight in the presence of various proteins / peptides in TH. The sedimentation was measured and the decrease in optical density (OD decrease) after 1 hour was calculated and compared to the sedimentation of AP1 in TH alone. The average value from at least two experiments was taken.

[0083] APP-related sequences in various M and M-like proteins, including the Ml protein, were identified (Figure 7). Among the M serotypes in which APP-related sequences were identified, M
When three strains of the serotype 4, 12, 49 were tested, they were found to aggregate, but this aggregate did not block protein H (data not shown). Many M and M-like proteins remain unsequenced,
The proteins having the APP-related sequences listed in FIG. 7 have been found in some of the most ubiquitous and important streptococcal strains, especially in S. pyogenes strains. The serotypes associated with those shown in FIG. 7 account for 46% of Streptococcus pyogenes clinically isolated in the United Kingdom during 1980-1990 (Colman et al, J. Med Microbiol). 1993 Vol.
39 165-178). The fact that APP-related sequences are universal to clinically isolated ones explains that aggregation affects the virulence of Streptococcus pyogenes and other streptococci.

[0084] Adherence to mucosal surfaces is an important early stage of S. pyogenes infection. We are AP
One strain and the M6 protein-expressing strain JRS4 deficient in protein H were used to analyze the importance of the APP-related sequence on the streptococcal surface, which affects aggregation and adhesion.

Detroit 562 human (cancer) pharyngeal epithelial cells (ATCC CCL 138) were obtained from ICN and contained the minimum required medium (MEM) containing Earle's salt.
), Glutamine (ICN) 0.1 mM, fetal calf serum (FCS) 10% obtained from Life Technologies, penicillin / streptomycin (5000 units /
mL; 5000 µg / mL, PEST; ICN) at 37 ° C, 5
The cells were cultured in a% CO ₂ atmosphere. Cells for evaluating adherence were cultured in 24-well tissue culture plates until they were almost confluent. Cells were washed three times with antibiotic-free medium prior to use. Bacteria were cultured overnight on TH, collected by centrifugation, washed once in PBS and resuspended in HEM supplemented with 10FCS. 2x10 ⁷
Individual bacteria were added to the wells and the plate was incubated for 2 hours at 37 ° C. Wells in PBS
To remove unattached bacteria and 0.1 mL of trypsin (2.5 mg / mL in PBS) and Triton X-100 (0. 0 in PBS) to lyse epithelial cells.
(025%) 0.5 mL was added to each well. The necessary dilutions were made so that each well was triplicated on the TH plate to determine the number of live bacteria.

As shown in FIG. 8, both wild-type strains have strong agglutination and adhere well to epithelial cells. BM27.6 is a variant of AP1 and does not express protein H, but BJM7
1 lacks both protein H and M1 proteins. The data in FIG. 8 show that when protein H is deficient from the surface of bacteria, the cohesion and adhesion of bacteria are significantly reduced,
This indicates that the cohesiveness is further reduced when one protein is removed. Results with the M6 protein-deficient variant JRS145 also suggest that the presence of the APP-related sequence on the surface promotes aggregation and attachment.

The strain AP1 that was the subject of this study, a strain of S. pyogenes that expresses the M protein, survives and grows in human whole blood. This is in contrast to the non-aggregating BMJ71 strain that died quickly. As mentioned above, both soluble protein H and APP inhibit the aggregation of AP1. This peptide should inhibit the growth of AP1 bacteria in human blood if aggregation contributes to the antiphagocytic effect.

Bacteria grow in a fast semi-log form (OD ₆₂₀ = 0.15) and are strongly diluted in TH. 100 μL of bacterial solution is incubated with the protein or peptide for 30 minutes on ice, mixed with 1 mL of heparinized blood collected from different donors, at 37 ° C.
To continuously invert. A sample (100 μL) was collected every hour, TH containing 0.5% agar was added, developed on a TH-agar plate, and cultured at 37 ° C. overnight.

As shown in Table 2 below, when H or APP was added but no irrelevant peptide was added, the growth of AP1 bacteria in human whole blood was inhibited. APP-mediated aggregation suggests that S. pyogenes is protected from phagocytosis. Table 2 Survival of AP1 in human blood Culture time (hours) O5 CFU / mL blood ¹ inhibition% AP1 + PBS 88 ± 67 126476 ± 90500 AP1 + protein H 72 ± 86 52632 ± 60627 57.4 ± 29.4 (1.25 nmol) AP1 + APP 88 ± 82 43938 ± 39205 65.9 ± 25.1 (1000 nmol) AP1 + protein L 173 ± 118 256333 ± 102372 0 (1.67 nmol) ¹ Values are the mean ± SD of five experimental values using blood collected from different donors

Example 6 Immune Tests A test was then performed to determine the potential of APP for the production of antibodies showing potential as vaccine candidates.

A. A 19-mer APP peptide conjugated to KLH (Pierce) was used to increase rabbit immunity. 0.4 mg of 19-mer APP peptide and 0.1 m of KLH
g to a volume of 0.5 mL and Freund's adjuvant (Sigma, St. Louis, MO).
It was mixed with 5 mL (1 part complete and 2 parts incomplete) and 10 × 0.1 mL was injected subcutaneously on the back of the rabbit. Immunization was performed on the first day, and the same amount of the protein-based immunopotentiator was injected on days 30 and 60 in the same manner.

Protein H, APP-peptide, protein L-peptide (Actigen, Cambr
idge, UK) with each binding buffer (Na ₂ CO ₃ 0.016 M, NaHCO ₃ 0.035 M, pH
200 μL of the 1 μg / mL solution diluted in 9.6) was added to a 96-well plate (Nunc,
Maxisoap) and left overnight. After washing with PBS, each well was incubated with 200 μL of pre-immune serum or antiserum at 37 ° C. for 1 hour. The sera is PBS + 0.05% Tween®-20.2% bovine serum albumin (BSA) 1: 1000; 1: 2000; 1: 4000; 1: 8000; 1: 16000; 1: 32000; 64000; 1
: 128000 diluted.

A secondary antibody, goat anti-rabbit HRP-labeled (BioRad, Hercules, CA) was added to PBS + 0
. A 1: 3000 dilution in 05% Tween®-20.2% BSA was added for activity detection. 200 mg of ABTS (2,2′-azino-di (3-ethylbenzothiazoline sulfonate (NH ₄ ) ₂ -salt) was dissolved in 10 parts of water.
Substrate buffer (citric acid 0.1 M, Na ₂ HPO ₄ x2H ₂ O 0.1 M, pH 4.5)
) 40 mL, was prepared a colored reagent mixture composed of ABTS solution _{2mL, 30% H 2 O 2} 0.8mL.

[0094] 200 µL of the coloring reagent mixture was added to each well, developed, and cultured at 37 ° C for 30 minutes. Plates were read at 405 nm using an ELISA reader.

As shown in FIG. 9, it was proved that APP was bound to the antibody to form an anti-APP antigen.

B. Sheep were fed Spy-pH YQE33 and conjugated to keyhole limpet hematocyanin (KLH) (YQWQLQKQQQLETEKQISEA
(SRKSLSRDLEASRC-COOH) dissolved in Freund's complete adjuvant was subcutaneously administered to six places. On days 28, 56 and 82 after the first immunization, 350 μg of Spy-PH YQE33-KLH conjugate dissolved in Freund's complete adjuvant was administered to six places to enhance immunity. Animals were bled 14 days after the last boost of immunity to prepare serum for measuring anti-Spy-PH-YQE33 antibody by ELISA.

A microtiter plate (96 wells) was filled with 0.05M bicarbonate buffer (pH 9).
. Spy-PH-Y was added to 6) by adding a solution in which the peptide was dissolved at 5 μg / mL.
The cells were coated with the QE33 peptide and cultured (60 minutes, 37 ° C.). 0.05% Tw in PBS
een-20 (PBS-Tween) and bovine serum albumin (BS).
The plate was blocked with 1% of A) to minimize background binding. The sheep immune serum prepared as described above was mixed with PBS-Tween for 1 hour.
: Diluted to 10,000 and pre-cultured with 100 μg / mL of Spy-PH-QKQ19 (QKQQQLETEKQLSEASRKSC-COOH) related peptide or 100 μg / Ml of Spy-LP-KEY17 (CKEY TDKLDKLDKESKDK-COOH) unrelated peptide (60 min, 37 ° C). Control sera were cultured without peptide. Pre-cultured serum was added to the plate and incubated at 37 ° C for 60 hours.
The cells were cultured separately. Donkey anti-sheep IgG conjugated to horseradish peroxidase
(1: 1000 dilution in PBS-Tween) to the plate (60 min, 37 ° C.)
), And an enzyme substrate solution containing 3,3 ′, 5,5′-tetramethyl-benzidine in phosphate citrate buffer at pH 5.0 was added (10 min, RT). The colorimetric reaction was stopped by adding 2 M HCl and the optical density was measured at 450 nm. Plate is ELI
Washed with PBS-Tween at least three times between each step of SA.

The results are shown in FIG.

C. The recombinant protein H was prepared according to the method of Berge et al. (Berge et al., (1997) J. Bi.
ol. Chem. 272, 20774-20781) from E. coli expression. Protein H is human I
Purified from E. coli lysate by monoaffinity chromatography using a gG-Sepharose column. The bound protein H was eluted with 3M KSCN and
S was removed by dialysis.

A protein extract was prepared from Streptococcus pyogenes by partially digesting the cell wall using lysozyme and immunolysine. About 2 mL of cells grown to an OD _{600 nm} of about 1.0 is pelleted in a low-speed centrifuge, and 10 m containing newly added lysozyme (Sigma) 5 mg / mL and immunolysine (Sigma) 100 U / mL.
It was redispersed in 100 μL of MTris-HCl. After culturing at 37 ° C. for 10 minutes, the cells were collected by centrifugation, and 1mM of 10 mM Tris-HCl (pH 8.0).
L and washed twice with NuPage sample buffer (Novex) containing 50 mM DDT.
) 150 μL was added for lysis.

The proteins were separated by electrophoresis, and 10% NuPage Bis-Tris-HCl buffered acrylamide gel and NuPage MES SDS running buffer were used so that the optimum separation could be performed in the molecular weight range of 39 to 60 kDa. Details of the NuPage electrophoresis system can be found at Novex Electrophoresis GmbH (Brueningstrasse 50, Building C584,
D-65929 Frankfurt / M). The protein was transferred to a PVDF membrane filter using the Novex XCell II drip device and the method recommended by the manufacturer. After transferring to a PVDF filter, 0.05% v / v Tween 20 (PBST
) At room temperature with a 1: 1 mixture of UHF skim milk containing
Blocked for 0 minutes. The filter is made up of blocking buffer (1 part skim milk and PBST).
The cells were incubated with the primary antibody serum (1: 1000) for 1 hour at room temperature in 3 parts) and washed three times with blocking buffer for 5 minutes each. Filters were incubated with anti-bovine IgG alkaline phosphate conjugate diluted 1: 5000 for 30 minutes at room temperature. The filter was washed twice with PBST for 10 minutes and 10 mM Tris-HCl (pH 8.0) before adding bromo-chloro-indolyl phosphate / nitro-blue tetrazolium (BCI / MBT substrate kit, Sigma), a colored substrate of alkali phosphate. ), 150 mM Na
Two final washes were performed with cl for 5 minutes.

Recombinant protein H (800 ng) and M6 form of Streptococcus pyogenes (protein H
Immunostaining was performed using protein extracts from non-phenotype) and AP1 (M1 protein H phenotype). The main band was detected in the track bearing the purified recombinant protein H, and the mild high molecular weight band was detected in the track bearing AP1.
This indicates that a small amount of protein H was enzymatically released from the cell wall of the AP1 strain.

The results show that immunization with the APP peptide, which also acted on the carrier tanapac KLH, resulted in the purified recombinant protein H and the wild-type protein H from Streptococcus pyogenes.
Elicits IgG serum antibodies bound to. The difference in molecular weight between purified recombinant protein H and wild-type protein H from Streptococcus pyogenes is determined by the fact that recombinant protein H is shorter by 30 amino acids in length.

Example 7 Bactericidal Analysis S. pyogenes Streptococcus A group (AP1) bacteria were Todd Hewitt broth (TH)
It grows in an early semilogarithmic form (OD ₆₂₀ = 0.15) in (Difco Laboratories, Detroit, MN) at 37 ° C., 5% CO ₂ atmosphere. Five steps of serial dilution were performed with TH bouillon by adding 4.6 mL of TH-bouillon to 0.4 mL of the bacterial solution. 100 μL of the fourth or fifth dilution was added to 100 μL of undiluted pre-immune serum (pre-APP).
The cells were cultured on ice or 100 μL of undiluted antibody serum (anti-APP), 100 μL of 1 mg / mL pre-immune serum (pre-IgG) or antiserum (anti-IgG) for 30 minutes on ice. Heparin-treated human blood (1 mL) was added to the mixture (200 μL), and the mixture was continuously inverted at 37 ° C.

A 100 μL sample was withdrawn at each time and TH2.5m containing 0.5% agar was used.
L was added thereto, developed on a TH-agar plate, and cultured at 37 ° C. overnight. The number of colonies generated was counted. The IgG fractions from the pre-immune serum and the antiserum were each purified on the protein LG-Sepharose.

The results are shown in Table 3 below, and show that anti-APP serum inhibits the survival of AP1 in human blood. APP has its own inhibitory activity, and it can be seen from an overview of the vitality of APP that it is not only a vaccine candidate but also an aggregation inhibitor. Table 3 people AP1 survival inhibitor inhibitor inhibits% before -APP serum ¹ 30.9 ± 24.8 anti -APP serum ¹ 35.5 ± 15.9 before IgG ² analog purified from -APP serum 50.3 ± 28.9 anti -APP serum of in the blood IgG ² analog purified from 80.2 ± 11.8 ¹ Average from 5 experiments using blood from 3 different donors ² Average from 3 experiments using blood from ² different donors

Example 8 Challenge with APP Peptide and Streptococcus pyogenes Challenge The efficacy of APP in increasing survival due to anti-aggregation properties was examined.

S. pyogenes strain AP1 of the M1 serotype was grown at 37 ° C. in 5% C in Todd Hewitt medium.
O were grown overnight in ₂ atmosphere. The cells were collected by centrifugation at 3,800 × g for 10 minutes. The resulting bacterial pellet is washed twice and then washed with 1 × phosphate buffered saline, P
Resuspended in BS. Dissolve 2 mg of 19-mer APP-peptide in PBS and add
Units forming 10 ⁶ colonies of one bacterium were mixed to make a total of 0.2 mL. The mixture was injected subcutaneously into mice using a so-called air-swallowing model. In this method, 0.8 mL of air and 0.2 mL of the bacterial / peptide mixture are filled into a syringe and mixed. This is the so-called air intake / drink model. The mice are left in the cage and the number of survivors is counted over time. Table 4 shows the results. Table 4 Bacteria (cfu / mL) Bacteria + APP-peptide Surviving number 10 ⁶ -All dead 10 ⁶ 2 mg Total surviving

All mice receiving bacteria only died within 24 hours, while bacteria + A
All the mice to which the PP-peptide was administered were still alive after 4 weeks.

[Brief explanation of the figure]

FIG. 1. Sedimentation analysis of AP1 bacteria. AP1 bacteria are TH (○), TH (●) containing 10% human serum, 1.4 mg /
Grow overnight at 37 ° C. in TH (△) containing mL of human IgG. Bacteria were redispersed and allowed to settle at room temperature. Sedimentation velocity was determined by plotting the optical density at 620 nm against time.

FIG. 2 Lysis of AP1 surface protein (A) AP1 bacteria were cultured with PBS (ＰＢＳ), papain (イ ン), streptococcal cysteine proteinase (△) or CNBr (（). The bacteria were digested, centrifuged, washed, re-dispersed, and subjected to sedimentation analysis. (B) The above (A) having a concentration of 2 × 10 ⁹ bacteria / mL was serially diluted and then subjected to a binding test with ¹²⁵ I-labeled IgG. (C) SDS-PAGE analysis of CNBr-solubilized material from AP1 bacteria. The fragments generated by CNBr are represented by graphical representations of the H and M1 proteins. NH ₂ - terminal signal sequence (Ss) is displayed, the protein is bound to the bacterial cells by COOH- terminal D-domain. The sequences of the Ss, C and D domains show a high degree of homology. IgGFc-binding is located in the AB domain of the H protein and the S domain of the M1 protein. The numbers in the figure indicate the positions of the amino acid residues. The H protein used here had a truncated form lacking the 27-COOH-terminal amino acid residue that binds to the bacterial cell wall (
42-349), whereas the M1 protein covers residues 42-484.

FIG. 3: Protein H bound to AP1 bacteria and purified protein H. (A) 2 × 10 ⁹ cells / mL of AP1 and AP6 bacteria were serially diluted and then labeled with ¹²⁵ I with protein H (●) or M1 protein (△). Was subjected to a binding test. ^{12 5} I-labeled IgG (○) were used as a positive control sample. (B) Various amounts of protein H and M1 proteins were applied to PVDF filters. The filters were ¹²⁵ H-labeled protein H or M1 protein (2 ×
(10 ⁵ cpm / ml), followed by autoradiography for 3 days.

FIG. 4. Mapping of the self-association region of protein H. (A) Binding of ¹²⁵ H-labeled protein H (○), protein H fragment AB (△) or fragment AB (□) to 4AP1 bacteria. (B) The binding of ¹²⁵ I-labeled protein H to AP1 bacteria (2 × 10 ⁹ cells / mL) was determined by varying amounts of unlabeled protein H (）) or protein H fragments AB (△) and A (□). ). (C) Illustration of protein H. The various fragments of protein H are shown below the figure along with the sequence of the cohesive protein H peptide (APP). The numbers are for amino acid residues.

FIG. 5: Radiolabeled protein H has affinity for protein H-Sepharose. Protein H (10 ⁶ cpm) labeled with ¹²⁵ I was applied to a Protein H-Sepharose column. The column was washed thoroughly with PBSAT before the human IgG (5
mg / mL) and a second wash with PBSAT followed by 3M KSC
Finally eluted with N. The radioactivity of the fractions was plotted against the elution volume.

FIG. 6. APP is cross-linked to protein H. Protein H (30 p) in the presence of ¹²⁵ I-labelled APP (about 450 pmol)
0 pmol) was cross-linked with DSS. One of the gels was stained and the other was dried and autoradiographed to analyze the sample by SDS-PAGE. Lane 1
: Protein H without crosslinking agent; Lane 2: Protein H with crosslinking agent; Lane 3
: Protein H cross-linked in the presence of ¹²⁵ I-APP; Lane 4: ¹²⁵ I-APP
H and excess unlabeled APP (450 n
mol); Lane 5: Protein H cross-linked in the presence of the ¹²⁵ I-labeled B1 domain of Protein L.

FIG. 7. APP-related sequences are found on some M or M-like proteins. Sequences of sequences associated with APP can be found in databases. The identity of APP at the amino acid and nucleotide levels has also been obtained with the M serotype from which the protein is derived. Various proteins M49, Sir22, ML2.1, M
1, M5, M12, Arp4 and M6 are also found at APP-related sequence positions.

FIG. 8: Sedimentation analysis and attachment of Streptococcus pyogenes. (A) Wild-type AP1, mutant deficient in protein H (BM27.6), mutant deficient in both protein H and M1 protein (BMJ71), wild-type M6 Stock (J
RS4) (not showing protein H), M6 protein deficient mutant (JRS14
The cohesion test of 5) was performed. Sedimentation was determined from the decrease in optical density at 620 nm after 1 hour. The average value ± SD is determined. (B) The wild strain and the mutant strain were examined for their adhesion to human epithelial cells. One hundred percent attachment is 1.49 × 10 ⁶ ± 0.5 AP 1 bacteria / epithelial cell layer or 0.27 × 10 ⁶ ± 0
. Corresponds to 04 × 10 ⁶ JRS4 bacteria / epithelial cell layer. The AP1 mutant was compared to AP1, and the JRS145 mutant was compared to JRS4. Values are means ± SD.

FIG. 9: ELISA results using antiserum prepared by injecting rabbits with KLH-applied APP together with an adjuvant.

FIG. 10. Nucleotide sequences of APP-peptide (33-mer) and other M protein homologs.

FIG. 11: Positioning of APP-related sequences using APP-peptide (33 mer).

FIG. 12: Effect of peptide preadsorption on detection of antibodies induced by Spy-PH-YQE33.
Sheep were primed and then boosted with the protein H-derived peptide Spy-PH-YQE33, which was commensurate with the KLH carrier protein. Serum after immunization (1: 1
(0000 dilution) were incubated with 100 μg / mL of an irrelevant peptide (Spn-LP-KEY17) or 100 μg / mL of a relevant peptide (Spy-PH-QKQ19) (60 min, 37 ° C.). Control samples were cultured without peptide. Antibodies against the Spy-PH-YQE33 epitope were thus measured by ELISA. Result 450
Expressed as the optical density measured in nm (mean, n = 4 wells).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/12 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZWF term (reference) 4B024 AA01 BA31 CA04 DA06 EA04 GA11 HA01 4C084 AA02 AA07 BA01 BA17 BA18 BA19 CA04 MA52 MA66 NA14 ZB351 ZB352 4C085 AA03 BA14 BB11 CC07 CC04 DD62GG08 AA10 AA11 AA30 BA17 BA18 CA11 DA76 DA86 EA29 FA72 FA74 GA26

Claims (16)

[Claims] 1. A polypeptide of up to 50 amino acids in length, adapted for use as a vaccine against streptococcal infection, comprising: (a) S. pyogenes having the sequence of QKQQQLETEKQISEASRKS
(B) an amino acid sequence of an outer membrane protein of a streptococcus strain corresponding to sequence (a); (c) a sequence consisting of 6 or more amino acids (a) or (b) Or (d) a sequence (a) denatured by deletion, insertion, substitution, or rearrangement;
The above polypeptide consisting of the sequence consisting of (b) or (c). 2. The polypeptide according to claim 1, wherein the sequence (a) is the sequence YQEQLQKQQQLETEKQISEASRKSLSRDLEASR. 3. The polypeptide according to claim 1, wherein the sequence (b) comprises an amino acid sequence of an outer membrane protein of Streptococcus pyogenes. 4. The polypeptide according to claim 3, wherein the sequence (b) comprises the amino acid sequence of Streptococcus pyogenes M or M-like protein. 5. The polypeptide according to any one of claims 1 to 4, wherein the length of the polypeptide is up to 40 amino acids, preferably up to 30 amino acids. 6. The polypeptide according to claim 1, wherein the fragment of sequence (a) or (b) is 10 or more amino acids in length, preferably 18 amino acids in length. peptide. 7. The sequence (a) is substantially the sequence QKQQQLETEKQISEASRKS or the sequence Y
The polypeptide according to claim 1, which is QEQLQKQQQLETEKQISEASRKSLSRDLEASR. 8. A chimeric protein comprising the first polypeptide according to any one of claims 1 to 7 and a second polypeptide which is not adjacent to the first polypeptide in nature. 9. A polynucleotide encoding the peptide according to any one of claims 1 to 8. 10. An expression vector comprising a polynucleotide according to claim 9 and a regulatory sequence capable of being operably linked to the polynucleotide for expression of a polypeptide encoded by the polynucleotide. 11. A host cell transformed with the expression vector according to claim 10. 12. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 13. A vaccine composition comprising the polypeptide of claim 1, an adjuvant and a pharmaceutically acceptable carrier. 14. A vaccine composition comprising the polynucleotide of claim 9 and a pharmaceutically acceptable carrier for said polynucleotide. An antibody against the polypeptide according to any one of claims 1 to 7. 16. A polypeptide having a length of up to 50 amino acids, comprising: (a) amino acids 150 to 168 of protein H of S. pyogenes having the sequence of QKQQQLETEKQISEASRKS; (b) sequence An amino acid sequence of an outer membrane protein of a streptococcal strain corresponding to (a), wherein the sequence has a function of inhibiting aggregation or adhesion of the streptococcal strain; (c) six or more amino acids A fragment of the sequence (a) or (b) consisting of: a fragment having a function of inhibiting streptococcal aggregation or adhesion; and a),
The above polypeptide, which is a sequence consisting of (b) or (c), which has a function of inhibiting aggregation or adhesion of streptococci.