JP2002525041A - Streptococcal Cβ protein composition - Google Patents

Abstract

  (57) [Summary] The present invention relates to a protein fragment or peptide that elicits an antibody against Gram-positive bacteria that is bactericidal using complement alone without involvement in the opsonophagocytic mechanism. (Including an immunogenic IgG binding domain comprising a peptide sequence of at least 8 amino acids) and vaccines and conjugates comprising the same. The present invention also relates to polynucleotide molecules encoding the protein fragments or peptides, vectors containing such polynucleotide molecules, and host cells transformed with the vectors. The present invention relates to a process for obtaining substantially pure Cβ protein or fragments and / or variants thereof. The present invention also relates to a method of inducing an immune response in an animal, the method comprising administering to the animal a therapeutically effective amount of a vaccine of the present invention in a pharmaceutically acceptable carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention raises antibodies that have complement alone and are bactericidal against Gram-positive bacteria (ie, their activity does not depend on the opsonophagocytic mechanism). Protein fragments or peptides that The present invention also relates to fragments of the group B streptococcal β antigen and variants thereof with reduced or eliminated ability to bind human IgA. These fragments retain useful immunological properties to formulate conjugate vaccines against group B streptococci. The invention also relates to the isolation and purification of group B streptococcal Cβ proteins and variants and / or fragments thereof.

Related Art Streptococci are a large and diverse set of Gram-positive bacteria that have been arranged into groups based on the antigenicity and structure of their cell wall polysaccharides (Lanc.
efield, R .; C. , J. et al. Exp. Med. 57: 571-595 (193)
3); Lancefield, R .; C. Proc. Soc. Exp. Biol
. and Med. 38: 473-478 (1938)). Two of these groups have been associated with severe human infections. Those classified as group A streptococci are the most common bacteria, and "strep throats"
Is the organism that causes it. Group A streptococcal organisms have also been associated with more severe infections of rheumatic fever, streptococcal impetigo, and sepsis.

[0003] Group B streptococci were not known as human pathogens in standard medical texts until the early 1970's. Since that time, studies have shown that group B streptococci are important perinatal pathogens in both the United States and developing countries (Smith, AL and Haas, J. Infections).
of the Central Nervous System, Raven
Press, Ltd. , New York, pp. 313-333 (1991)).
. Systemic group B streptococcal infection during the first two months of birth affects 3 newborns per 1000 newborns (Dillon, HC, Jr. et al., J. Ped.).
iat. 110: 31-36 (1987)), which produces 11,000 cases each year in the United States. These infections cause symptoms of congenital pneumonia, sepsis, and meningitis. A substantial number of these infants die or have permanent neurological sequelae. In addition, these group B streptococcal infections can be associated with high pregnancy-related morbidity, which occurs in nearly 50,000 women each year. Additionally, those at risk for group B streptococcal infection include those who have altered their immune response either congenitally, chemotherapeutically, or by other means.

[0004] Group B streptococci further comprise at least nine different types (eg, Ia, Ib, II, III, IV, V, VI, VII, and VII) based on bacterial capsular polysaccharides.
I). These different types, which are the most pathologically important, are Ia, Ib, I
Streptococci with type I, III, and V capsular polysaccharides. These four types of group B streptococci represent more than 90% of all reported cases. The structure of each of these various polysaccharide types has been solved and characterized (
Jennings, H .; J. Et al., Biochemistry 22: 1258-
1263 (1983); Jennings, H .; J. Et al., Can. J. Bioc
hem. 58: 112-120 (1980); Jennings, H .; J et al., P
rc. Nat. Acad. Sci. USA. 77: 2931-2935 (19
80); Jennings, H .; J. et al. Biol. Chem. 258: 17
93-1798 (1983); Wessels, M .; R. J. et al. Biol. C
hem. 262: 8262-8267 (1987)). As found in many other human bacterial pathogens, group G streptococcal capsular polysaccharides, when used as vaccines, are very effective, potent against infection by these bacteria It has been shown to provide protection. This is because the first Lancefield (
Lancefield, R .; C. J. et al. Exp. Med. 142: 162-1
70 (1975)), and more recently, a number of studies by Kasper and co-workers (Baker, CJ et al., N. Engl. J. Med. 3).
19: 1180-1185 (1988); Baltimore, R .; S. J
. Infect. Dis. 140: 81-86 (1979); Kasper, D.
. L. J. et al. Exp. Med. 149: 327-339 (1979); Mad.
off, L .; C. J. et al. Clin. Invest. 94: 286-292 (1
994); Marques, M .; B. Et al., Infect. Immun. 62: 1
593-1599 (1994); Wessels, M .; R. J. et al. Clin.
Invest. 86: 1428-1433 (1990); Wessels, M .;
R. Et al., Infect. Immun. 61: 4760-4766 (1993);
Wyle, S.M. A. J. et al. Infect. Dis. 126: 514-522 (
1972)). However, many other capsular polysaccharide vaccines (An
Derson, P .; J. et al. Clin. Invest. 51: 39-44 (19
72); Gold, R.A. J. et al. Clin. Invest. 56: 1536-1
547 (1975); Gold, R.A. J. et al. Infect. Dis. 136S
S31-S35 (1977); Gold, R .; M. J. et al. Infect. D
is. 138: 731-735 (1978); Makela, P .; R. H. Et al.,
J. Infect. Dis. 136: S43-50 (1977); Peltol
a, A. Et al., Pediatrics 60: 730-737 (1977); Pe.
ltola, H .; Et al. Engl. J. Med. 297: 686-691 (1
977)), vaccines formulated from pure type Ia, Ib, II, and III capsular carbohydrates are relatively weak immunogens and have little efficacy in children less than 18 months of age No (Baker, CJ and Kasp)
er, D .; L. Rev .. Inf. Dis. 7: 458-467 (1985);
Baker, C.E. J. Et al. Engl. J. Med. 319: 1180-11
85 (1988); Baker, C .; J. Et al., New Engl. J. Med.
322: 1857-1860 (1990)). These pure polysaccharides are classified as T-cell dependent antigens. Because they induce a similar immunological response in animals lacking T lymphocytes (Howard, JG et al.
Cell. Immunol. 2: 614-626 (1971)). These polysaccharides are not expected to elicit a secondary booster response. Because they do not interact with T cells and therefore do not trigger a subsequent "helper response" by secreting various cytokines. For this reason, each successive administration of the polysaccharide as a vaccine releases a certain amount of antibody, whereas the T cell-dependent antigen elicits increasing concentrations of antibody each time it is administered .

[0005] Goebel and Avery reported in 1931 that by covalently attaching pure polysaccharides to proteins, they could elicit an immune response against that polysaccharide that could not be achieved by using the polysaccharide alone. (Avery,
O. T. And Goebel, W .; F. , J. et al. Exp. Med. 54: 437-
447 (1931); Goebel, W .; F. And Avery, O .; T. , J
. Exp. Med. 54: 431-436 (1931)). These observations have begun and have formed the basis of current conjugate vaccine technology. Numerous studies have followed, which show that when the polysaccharide binds to the protein before being administered as a vaccine, the immune response to the polysaccharide changes from a T-independent response to a T-dependent response. For a review, see Dick, W .; E. FIG. , Jr. And Beurr
et. , Glycoconjugates of bacterial c
arbohydrate antigens: Contributions t
o Microbiology and Immunology. Krouse et al., Eds. 48-114 (1989); Jennings, H. et al. J. And Good,
R. K. , Neoglycoconjugates: Preparation
and Applications. Lee, Y .; C. And Lee, R.A. T.
Ed., Academic Press, New York, pages 325-371 (1
994); and Robbins, J. et al. B. And Schneerson, R .;
, J. et al. Infect. Dis. 161: 821-832 (1990). Currently, most of these polysaccharide protein conjugate vaccines are formulated with well-known proteins (eg, tetanus toxoid and diphtheria toxoid or variants thereof). These proteins were originally used. Because they have already been approved for human use and have been well characterized. However, as more and more polysaccharides are attached to these proteins and used as vaccines, interference between different vaccines using the same protein has become prominent. For example, when several different polysaccharides are conjugated to tetanus toxin and given sequentially, their immune response to the first administered polysaccharide conjugate is much greater than the last administered. However, if each of the polysaccharides is conjugated to a different protein and is administered sequentially, the immune response to each of the polysaccharides will be the same. Carrier suppression is the term used to describe this observed phenomenon. One approach to overcoming this problem is to match proteins and polysaccharides such that they are from the same organism.

[0006] Of the various antigens used to classify and subgroup group B streptococci,
One was a protein known as the Ibc antigen. This protein antigen was first described by Wilkinson and Eagon in 1971 (Wilkinson, HW and Eagon, RG, Infect.
Immun. 4: 596-604 (1971)) and was known to be composed of two different proteins, termed α and β. Then Ib
The c antigen has been shown to be effective when used as a vaccine antigen in a mouse model of infection by Lancefield and coworkers (
Lancefield, R .; C. J. et al. Exp. Med. 142: 165-1
79 (1975)).

[0007] Its isolation, purification, and functional characterization of the β antigen (Cβ) protein of group B streptococci has been achieved by Russell-Jones et al. (Russel-
Jones, G .; J. And Gotschlich, E .; C. , J. et al. Exp. M
ed. 160: 1476-1484 (1984); Russel-Jones,
G. FIG. J. J. et al. Exp. Med. 160: 1467-1475 (1984))
.

In testing the IgA binding activity of surface proteins from various GBS strains,
Russel-Jones et al. First described Triton® X-100.
Cβ protein was extracted using the detergent method (Ruddell-Jone)
s, G .; J. J. et al. Exp. Med. 160: 1467-1475 (1984)
)). They identified a 130 kD protein that was consistently associated with IgA binding activity, but the protein was always contaminated with smaller proteins, only some of which bound to IgA. They are,
These lower molecular weight proteins were expected to be degradation products from uncharacterized bacterial proteases. Russel-Jones et al. Reduced proteolytic activity by extracting Cβ protein in hot SDS, and then purified the protein by SDS gel chromatography. They obtained a substantially pure protein, but this method yielded the final product,
Not provided in sufficient quantity and purity for subsequent binding to bacterial polysaccharide.

[0009] Madoff et al. Obtained Cβ protein using preparative SDS-PAGE and electroelution of Cβ protein (Madoff et al., Infect. Immun.
. 60 (12): 4989-4994 (1992)). This method also did not help scale up. (1996) obtained fragments of the Cβ protein from inclusion bodies produced in cells that overexpressed the Cβ protein fragment (Jerlsrom et al., Infect. Immun.
64 (7): 2787-2793 (1996)). The inclusion bodies were first solubilized in 8M urea. The resulting supernatant was then subjected to FPLC using a MonoQ® ion exchange column. Yield and purity were not described in detail, but it appears that some degree of purification was achieved simply by isolating the inclusion bodies.

[0010] Russel-Jones et al. Reported that one of the properties of the Cβ protein was that human IgA
It was demonstrated that the binding was specific for immunoglobulin. The binding site on the IgA molecule was located in the Fc portion of the immunoglobulin heavy chain. They further showed that the Cβ protein consists of a single polypeptide with a predicted molecular weight of 130 kD. The gene responsible for the expression of Cβ protein was cloned (Cl
eat, P.E. H. And Timmis, K .; N. , Infect. Immun.
55: 1151-1155 (1987)), and sequenced by a group led by Timmis (Jerlsrom, PG et al., Mol.
ec. Microbiol. 5: 843-849 (1991)). Their later studies demonstrated that IgA binding activity could be ascribed to a 746 bp DNA fragment of the gene defined by being initiated at the BglII restriction endonuclease cleavage site and wrapped at the HpaI restriction endonuclease cleavage site.

As previously mentioned, Lancefield's 1975 study showed that the Ibc antigen is an effective vaccine antigen in a mouse model of group B streptococcal infection (Lancefield, RC et al. J. Exp.
165-179 (1975)). It is not clear at this time that the α or β protein component of the Ibc antigen is responsible for this production. Madoff et al. Illuminated the problem and demonstrated that purified Cβ protein used as a vaccine protects infant mice from experimental infection with group B streptococci expressing this protein (Madoff et al.). , LC, et al., Infect.
0: 4989-4994 (1992)). Madoff et al. Also noted that they
When the streptococcal capsular polysaccharide was conjugated to the Cβ protein, the vaccine could be used to immunize infant mice with type III group B streptococci (which do not express Cβ) or type Ib group B streptococci (which express Cβ but have (Lacking type capsular polysaccharides) (Madoff, LC et al., J. Clin. Invest.
94: 286-292 (1994)). Thus, this Cβ protein conjugate vaccine serves several functions: the polysaccharide elicits protective antibodies against the polysaccharide capsule, and the Cβ protein elicits protective antibodies against the protein, Was altered from a T-independent response to a T-dependent response.

This polysaccharide Cβ protein conjugate strategy works well in mice.
Clearly, however, its purpose is to protect humans from group B streptococcal infection. The only caveat in using this same strategy in humans is that
The Cβ protein binds non-specifically to human IgA immunoglobulin (Cβ does not specifically bind to mouse IgA). This human IgA binding activity of Cβ may reduce the efficacy of a polysaccharide Cβ protein conjugate vaccine against humans. This is because the antigen bound to IgA can be cleared from the system so quickly that no antigen-specific antibody response is generated. In addition, potentially protective epitopes on the Cβ protein may be masked when human IgA binds to the Cβ molecule. Therefore, a mutant Cβ that lacks IgA binding capacity but retains the native structure as much as possible
It is advantageous to obtain proteins.

With this aim in mind, several groups have attempted to determine the IgA binding region of the Cβ protein. Brady and Boyle (Infet. Im
mun. 57: 1573-1581 (1989)) identified a particular form of group B streptococcal β antigen that does not bind to the Fc region of IgA. Brady and Boyl
e also observed that certain strains tested in their study secreted low molecular weight forms of the antigen. HG806 secreted a form of approximately 38 kD that did not show human IgA-Fc binding. Two other strains, 2AR and DL471B, secreted the approximately 55 kD and 53 kD forms, respectively. However, these forms showed IgA-Fc binding.

Further, Jerlstrom et al. (Molec. Microbiol. 5:84
3-849 (1991)) used experiments in which subfragments of the Cβ protein were expressed as fusion proteins to identify two regions of the Cβ protein that could bind IgA. These experiments show that the IgA binding domain is a 747 bp BglII-HpaI fragment of the Cβ protein and a 1461 bp fragment.
HpaI-HindIII fragment.

US Pat. Nos. 5,595,740 and 5,766,606 describe deletion mutants of the Cβ protein that lack a larger region encompassing this domain and do not bind IgA. I do. Furthermore, the international application PCT / US97 / 1531
9 describes a 12 amino acid domain of the Cβ protein;
Binding has been reduced or eliminated.

SUMMARY OF THE INVENTION The present invention raises an antibody that is bactericidal against Gram-positive bacteria using complement alone (ie, its activity is opsonophagocytic).
totic) not protein-dependent) protein fragments or peptides.

[0017] The present invention also relates to a protein fragment or peptide related to the group B streptococcus (GBS) β antigen, which comprises the immunogenic IgG binding domain of the protein. Preferably, the IgA bound by the protein fragment or peptide is reduced or eliminated.

In particular, the invention relates to protein fragments or peptides comprising a proline-rich region, wherein at least every third residue is proline. In a preferred embodiment, the protein fragment or peptide has the formula:-[P-
_{Y 1 -Y 2 -P-Y 1} -Y 2] r - or - [Y ₁ -Y include _{2 -P-Y 1 -Y 2 -P} ] successive having _r (repeat) the amino acid sequence, wherein Y ₁ represents either an acidic amino acid residue or a basic amino acid residue, Y ₂ represents a neutral amino acid, and r is an integer that can range from 1-5. More preferably, Y ₁ is D or K and Y ₂ is V or L. More preferably, r is 4. Most preferably, Y ₂ is a hydrophobic amino acid.

The present invention also relates to a protein fragment or peptide of the invention having the formula XYZ, wherein X is the amino acid 827 and 1027 of FIG. 1 (SEQ ID NO: 2) (wild-type Cβ protein). Y represents a hydrogen bonded to X or the N-terminal sequence of FIG. 1 (SEQ ID NO: 2) or an N-terminal fragment or variant thereof bonded to X; Z represents a hydrogen bonded to X or a C-terminal sequence of FIG. 1 (SEQ ID NO: 2) bonded to X or a C-terminal fragment or a mutant thereof, provided that when it is present in Y, FIG. At least one of amino acids 1-164 of SEQ ID NO: 2 is non-wild type, with the proviso that this protein further comprises the N-terminal fragment of the amino acid sequence of FIG. 1 (SEQ ID NO: 2). Or at least one of the C-terminal fragment.

The present invention also relates to a peptide or protein fragment of the invention as described above, wherein Y is AX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ -B, wherein A is amino acids 1-16 of the sequence shown in FIG. 1 (SEQ ID NO: 2)
4 or its N-terminal fragment, B represents a sequence starting at amino acid 177 and ending at the amino acid linked to X, and X ₁ -X ₁₂ are each A
independently selected from the group consisting of la, Val, Leu, Ile, Pro, Met, Phe, Trp, binding, and the wild-type amino acid found at the corresponding position in the sequence shown in FIG. 1 (SEQ ID NO: 2); Where the amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding this protein,
However, at least one of X ₁ to X ₁₂ (including both ends) is different from the wild-type amino acid.

The present invention also relates to polynucleotide molecules encoding the peptides or protein fragments of the present invention, as well as vectors containing such polynucleotide molecules, and host cells transformed with the vectors.

The present invention also relates to conjugates comprising a peptide or protein fragment of the invention covalently linked to a capsular polysaccharide. Alternatively, the peptide or protein fragment can be conjugated or complexed with a porin protein, proteosome, or other carrier protein.

The invention also relates to a vaccine comprising a peptide or protein fragment of the invention and a pharmaceutically acceptable carrier.

The present invention also relates to a method of inducing an immune response in an animal, the method comprising the step of administering to the animal an effective amount of the vaccine of the present invention.

The present invention also monitors a process for obtaining a substantially pure Cβ protein or a fragment and / or variant thereof, the method comprising the following steps: (a) obtaining the Cβ protein in a cell extract (B) subjecting the Cβ protein to ion exchange chromatography and collecting the Cβ protein-containing fraction; (c) pooling and diluting the Cβ protein-containing fraction; and (d) Subjecting the diluted Cβ protein-containing fraction to ligand affinity chromatography or gel filtration chromatography and collecting the fraction; whereby substantially pure Cβ protein or fragments and / or variants thereof The body is obtained.

The present invention further relates to a process for obtaining a Cβ protein or fragment and / or variant from a bacterial cell, wherein the bacterial cell is transfected with a nucleotide sequence encoding the Cβ protein or fragment and / or variant thereof. Wherein the cell overexpresses the Cβ protein or a fragment and / or variant thereof.

The present invention also relates to a process for obtaining Cβ protein from bacterial cells that naturally produce Cβ protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The antigenic protein fragments and peptides of the present invention have been found to elicit antibodies that can kill streptococci using complement alone. This bactericidal activity is different from opsonophagocytosis, in which the bacteria are killed in leukocytes, and has not been observed at all in Gram-positive bacteria using antibodies specific to it. In fact, antibodies elicited by streptococcal capsular polysaccharide expressing the Cβ protein do not kill streptococci in the absence of leukocytes.

Examination of the antigenic characteristics of the Cβ protein has led to the discovery of antigenic regions with shared characteristics among Gram-positive bacteria. Antibodies raised against protein fragments and peptides based on the amino acid sequence of this region of the Cβ protein have bactericidal activity, which is independent of the opsonophagocytic mechanism.
Accordingly, the present invention relates to protein fragments or peptides that elicit antibodies that are bactericidal against Gram-positive bacteria by themselves (ie, an activity that is independent of the opsonophagocytic mechanism).

The antigenic protein fragments and peptides of the present invention are derived from the Gram-positive amino acid sequence at the C-terminus of the native protein and are believed to be embedded in the bacterial cell wall. In native bacterial proteins, the antigenic regions corresponding to the protein fragments and peptides of the invention are exposed to antibody binding during a cell division event. In the area where one bacterium begins to form two bacteria, the cell wall splits into small pieces and reforms. This area is
Also known as the septum. It is correct that in this region during the division, the antigenic region is available for antibody binding and activation of complement. Confocal microscopy results (Example 13) show that antibodies generated against the protein fragment specifically bind to septum areas, and that more antibody is not non-dividing cells, It clearly shows binding to actively dividing cells. FACS
The results of the can analysis confirmed these results and added further evidence for this observation. In addition, Coleman et al. Had previously made similar observations.

Coleman et al. Studied the immunoglobulin binding site and Cβ antigen in group B streptococci using colloidal gold immunolabeling. Coleman et al., Infe
ct. Immun. 58: 332-340 (1990). Colloidal gold, Ig
A was conjugated to characterize IgA binding properties, and conjugated to IgG to characterize IgG binding. Coleman et al.'S electron micrograph shows that non-immune human IgA binding is distributed over the bacterial surface, but anti-Cβ-specific IgG binding is localized to the area defining the septum of dividing bacterial cells. It had been turned into.

It was therefore an object of the present invention to determine whether a particular site in a Cβ protein is responsible for raising an antibody capable of performing this unique bactericidal activity. Identification of such sites can ensure that the vaccine for group B streptococci includes the Cβ protein.

[0033] In particular, the present invention relates to a protein fragment or plasmid comprising a proline-rich region.
About the peptide. Here, proline is present at least every three residues and
Antibodies raised against the protein fragment or peptide of
Is bactericidal against Gram-positive bacteria. In a preferred embodiment,
The present invention relates to a protein comprising a continuous (repeated) amino acid sequence having the formula:
For protein fragments or peptides:-[PY₁-Y_Two-P-Y₁-Y_Two] _r -Or-[Y₁-Y_Two-P-Y₁-Y_Two-P]_r− (Where Y₁Is an acidic amino
Represents either an acid residue or a basic amino acid residue;_TwoReplaces neutral amino acids
And r is an integer from 1 to 5). More preferably, Y₁Is D or
K and Y_TwoIs V or L. More preferably, r is 4
. Most preferably, Y_TwoIs a hydrophobic amino acid.

In another embodiment, the protein fragment or peptide comprises a continuous (repeated) amino acid sequence having the formula:-[P-DY ₃ -P
-K-L] _r - or - [K-L-P- D-Y 3 -P] r - ( wherein, Y ₃ represents V or A). More preferably, Y ₃ is V. More preferably also r
Is at least 3.

In another embodiment, the protein fragment or peptide has a sequence having the formula: [[S-PKY ₄ -P-E-A-P-Y ₅ -V-P-E] _r- ] Target (repeated) amino acid sequence. Here, Y ₄ represents T or A, and Y ₅ represents H or R. More preferably, Y ₄ is T and Y ₅ is H. Also, more preferably, r is at least 3.

The present invention also relates to the peptides or protein fragments of the present invention, which comprise an immunogenic IgG binding domain capable of eliciting the antibodies described above. Preferably, IgA binding by the protein fragment or peptide is reduced or eliminated. In a preferred embodiment, this domain has at least 8
The peptide sequence of the amino acid sequence is included between about positions 828 and 1027 of the amino acid sequence (SEQ ID NO: 2) shown in FIG.

In another preferred embodiment, the invention relates to a peptide or protein fragment according to the invention, comprising an amino acid sequence having the formula YZX. Where X represents at least eight consecutive amino acid residues between amino acids 828 and 1027 (inclusive) of FIG. 1 (SEQ ID NO: 2), and Y is hydrogen or hydrogen bonded to X. 1 represents the N-terminal amino acid sequence of FIG. 1 (SEQ ID NO: 2), or an N-terminal fragment and / or variant thereof, and Z is hydrogen or X-attached C-terminal amino acid sequence of FIG. Stands for C-terminal fragment and / or variant, provided that amino acids 1 to 16 of FIG. 1 (SEQ ID NO: 2)
When at least one of the four is present in Y, it is non-wild-type, and furthermore, the protein is at least one of the N-terminal or C-terminal fragments of the amino acid sequence of FIG. 1 (SEQ ID NO: 2). More preferably, Y does not include at least amino acids 1-176 of FIG. 1 (SEQ ID NO: 2). Also, more preferably, Z comprises at least amino acid 901 of FIG. 1 (SEQ ID NO: 2). Also, more preferably, the peptide or protein fragment of the present invention has SEQ ID NO: 3
Not one amino acid sequence. Also, more preferably, the peptide or protein fragment of the present invention comprises a polypeptide of about 55 kD, which is secreted from group B streptococcus strain 2AR, DL471B or HG806, respectively.
Neither the 3 kD polypeptide nor the approximately 38 kD polypeptide.

[0038] Preferably, the epitope sequence X is selected from the group consisting of: PPK
TPDVP (SEQ ID NO: 32), PDVPKLPD (SEQ ID NO: 33), KLPDV
PKL (SEQ ID NO: 34), VPKLPDVP (SEQ ID NO: 35), KLPDAPK
L (SEQ ID NO: 36), APKLPDGL (SEQ ID NO: 37), ETPDTPKI (
SEQ ID NO: 38), RTVRLALG (SEQ ID NO: 39), and GGGTVVRVF
(SEQ ID NO: 40). In a preferred embodiment, X is 8, 9, 10, 11, between amino acids 828 and 1027 (inclusive) of Figure 1 (SEQ ID NO: 2).
Represents any one of 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive amino acid residues.

In another embodiment, the present invention relates to any 8 of the amino acid sequence between amino acids 828 and 1027 (inclusive) of the sequence shown in FIG. 1 (SEQ ID NO: 2).
A peptide or protein fragment comprising an amino acid sequence consisting of three consecutive residues. More preferably, the eight consecutive residues are selected from the group consisting of: PPKTPDVP (SEQ ID NO: 32), PDVPKLPD (SEQ ID NO: 33), KLPDVPKL (SEQ ID NO: 34), VPKLPDVP (SEQ ID NO: 35), KLPDAPKL (SEQ ID NO: 36), APKKLPDGL (SEQ ID NO: 37)
), ETPDTPKI (SEQ ID NO: 38), RTVRLALG (SEQ ID NO: 39),
And GGGTVRVF (SEQ ID NO: 40).

In another embodiment, the invention relates to a protein fragment or peptide having the formula YZX. Where X represents any 20 consecutive amino acid residues between amino acid 828 and amino acid 1027 (inclusive) of FIG. 1 (SEQ ID NO: 2), and Y is hydrogen bonded to X Or represents the N-terminal amino acid sequence of FIG. 1 (SEQ ID NO: 2), or an N-terminal fragment and / or variant thereof, and Z is a C-terminal amino acid of FIG. 1 (SEQ ID NO: 2) bonded to hydrogen or X A sequence or a C-terminal fragment and / or variant thereof, provided that at least one of amino acids 1-164 of FIG. 1 (SEQ ID NO: 2) is present in Y, is non-wild-type, and furthermore, the protein is , At least one of the N-terminal fragment and the C-terminal fragment of the amino acid sequence of FIG. 1 (SEQ ID NO: 2). More preferably, X is selected from the group consisting of: SPKT
PEAPKIPEPPKTPDVP (SEQ ID NO: 41), PEAPKIPEPPPK
TPDVPKLPD (SEQ ID NO: 42), KIPEPPPKTPDVPKLPDVP
KL (SEQ ID NO: 43), PPKTPDVPKLPDVPKLPDVP (SEQ ID NO: 44), PDVPKLPDVPKLPDVPKLPD (SEQ ID NO: 45) and K
LPDVPKLPDVPKLPDAPKL (SEQ ID NO: 46). Also, more preferably, Y does not include at least amino acids 1-176 of FIG. 1 (SEQ ID NO: 2).

In another embodiment, the present invention relates to any one of the amino acid sequences between amino acids 828 and 1027 (inclusive) of the sequence shown in FIG. 1 (SEQ ID NO: 2).
A peptide or protein fragment containing 0 consecutive residues. More preferably, the 20 residues are selected from the group consisting of: SPKTP
EAPKIPEPPPKTPDVP (SEQ ID NO: 41), PEAPKIPEPPPKT
PDVPKLPD (SEQ ID NO: 42), KIPEPPPKTPDVPKLPDVPK
L (SEQ ID NO: 43), PPKTPDVPKLPDVPKLPDVP (SEQ ID NO: 4
4), PDVPKLPDVPKLPDVPKLPD (SEQ ID NO: 45) and KL
PDVPKLPDVPKLPDAPKL (SEQ ID NO: 46).

In another embodiment, the present invention relates to a peptide or protein fragment having the formula YXZ. Where X represents at least 21 contiguous amino acid residues between amino acids 828 and 1027 (inclusive) of FIG. 1 (SEQ ID NO: 2), and Y is hydrogen bonded to X Or Figure 1 (SEQ ID NO: 2)
And Z represents a C-terminal amino acid sequence of FIG. 1 (SEQ ID NO: 2) or a C-terminal fragment and / or a mutation thereof bonded to hydrogen or X. 1 is non-wild-type if at least one of amino acids 1-164 of FIG. 1 (SEQ ID NO: 2) is present in Y, and furthermore, the protein has
At least one of the N-terminal fragment or the C-terminal fragment of SEQ ID NO: 2)
One. More preferably, X is amino acid 8 of the sequence shown in FIG. 1 (SEQ ID NO: 2).
It consists of an amino acid sequence between 28 and amino acids 1027 (inclusive). Also, more preferably, Y does not include at least amino acids 1-176 of FIG. 1 (SEQ ID NO: 2).

[0043] In another embodiment, the present invention relates to at least 21 contiguous residues of the amino acid sequence between amino acid 828 and amino acid 1027 (inclusive) of the sequence shown in FIG. 1 (SEQ ID NO: 2). A peptide or protein fragment comprising an amino acid sequence consisting of: More preferably, the peptide or protein fragment of the invention has the amino acid sequence PDVPKLPDVPKLPDVPKLPD
Includes an amino acid sequence consisting of APKL (SEQ ID NO: 47). Most preferably, the peptide or protein fragment of the invention has the sequence shown in FIG.
)) And an amino acid sequence consisting of the amino acid sequence between amino acids 828 and 1027 (inclusive).

Jerlsoroem, P .; G. FIG. Et al., Molec. Microbio. 5:
Although the numbering system of 843-849 (1991) has been utilized in FIG. 1 and elsewhere in reference to the sequences of the invention, the peptides, protein fragments and nucleic acids of the invention have also been described in Heden, L. -O. Et al., Eur. J.
Immunol. 21: 1481-1490 (1991), it should be noted that it relates to the wild-type Cβ protein sequence.

Although the sequences disclosed by Jerlstorem et al. And Heden et al. Show only minor variants that are related to one another, differences in the two amino acids are specifically noted as examples of variants encompassed by the present invention. The Jellstörm et al. Sequence shown in FIG. 1 shows a Lys at position 878, whereas the corresponding amino acid in the Heden et al. Sequence is Glu. Also, Jellstroem et al. Show three perfect repeats of PVDPKL (SEQ ID NO: 51), while Heden et al. Show only two perfect repeats of the sequence of its six residues ("3 'Th' iteration is Her
with Ala in place of Val corresponding to position 897 of the sequence of lström et al.). Such a variant, when reading a repetitive sequence as described herein, has a D
It may be due to NA polymerase "wobble" or slip strand synthesis. Thus, the proteins and nucleic acids of the invention include such minor variants as exist between the sequences disclosed by Jerlstroem et al. And Heden et al.

The mutation in the region of the Cβ protein located between about amino acid residue 163 and about amino acid residue 176 of the wild-type Cβ sequence shown in FIG.
This results in protein fragments or peptides that have reduced or eliminated their binding properties but retain their three-dimensional structure sufficiently to retain most of their antigenicity (Examples 4 and 5). Substitutions or deletions of amino acids in this region
Reduce or eliminate gA binding, but maintain the antigenicity of the protein. Thus, the amino acid sequence of the Cβ polypeptide can be altered to complete a protein that does not bind IgA. Suitable amino acid substitutions that remove IgA binding include substituting one or more residues with amino acids having different properties. For example, a strong hydrophilic amino acid can be replaced by a strong hydrophobic amino acid. Amino acids that can be grouped together include the aliphatic amino acids Ala, Val, Le.
u and Ile, hydroxyl residues Ser and Thr, acidic residues Asp and Glu, amide residues Asn and Gln, basic residues Lys and Arg, and aromatic residues Phr and Tyr. Thus, one of skill in the art would be aware of the need to reduce or eliminate IgA binding to approximately residue 16 in the Cβ protein.
Understand how to determine the appropriate amino acid substitution or deletion in the region between 3 and residue 176.

In addition, guidance on which amino acid changes appear to have significantly detrimental effects on function can be found in Bowie, J. et al. U. "Deciphering
the Message in Protein Sequences: Tol
erance to Amino Acid Substitutions ",
Science 247: 1306-1310 (1990).

Thus, in another aspect, the present invention also relates to the above-described and lacking IgA binding.
For peptides or protein fragments, in particular, Y is AX₁X_TwoX_ThreeX_Four X_FiveX₆X₇X₈X₉X_TenX₁₁X₁₂Represents an amino acid sequence comprising -B, wherein A is
Includes amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2) or its N-terminal
B, including the end fragment, begins at amino acid 177 and is linked to X.
Represents a sequence ending with a amino acid, and X₁~ X₁₂Are independently of each other Ala, V
al, Leu, Ile, Pro, Met, Phe, Trp, binding and shown in FIG.
From the wild-type amino acid found at the corresponding position in the
Group. Here, the position of the amino acid is
Numbered from the first amino acid in the amino acid sequence of Tib. Where X₁~ X_{1 Two} At least one (including both ends) is other than a wild-type amino acid.

The present invention also provides a peptide or protein fragment as described above and lacking IgA binding.
Regarding the fragmentation, in particular, Y is AX₁X_TwoX_ThreeX_FourX_FiveX₆X₇X₈X₉X_TenX₁₁X_{1 Two} Represents an amino acid sequence containing -B. In a particularly preferred embodiment, the amino acid X₇ And X₁₂Is Ala (SEQ ID NO: 3). In another preferred embodiment,
Amino acid X_FourAnd X₁₁Is Pro (SEQ ID NO: 4). Another preferred embodiment
The amino acid X₇Is Thr and amino acid X₁₂Is Leu
(SEQ ID NO: 5). In a more preferred embodiment, the amino acid X_Five, X₇, X₈,
X_Ten, X₁₁And X₁₂Are each replaced by a bond (SEQ ID NO: 6).

In a preferred embodiment, the protein fragments and peptides of the present invention are substantially pure.

[0051] IgA binding activity of Cβ may require dimerization of Cβ. Therefore, I of Cβ
Even if the gG binding region is not mutated as described above, mutation of a region of Cβ, which may be necessary for dimerization, may result in a form of Cβ that cannot bind IgA. Deletion of the Cβ portion from residue 729 to the C-terminus of the sequence shown in FIG. 1 (SEQ ID NO: 2) eliminates Cβ dimerization. Experimental results supporting this finding can be found in Table 1. Several fragments of Cβ were inserted into each of two different vectors. If the array shown in the table is before or after the outer parenthesis, this means:
Indicates that the Cβ sequence does not extend further at the end of the fragment, ie, the nucleotide sequence inserted into the vector is only the indicated amino acids of the Cβ sequence and does not encode any other. If the sequence shown in the table is an abbreviation (...
. ) Before or after, indicates that the remainder of the Cβ sequence at that end of the fragment is also included in the vector. The nucleotide sequence encoding the peptide shown in the upper half of the table was inserted into either vector pTOPE or vector pET17b. Both of these vectors allow expression of the inserted fragment from the T7 promoter, and both
A fusion protein comprising the amino acid sequence encoded by the insert is generated from the N-terminal fragment of the 10 capsid protein. However, while pET17b encodes only 8 amino acids of the φ10 protein, pTOPE encodes a 288 amino acid fragment of the φ10 protein.

[0052]

[Table 1] As shown in Table 1, certain fragments of Cβ generated from pET17b show reduced IgA binding, while the same fragments generated by pTOPE can bind IgA. The fragments tested lack the region of Cβ predicted to be involved in dimerization but do not contain any mutations in the putative IgA binding domain (the Cβ fragment inserted into the vector pET24b at the end of Table 1 Note that it contains a putative dimerization region, but nevertheless exhibits reduced IgA binding due to mutations in the IgA binding domain as described above).
These Cβ fragments are presumed to bind to IgA when produced from pTOE. This is because a 288 amino acid fragment of the φ10 protein allows for dimerization of the Cβ fragment. This may be due to the fact that the φ10 capsid protein usually forms oligomers; thus, the region responsible for oligomerization allows dimerization of the inserted Cβ fragment,
Thus allowing IgA binding. Accordingly, the present invention also relates to peptides or protein fragments having a mutation in the dimerization domain of Cβ, wherein the mutated Cβ protein is unable to bind IgA. Of course, for the purpose of producing non-IgA binding peptides or fragments that maintain as much of the antigenicity of the wild-type Cβ protein as possible, Cβ dimerization should not be prevented.

In a preferred embodiment, at least one amino acid residue 521 to 541 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2) is (a) deleted or (b)
Modified, so that the protein has Cβ of E, co
When produced in li, it is not cleaved in this region. In a more preferred embodiment,
At least one of amino acid residues 533 to 541 of the sequence shown in FIG. 1 (SEQ ID NO: 2) is either (a) deleted or (b) modified.
In a much more preferred embodiment, at least one of amino acid residues 537 and 538 is either (a) deleted or (b) modified. Of course, those skilled in the art will recognize other suitable amino acid substitutions by routine experimentation and by
on Heijne (Nucleic Acids Res. 14: 4683-
4690 (1986).

Since the Cβ protein is membrane-bound in the wild-type state, purification of the Cβ protein, mutant, and / or fragment described above removes hydrophobic residues of the transmembrane domain of the Cβ protein. (This transmembrane domain is shown by residues 1095 to 11 of the sequence shown in FIG. 1 (SEQ ID NO: 2)).
27). This may be by substituting non-hydrophobic residues for hydrophobic residues (residues 1108-1116 of the sequence shown in FIG. 1 (SEQ ID NO: 2)) or by deleting those hydrophobic residues. Can be achieved by: Purification of membrane-bound Cβ requires the use of detergents, whereas mutant Cβs lacking the hydrophobic transmembrane region can be purified without the use of detergents. Thus, the invention also relates to peptides or peptide fragments in which the nine hydrophobic residues constituting the transmembrane domain have been deleted or replaced by non-hydrophobic amino acids.

E. Production of Cβ protein from E. coli can be problematic. This is because this protein is cleaved at a specific region. This is probably due to E.
coli signal peptidase. This cleavage results in a truncated protein. This may not be ideal for a vaccine. Because
This is because it may lack the antigenic epitope of the wild-type Cβ protein. The cleavage site was determined by sequence analysis and by matrix-assisted laser desorption onset flight time (
laser desorption initiated time of f
light) (MALDI-TOF) mass spectrometry (von Heijne, Nu
cleic Acids Res. 14: 4683-4690 (1986)). The cleavage site is between amino acid residues 538 and 539 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2) (after alanine and before glutamine). This signal peptidase recognition site is located within a 20 amino acid stretch located between residues 521 and 541 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). Thus, by deleting this region, the protein becomes E. coli. E. coli can be successfully produced. In addition, because signal peptidases have very stringent sequence specificity, changes in the signal peptidase recognition sequence (including even single conservative amino acid substitutions in this region) are likely to occur in E. coli. co
The cleavage of the protein by li can be eliminated. The recognition sequence required for cleavage by this signal peptidase is GluLeuIleLysSerAlaG
It is believed to be lnGlnGlu (SEQ ID NO: 11), which corresponds to amino acid residues 533-541 of the sequence shown in FIG. 1 (SEQ ID NO: 2). Changes in this sequence, either deletions or non-conservative substitutions, at either serine or alanine residues are expected to eliminate signal peptidase cleavage. Of course, ideally, mutagenesis of the Cβ sequence, for the purpose of eliciting an immunogenic response,
Minimized to preserve the tertiary structure of the wild-type antigen.

The present invention also relates to polynucleotide molecules encoding the peptides and protein fragments of the present invention, vectors containing these nucleotide molecules, and host cells transformed with the vectors.

The present invention also relates to the expression of the protein fragments and peptides of the present invention in a cellular host. Prokaryotic hosts that can be used to clone and express the proteins or peptides of the invention are well known in the art. Vectors that replicate in such host cells are also well known.

Preferred prokaryotic hosts include Escherichia, Bacillus
, Streptomyces, Psudomonas, Salmonella,
Bacteria of the genus such as Serratia, Xanthomonas, but are not limited thereto. Two such prokaryotic hosts are E. coli. coli DH1
OB and DH5αF′IQ (available from LTI, Gaithersburg, MD). The most preferred host for cloning and expressing the protein fragments and polypeptides of the present invention is E. coli. coli BL21 (N
ovagen, Inc. , Madison, WI). This E. E. coli is lysogenic for DE3 phage.

The present invention also relates to vectors comprising isolated DNA molecules encoding the peptides and protein fragments of the present invention, host cells genetically engineered with recombinant vectors, and proteins or polypeptides of the present invention produced by recombinant techniques. For the production of peptides.

[0060] Host cells can be engineered to take up nucleic acid molecules and express protein fragments or peptides of the invention. For example, the recombinant construct
It can be introduced into host cells using well-known techniques of infection, transduction, transfection, and transformation. Polynucleotides can be introduced alone or with other polynucleotides. Such other polynucleotides can be introduced independently, co-introduced, or introduced in conjunction with a polynucleotide of the invention.

Thus, for example, a polynucleotide can be ligated to a vector containing a selectable marker for propagation in a host. Vector constructs can be introduced into host cells by the techniques described above. Generally, a plasmid vector is introduced as DNA in a precipitate (eg, a calcium phosphate precipitate) or in a complex with a charged lipid. Electroporation can also be used to introduce a polynucleotide into a host. If the vector is a virus, it can be packaged in vitro or introduced into a packaging cell, and the packaged virus can transduce the cell. A wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells according to this aspect of the invention are well known and routine to those of skill in the art. Such a technique is disclosed in Molecular Cloning: A Laboratory.
Manual, 2nd edition, edited by Sambrook et al., Cold Spring Ha.
rbor Laboratory (1989). This is an example of a number of laboratory manuals detailing these techniques.

According to this aspect of the invention, the vector may be, for example, a plasmid vector,
Single- or double-stranded phage vector, or single- or double-stranded RNA
Alternatively, it may be a DNA virus vector. Such vectors can be introduced into cells as polynucleotides, preferably DNA, by well-known techniques for introducing DNA and RNA into cells. Vectors may also, and in the case of phage and viral vectors, be introduced into cells as packaged or encapsulated viruses by well-known infection and transduction techniques, and preferably (indeed). Viral vectors can be replication competent or replication deficient. In the latter case, virus growth is generally
Occurs only in complementing host cells.

Preferred among the vectors in certain respects are vectors for the expression of the protein fragments or peptides of the invention. Generally, such vectors include a cis-acting control region operably linked to the polynucleotide to be expressed and effective for expression in a host. Suitable trans-acting factors are either supplied by the host, or by a complementing vector, or upon introduction into the host, by the vector itself.

In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression, or may be expression in only certain cell types, or may be both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate (eg, temperature and nutrient additives). Various vectors suitable for this aspect of the invention include constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, which are well known and commonly used by those of skill in the art. Engineered host cells can be cultured in conventional nutrient media (US Pat. No. 5,464,758). This nutrient medium is
In particular, it may be modified as appropriate for activating a promoter, selecting a transformant, or amplifying a gene. Culture conditions (eg, temperature, pH, etc.) previously used with the host cell selected for expression will generally be appropriate for expression of a protein fragment or peptide of the invention, as is well known to those skilled in the art. It is.

[0065] Many different expression vectors can be used to express the protein fragments or peptides of the present invention. Such vectors include: chromosome derived vectors, episomal derived vectors, viral derived vectors (eg, bacterial plasmid derived, bacteriophage derived, yeast episomal derived, yeast chromosomal element derived, viral (eg, baculovirus) , Papovavirus (eg, SV40), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retrovirus), and vectors derived from combinations thereof (eg, vectors derived from plasmids and bacteriophage genetic elements (eg, For example, cosmids and phagemids)).
These can all be used for expression according to this aspect of the invention. Generally, any vector suitable to maintain or propagate a polynucleotide in a host, or to express a polypeptide or protein, can be used for expression in this regard.

A suitable DNA molecule can be inserted into a vector by any of a variety of well-known and conventional techniques. Generally, the DNA molecule for expression is ligated to the expression vector by cutting the DNA sequence and the expression vector with one or more restriction endonucleases, and then ligating the restriction fragments together using T4 DNA ligase. Restrictions and procedures for ligation that can be used for this are well known and routine to those skilled in the art. Procedures appropriate in this regard, and for constructing expression vectors using alternative techniques, are also well known and
It is routine to those skilled in the art and is described in great detail in Sambrook et al., Supra.

[0067] The DNA molecule inserted into the expression vector may contain appropriate expression control sequences (eg, m
(Including a promoter that directs RNA transcription). As representatives of such promoters, simply listing a few well-known promoters,
phage lambda PL promoter, E. E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the retroviral LTR promoter. Numerous promoters not mentioned are well known to be suitable for use in this aspect of the invention and are readily available to those of skill in the art in the manner set forth by the discussion and examples herein. It is understood that this can be done.

In general, expression constructs will contain transcription initiation and termination sites, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct contains a translation initiation AUG first, and a stop codon appropriately located at the end of the polypeptide to be translated.

In addition, the construct may include regulatory regions that regulate as well as engender expression. In general, according to many commonly used procedures, such regions operate by controlling transcription (eg, inter alia, repressor binding sites and enhancers).

[0070] Vectors for propagation and expression generally include a selectable marker. Such markers may also be suitable for amplification, or the vector may include additional markers for this purpose. In this regard, the expression vector is preferably to provide a phenotypic trait for selection of transformed host cells,
It contains one or more selectable marker genes. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and E. coli.
For culture of E. coli and other bacteria, include the tetracycline or ampicillin resistance gene.

A vector containing the appropriate DNA sequence as described elsewhere herein, as well as the appropriate promoter and other appropriate control sequences, may comprise a vector within the desired polypeptide or protein. It can be introduced into a suitable host using a variety of well-known techniques suitable for expression. Representative examples of suitable hosts include bacterial cells (eg,
E. FIG. coli, Streptomyces, and Salmonella ty
phimurium cells). Hosts for many different expression constructs are well known, and one of skill in the art, with the present disclosure, can readily select a host for expressing a protein or fragment according to this aspect of the invention.

More particularly, the present invention also includes recombinant constructs (eg, expression constructs) comprising one or more of the above sequences. This construct includes a vector (eg, a plasmid or viral vector) into which such a sequence of the invention has been inserted. Sequences can be inserted in a forward or reverse orientation. In certain preferred embodiments in this regard, the construct further comprises a regulatory sequence operably linked to the sequence (eg, including a promoter). Large numbers of suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention.

The present invention also relates to the enrichment of polypeptides or proteins with reduced or eliminated ability to bind to human IgA, and therefore, the present invention relates to the use of the in vitro mutagenesis method to produce the protein of the present invention. Generating fragments or peptides. Numerous in vitro mutagenesis methods are well known to those of skill in the art; some are provided herein by way of example.

One such method is to insert the plasmid into the plasmid by digesting the plasmid either partially or completely with appropriate restriction enzymes and then ligating the ends to produce the plasmid again. Deletions or insertions are introduced into the polynucleotide molecule. Very short deletions can be made by first cutting the plasmid at restriction sites and then subjecting the linear DNA to controlled nuclease digestion to remove a small number of bases at each end. Precise insertions can also be made by ligating a double-stranded oligonucleotide linker to a plasmid that has been cut at one restriction site.

[0075] Chemical methods can also be used to introduce mutations into single-stranded polynucleotide molecules. For example, one base pair change at a cytosine residue can be made using a chemical such as bisulfite. This chemical deaminates cytosine to uracil, thereby converting GC base pairs to AT base pairs. Preferably, D
Oligonucleotide-directed mutagenesis is used so that all possible classes of base pair changes can be made at any determined site along the NA molecule. The technique generally involves annealing oligonucleotides complementary (excluding one or more mismatches) to the single-stranded nucleotide sequence of interest. The mismatched oligonucleotide is then extended by a DNA polymerase to produce a double-stranded DNA molecule that contains the desired change in the sequence of one strand. Sequence changes can, of course, result in amino acid deletions, substitutions, or insertions if the change is made in the coding region of the gene. The double-stranded polynucleotide can then be inserted into a suitable expression vector, and thus, a mutant polypeptide can be generated. The above oligonucleotide-directed mutagenesis can of course be performed via PCR. An example of such a system is the Ex-Site® PCR site-directed mutagenesis technique used in Example 4 (Stratagene, CA).

Using the Ex-Site® PCR site-directed mutagenesis technique, several different oligonucleotides induced different changes in the DNA sequence in the region of interest. In one particular example, overlapping primers were obtained. Both primers contained the sequences necessary to change lysine to alanine at amino acids 170 and 175 in the sequence shown in FIG. 1 (SEQ ID NO: 2) (Table 1). Forward primer (
Cβ613) has the following sequence (SEQ ID NO: 6):

[0077]

Embedded image And the reverse primer (designated Cβ642R) had the following sequence:

[0078]

Embedded image (This substitution is shown in bold). These oligonucleotides were converted to pNV22
Two templates were combined, which consisted of the Cβ gene inserted into the pSP76 vector. PCR was performed and the product was ligated and coli DH5
The strain was introduced into the α strain, thereby generating a clone containing the mutant Cβ gene.

The following vectors are commercially available and can be used in the practice of the present invention. Among the preferred vectors for use in bacteria are pQE70, pQ
E60, and pQE-9 (available from Qiagen); pBS vector, P
harscript vector, Bluescript (registered trademark) vector, pNH8A, pNH16a, pNH18A, pNH46A (Stratum
ne); ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 (available from Pharmacia); pUC18,
pUC19, and pPROEX-1 (available from LTI); and pTO
PE, pET17b, and pET24a (Novagen, Inc., Mad
ison, WI). These vectors are merely listed as illustrative of the many commercially available and well known vectors available to those of skill in the art for use in accordance with this aspect of the present invention. It is understood that any other plasmid or vector suitable for, for example, introducing, maintaining, propagating, or expressing a polynucleotide, protein fragment, or peptide of the invention in a host may be used in this aspect of the invention. You.

The promoter region may be a reporter transcription unit lacking a promoter region downstream of a restriction site (eg, chloramphenicol acetyltransferase (“CAT”) to introduce a candidate promoter fragment (ie, a fragment that may contain a promoter). ) Transcription unit) can be used to select from any desired gene. As is well known, introduction of a promoter-containing fragment into a vector at a restriction site upstream of the CAT gene results in the generation of CAT activity, which can be detected by standard CAT assays. Vectors suitable for this are well known and are readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of the polynucleotides of the present invention include not only well-known and readily available promoters, but also promoters that can be readily obtained by the techniques described above using reporter genes.

Among the known bacterial promoters suitable for the expression of the polynucleotides and polypeptides according to the invention are E. coli. E. coli lacI and lacZ promoters,
There are T3 and T7 promoters, gpt promoter, λPR, PL promoter, and trp promoter.

Selection of suitable vectors and promoters for expression in a host cell
The procedures are well known and the necessary techniques for construction of expression vectors, introduction of the vectors into hosts, and expression in hosts are routine in the art.

The present invention also relates to host cells containing the above-described construct. A host cell can be a prokaryotic cell (eg, a bacterial cell).

The construct in a host cell can be used in a conventional manner to produce the gene product encoded by the recombinant DNA sequence. Alternatively, the protein fragments or peptides of the present invention may be produced synthetically by a conventional peptide synthesizer.

After transformation of an appropriate host strain and propagation of this host strain to an appropriate cell density, if the selected promoter is inducible, it may be subjected to appropriate means (eg, temperature changes, or chemical changes). Exposure to an inducer) and the cells are cultured for an additional period.

[0086] Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

The microbial cells used in protein expression can be disrupted by any conventional method, including repeated freeze-thaw, sonication, mechanical disruption, or use of cell lysing agents; Such methods are well-known to those skilled in the art.

Preferably, the peptides may be prepared using well-known methods of solid phase peptide synthesis. The term "peptide" is intended to have no more than 100 amino acids.
Proteins consist of more than 100 amino acids. Preferably, the peptide is
Contains 8 to 50 amino acids.

In another aspect, the present invention relates to a method for isolating and purifying the proteins and polypeptides of the present invention. It has been discovered that the methods of purifying proteins and polypeptides of the present invention yield large amounts of substantially pure proteins. By "substantially pure" is intended that the protein or polypeptide obtained by the process of the present invention is at least 85% pure, with negligible contamination of nucleic acids and polysaccharides. One advantage of the method of the present invention is that it is scalable. That is, the method can be readily adapted for purification of large amounts of protein. The columns used in the purification receive large volumes / concentrations of the sample, use a dilution step rather than a concentration step to apply the sample from one column to the next, and both columns use the same buffer as the mobile phase Use liquid. Furthermore, this purification method is based on recombinant E. coli. c
Cβ protein variants obtained from both oli and native streptococci and / or
Alternatively, it is applicable to the purification of fragments.

Further, the use of protease inhibitors and detergents in purification
Allows purification using ion exchange and heparin columns to minimize product degradation. Finally, the product yield is higher than that obtained by the gel filtration column chromatography procedure used previously (Russell-Jo
nes et al. Exp. Med. 160: 1467-1475 (1984): M
adoff et al., Infect. Immun. 59 (1): 204-210 (19
91)). This is because a larger amount of sample can be processed simultaneously. This large-scale process also minimizes the operating time involved in purification.

The present invention provides a method and a method for producing substantially pure Cβ protein, or a fragment and / or fragment thereof.
Alternatively, for a process for obtaining a mutant, this comprises the steps of: (a) obtaining a Cβ protein in a cell extract; (b) subjecting the Cβ protein to ion exchange chromatography;
collecting the β protein-containing fraction; (c) pooling and diluting the Cβ protein-containing fraction; and (d) ligand affinity chromatography or gel filtration chromatography of the diluted Cβ protein-containing fraction. And collecting the fractions, whereby substantially pure Cβ protein or a fragment and / or variant thereof is obtained.

In a preferred embodiment, an anion exchange medium is used. More preferably, the anionic medium is a tertiary amine, quaternary ammonium, quaternary alkylamine, quaternary alkylalkanolamine, diethylaminoethyl, diethyl- (2-hydroxypropyl) aminoethyl, trimethylaminohydroxypropyl , A functional group selected from the group consisting of trimethylbenzylammonium, dimethylethanolbenzylammonium and dimethylethanolamine. Even more preferably, this functional group is a quaternary ammonium group. Most preferably, the quaternary ammonium group is a trimethylaminomethyl group.

[0093] In a preferred embodiment, the ion exchange medium comprises the above functional groups immobilized on a solid support material (eg, selected from the group consisting of agarose, dextran, cellulose and polystyrene). More preferably, the solid support material is in bead form. Most preferably, the solid support material is cross-linked agarose.

[0094] In a preferred embodiment, a ligand-affinity medium is used.
Preferably, the ligand of the ligand-affinity medium is a heparin-like molecule. More preferably, the heparin-like molecule is a glycosaminoglycan. More preferably, the heparin-like molecule is chondroitin sulfate, dermatan sulfate,
It is selected from the group consisting of keratan sulfate and hyaluronate. Most preferably, the heparin-like molecule is heparin.

[0095] In a preferred embodiment, the ligand-affinity medium comprises a ligand immobilized on a solid support material (eg, selected from the group consisting of agarose, dextran, cellulose, and polystyrene). More preferably, the solid support material is in bead form. Most preferably, the solid support material is cross-linked agarose.

[0096] In another embodiment, the fraction containing the Cβ protein or fragment and / or variant thereof is subjected to gel filtration chromatography. More preferably, the gel filtration media is a cross-linked allyl dextran / N, N'-methylenebisacrylamide copolymer.

In a preferred embodiment, the Cβ protein or a fragment thereof and / or
Alternatively, the mutant is subjected to ion exchange chromatography or ligand affinity chromatography in a buffer containing an amphoteric surfactant. Preferably, the amphoteric surfactant is CHAPS (3-((3-cholamidopropyl) dimethylammonio) -1-propanesulfonate). Even more preferably, the amphoteric surfactant is Empigen® BB (Albright & W
ilson Americas Inc. Alkyldimethylamine Betaine available from Glen Allen, Va.). Most preferably, the amphoteric surfactant is N, N-dimethyl-3-ammonio-1-propanesulfonate (Zwitterg from Calbiochem, La Jolla, Calif.)
ent®) is selected from the group consisting of n-octyl, n-decyl, n-dodecyl, n-tetradecyl and n-hexadecyl derivatives. More preferably, the surfactant is at a concentration of about 1% to about 10%. Most preferably, the concentration of the surfactant is about 5%.

[0098] In a preferred embodiment, the Cβ protein, fragment and / or variant thereof is subjected to chromatography on a first medium in a buffer containing a protease inhibitor. Preferably, the protease inhibitor is a serine protease inhibitor. More preferably, the protease inhibitor is selected from the group consisting of DFP, PMSF and APMSF. Most preferably, the protease inhibitor is 4- (2-aminoethyl) benzenesulfonyl fluoride.HCl (Boehringer Mannheim).
PEFAB from Corporation, Indianapolis, IN
LO SC or PEFABLOC PLUS, which may also include tyramine).

[0099] In a preferred embodiment, the eluate comprising Cβ protein or fragments and / or variants thereof from the first chromatography medium is purified prior to ligand affinity chromatography or gel filtration chromatography on the second medium. About 3 times using a buffer containing about 10% to about 20% amphoteric surfactant.

In a preferred embodiment, the Cβ protein or a fragment thereof and / or
Alternatively, the mutant is first or second by applying an eluate comprising a salt gradient.
From the chromatography medium. More preferably, the salt gradient is a step gradient comprising about 0.0M, about 0.05M, 0.075M, and 0.09M salt steps. Most preferably, the salt gradient is a linear gradient of about 0.0M to about 0.1M salt. More preferably, the salt is NaCl or KCl. Most preferably, the salt is NaCl.

In a preferred embodiment, the process of obtaining a substantially pure Cβ protein or a fragment or variant thereof is performed at a pH from about 7.0 to about 8.0. More preferably, the process is performed at a pH from about 7.6 to about 7.8.

[0102] In a preferred embodiment, the eluate further comprises from about 0.1% to about 1.0% of an amphoteric surfactant. More preferably, the eluate contains about 0.5% amphoteric surfactant.

In another preferred embodiment, the Cβ protein or fragment and / or variant thereof is obtained from a bacterial cell transfected with a nucleotide sequence encoding the Cβ protein or fragment and / or variant thereof. . Here, the cells overexpress the Cβ protein or fragments and / or variants thereof. More preferably, the bacterial cells are E. coli. E. coli cells. Most preferably, the Cβ protein or fragment or variant thereof comprises: (a) disrupting the cells; (b) ethanol / to a concentration of about 20% (v / v) ethanol / 0.1 M CaCl _2. Precipitating the non-proteinaceous material from the cells by adding CaCl ₂ : (c) removing the precipitated nonproteinaceous material to obtain a solution; (d) a concentration of about 80% (v / v) Precipitating the protein from the solution by adding ethanol to and collecting the precipitated protein; and (e) precipitating the protein in a buffer solution containing about 3% to about 7% amphoteric surfactant. Re-suspending

In another preferred embodiment, the Cβ protein is obtained from a bacterial cell that naturally produces the Cβ protein. More preferably, the bacterial cell is Str
eptococcus agalactiae cells. Most preferably, the Cβ protein comprises: (a) boiling the bacterial cells in a buffer containing about 3% to about 7% amphoteric surfactant to obtain a solution; (b) Cooling in an ice bath; (c) adding a cold solution of CaCl ₂ / ethanol to about 20% ethanol (v
/ V) / about process giving a density of 0.1 M CaCl _2; ethanol to a concentration of (e) about 80% (v / v); (d) obtaining a precipitated non-proteinaceous material is removed solution Precipitating the protein from the solution by adding and collecting the precipitated protein; and (f) removing the precipitated protein in a buffer solution containing about 3% to about 7% amphoteric surfactant. Resuspending.

The present invention also relates to a vaccine comprising a protein fragment or peptide of the present invention together with a pharmaceutically acceptable carrier. In one aspect, the present invention relates to protein fragments or peptides that induce antibodies that are bactericidal against Gram-positive bacteria having complement alone. That is, this activity does not depend on the opsonophagocytic mechanism. These protein fragments and peptides of the invention are particularly well suited for the production of vaccines that are useful in patients with reduced cellular immunity (eg, chemotherapeutic patients and leukemia patients).

Because the proline-rich region of streptococci described herein is characteristic of Gram-positive bacteria, the vaccines of the present invention will provide an immune response against other Gram-positive bacteria, including but not limited to: It is expected to be useful for inducing:

[0107]

Embedded image In a preferred embodiment, the protein fragment or peptide is conjugated to a polysaccharide. Conjugates of the invention can be formed by reacting the reducing end group of a polysaccharide with a primary amino group of a protein (eg, the amino group of a lysine residue) by reductive amination. The polysaccharide can be conjugated to any or all of the primary amino groups of the protein. Reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both. Preferably, the protein fragment or peptide is Jenn.
ings et al., US Pat. No. 4,356,170, which involves the controlled oxidation of polysaccharides using periodate, followed by reductive amination of the proteins of the invention. Is conjugated to

In a preferred embodiment, the polysaccharide is one of a group B streptococcal capsular polysaccharide selected from type Ia, type II, type III and type V. Bak
er, C.E. J. And Kasper, D .; L. Rev., Rev .. Inf. Dis. 7:
458-467 (1985); Baker, C .; J. Et al. Engl. J. M
ed. 319: 1180-1185 (1988); Baker, C .; J. N
ew. Engl. J. Med. 322: 1857-1860 (1990). The vaccine may also comprise a protein fragment or peptide of the invention conjugated to group B capsular polysaccharide type Ia (protein-Ia); a protein fragment or peptide of the invention conjugated to group B capsular polysaccharide type II (Protein-II); a protein fragment or peptide of the present invention conjugated to group B capsular polysaccharide type III (protein-III); and a protein of the present invention conjugated to group B capsular polysaccharide type V It may be a combination vaccine comprising one or more protein fragments or peptide-polysaccharide conjugates selected from the group consisting of fragments or peptides (protein-V). Most preferably, the vaccine is a combination vaccine comprising Protein-Ia, Protein-II, Protein-III and Protein-V. Such combination vaccines include type Ia, type II, type III, type V and I
Induces antibodies against group b streptococcus (type Ib group B streptococci also
To express). Furthermore, the immune response of the combination vaccine against polysaccharides is a T-dependent response.

A vaccine of the invention may include a conjugate comprising a peptide or protein fragment of the invention covalently conjugated to a coated polysaccharide. Alternatively, the peptide or protein fragment can be conjugated or complexed with a porin protein, proteosome or other carrier protein. U.S. Patent No. 5,439,808, which describes a method for the preparation of outer membrane group B porin from N. meningitidis, and WO 95/03069 (
This is H. Influenzae describes the preparation of P2 porins).

[0110] The vaccines of the present invention include one or more protein fragment or peptide vaccines or conjugate vaccines in effective amounts depending on the route of administration. While the subcutaneous or intramuscular route of administration is preferred, the vaccines of the invention may also be administered by the intraperitoneal or intravenous route. Those of skill in the art understand that the amount to be administered for any particular treatment protocol can be readily determined without undue experimentation. For each conjugate, an appropriate amount is expected to be in the range of 2 micrograms protein fragment or peptide per kg body weight to 100 micrograms protein fragment or peptide per kg body weight.
In a preferred embodiment, the vaccine comprises about 2 μg of the protein fragment or peptide or an equivalent amount of the protein fragment or peptide-polysaccharide conjugate. In another preferred embodiment, the vaccine comprises about 5μ
g protein fragment or peptide or an equivalent amount of protein fragment or peptide-polysaccharide conjugate.

The vaccines of the present invention may be used for oral administration in the form of capsules, liquid solutions, suspensions or elixirs, or in the form of sterile liquids such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate buffered saline, or any such carrier for which the protein fragment or peptide or conjugate vaccine has appropriate solubility properties. The vaccine can be in the form of a single dose preparation or a multi-dose flask that can be used for a mass vaccination program. See Remington's Pharmaceutical for how to prepare and use vaccines.
al Sciences, Mack Publishing Co. , East
on, PA, Osol (eds.) (1980); and New Trends an
d Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, MD
(1978).

[0112] The vaccines of the present invention may further include an adjuvant to enhance the production of protein-specific antibodies. Such adjuvants include various oil formulations (eg,
Complete Freund's adjuvant (CFA)), stearyl tyrosine (ST, see US Pat. No. 4,258,029), a dipeptide known as MDP, saponin (see US Pat. No. 5,057,540). , Aluminum hydroxide and lymphoid cytokines.

[0113] Freund's adjuvant is an emulsion of mineral oil and water that is mixed with an immunogenic substance. Freund's adjuvant is potent, but it is not usually administered to humans. Alternatively, adjuvant alum (aluminum hydroxide) or ST may be used for administration to humans. The protein fragment or peptide vaccine or its conjugate vaccine can be absorbed onto aluminum hydroxide, from which it is released slowly after injection. This vaccine is also available in Fuller
ton, U.S. Patent No. 4,235,877.

In another preferred embodiment, the present invention provides a method of administering a vaccine of the present invention, produced according to the methods described, to an animal in an amount effective to induce an immune response. And a method of inducing an immune response in the method.

While the invention has been described generally herein, the same can be more readily understood by reference to the following examples. These examples are provided by way of illustration,
And this is not intended to limit the invention unless otherwise indicated.

EXAMPLES Example 1 Cloning and Expression of the Gene Encoding Cβ To locate the IgA binding site on the Cβ protein, two oligonucleotides were synthesized. The first oligonucleotide (oligo 1) corresponds to the 5 'end of the mature protein and has the sequence (SEQ ID NO: 8) 5'-AAGGATCCACAAG
It has TGAGCTTGTAAAGGGACGAT-3 ', which contains a BamHI site. The second oligonucleotide is just short at the 3 'end of the gene and has the sequence (SEQ ID NO: 9) 5'-AAAACTCGAGTTTCTTTT
Has CCGTTGTTGATGTA-3 ′ and contains an Xhol site. Oligonucleotides to the 3 'end of this gene were chosen to eliminate the LPXTG motif found in many Gram-positive cell wall proteins. This sequence motif has been shown to be part of these proteins involved in the processing of these tissue cell wall proteins and eventually becoming covalently linked to peptidoglycan (Navarre, WW). And Schnew
ind, O .; , Molec. Microbiol. 14: 115-121 (19
94); Schneewind, O .; Et al., Science 268: 103-1.
06 (1995)). Using chromosomal DNA from A909 strain group B streptococcus containing the gene for the Cβ protein as a template, and standard PCR procedures, about 3.2 k
The product of b was produced as observed when electrophoresed on a 1% agarose gel. The PCR product containing the Cβ protein gene was cut with the endonuclease restriction enzymes BamHI and XhoI. This BamHI-XhoI DNA
The fragment contained the sequence for the entire Cβ protein except for the last 33 amino acids at the carboxyl terminus, including the putative IgA binding site. Then, this DN
The A fragment was converted to an appropriately restricted T using the standard T4 ligase procedure.
7 expression plasmid pET17b (Novagen, Inc., Madison)
WI). This plasmid was then transformed into E. coli using the protocol suggested by the manufacturer (Novagen. Inc.). E. coli strain BL21 (DE3
). E. coli containing this plasmid E. coli cells at 50 μg / ml
Selection was performed on LB plates containing carbenicillin. These plates were incubated at 37 ° C.
For overnight. Transformant colonies were carefully placed on nitrocellulose filters saturated with IPTG. After 30 minutes, the bacteria were lysed by placing the filter in a chloroform vapor chamber for 15 minutes at room temperature.

After removing the filters from the chamber, they were added to a 20 mM Tris
-Colony side up (c) on Whatman® 3MM filters previously saturated with HCl, pH 7.9, 6 M urea and 0.5 M NaCl.
olone-side up). After 15 minutes, remove these filters from PB
Washed three times with S and incubated for 1 hour with purified human IgA in PBS-Tween®. These filters were then washed with PBS-T
Wash in Ween® and goat anti-human IgA alkaline phosphatase conjugate (Cappel Research Products, West)
(Chester, PA) using standard procedures (Blak).
e, M. S. Et al., Analyt. Biochem. 136: 175-17 (19
84)). Some colonies demonstrating high IgA binding activity were selected and grown overnight at 30 ° C. in 1 ml LB broth containing carbenicillin. These cultures were then diluted 1-100 with fresh LB-carbenicillin broth and incubated for an additional 6 hours at 30 ° C. Expression was then determined by IPT
Induced by the addition of G, and the culture was continued at 30 ° C. for an additional 2 hours. The cells were harvested by centrifugation, resuspended in water, and subjected to several freeze-thaw cycles. The cells were harvested once more by centrifugation and the supernatant was saved for testing for their IgA binding activity.

Example 2 Identification of the IgA Binding Domain of Cβ Once the specific stable plasmid producing the recombinant Cβ protein has been activated and the expressed protein binds human IgA, Novatope (
The IgA binding region of Cβ was located using a strategy similar to that of System® (Novagen, Inc.). This procedure was performed according to the manufacturer's instructions. Briefly, purified plasmids containing the Cβ gene were randomly cut with DNase I and electrophoresed in a 2% low melting point agarose gel. Fragments of DNA corresponding to sizes between 100-300 base pairs were excised from the gel, purified and resuspended in TE buffer.
A single dA was added to the fragment using the recommended reaction mixture, and the fragment was ligated into a pETOPET vector containing a single dT end. After standard ligation procedures, the plasmid is transferred to the competent NovaBlue
(DE3) cells (Novagen, Inc.) and 50 μg /
Plated on LB plates containing ml carbenicillin. The plates were incubated overnight at 37 ° C. The transformant colonies were tested for IgA binding activity as described in Example 1. Some clones were
Selection was based on these bindings to gA. Bacteria from each of these clones were separately inoculated onto fresh LB plates and their I.D.
Retested for gA binding capacity. Plasmid preparations were made from each by standard means and sequenced.

The nucleotide sequence of the cloned Cβ protein gene fragment was determined by the dideoxy method using a denatured double-stranded plasmid DNA template as described (Current Protocols in Molecular).
Biology, John Wiley & Sons, New York, N.W. Y
. (1993)). Sequenase II kit (United Stat
es Biochemical Corp. , Cleveland, OH)
Used according to manufacturer's instructions. D obtained to include part of the Cβ gene
The minimum fragment of NA is shown in FIG. Translation of this sequence is shown in FIG. 1 (SEQ ID NO: 2).
) Corresponds to amino acids 101 to 230 of the mature Cβ protein. This D
Attempts to further shorten the NA fragment did not give any IgA binding activity.

Example 3 ELISA Inhibition Assay: Peptide Binding Studies Several synthetic peptides corresponding to the amino acid sequences contained within this region of the Cβ protein were made. The peptide was converted to an ABI 430A peptide synthesizer (App
lied Biosystems, Foster City, CA)
Synthesized using t-butoxycarbonyl chemistry and deprotected. The peptide from the resin sample was detached from the resin by treatment with HF in the presence of anisole (0 ° C./1 hour). Preparative purification of these peptides was carried out using a C18 column (2.14).
(Dymax-Rainin, Woburn, Mass.). This peptide was converted to the Waters Picotag system (
Waters, Milford, Mass.) Using PTC amino acid analysis. The synthetic peptide eluted from the C18 column as a major peak, typically consisting of 75-85% of the overall elution profile. The amino acid composition of the purified peptide agreed well with the sequence used to synthesize the peptide. These peptides were used in an ELISA inhibition assay to block binding of human IgA to purified Cβ protein as follows. The addition of 0.1 ml of purified Cβ per well to a microtiter plate (Nunc-
Immuno PlateIIF, Vanguard Internationala
1, Neptune, NJ). The plate was incubated overnight at room temperature. The plate was washed with 0.9% NaCl, 0.05% Brij 3
Washed 5 times with 5, 10 mM sodium acetate (pH 7.0), 0.02% azide. Purified human IgA myeloma protein was purchased from Cappel Labora
Tries, diluted in PBS containing 0.5% Brij 35, added to the plate, and incubated for 1 hour at room temperature. The plate was washed again as before and the secondary antibody alkaline phosphatase conjugated goat anti-human IgA (T
ago Inc. , Burlingame, CA) was diluted in PBS-Brij and added to the plate and incubated for 1 hour at room temperature. The plate is washed as before and 0.1 M diethanolamine, 1 mM MgCl ₂ ,
_0. 1mM ZnCl _2, 0.02% azide (pH 9.8) solution of p- nitrophenyl phosphate (Sigma 104 (R) phosphate substrate) (1
mg / ml) was added. The plate is incubated at 37 ° C. for 1 hour,
The absorbance at 405 nm was measured using an Elida-5 microtiter plate reader (Physica, New York, NY). Control wells lacked either the primary and / or secondary antibodies. This was done to obtain titers of human IgA myeloma protein. This results in a half-maximum reading (half-maxima) in the ELISA assay.
l reading). This titer was used in the inhibition ELISA.
Microtiter plates were sensitized and washed as before. Purified synthetic peptide was added and diluted in PBS-Brij. A dilution of the human IgA myeloma protein that gave a half-maximum reading was then added. This mixture was then incubated for 1 hour at room temperature. The plate was washed again and the conjugated secondary antibody was added as described. The plate is then processed,
And read as described. The percentage of inhibition is calculated as follows: 1- (ELISA value with added peptide) / (ELISA value without added peptide). The peptide inhibited in this ELISA assay has the sequence Asn-Hi
s-Gln-Lys-Ser-Gln-Val-Glu-Lys-Met-Al
a-Glu-Gln-Lys-Gly (SEQ ID NO: 10). this is,
It was suggested that at least a portion of the IgA binding domain of Cβ was contained within the region of the protein containing this sequence.

Example 4 Oligonucleotide-Specific Mutagenesis of the Gene Encoding Cβ The importance of this region in the Cβ protein for IgA binding activity was confirmed, and the mutant protein ultimately used in vaccine formulations was identified. To begin making, the Ex-Site® PCR site-directed mutagenesis protocol (Strr
A modified version of Atagene (developed by Stratagene, CA) was used. The template used was a plasmid called pNV222. This plasmid was constructed using the pSP76 vector (Promega, Madison).
, WI). The DNA oligonucleotide is A
Applied Biosystems Model 292 DNA Synthesizer (Fost
er City, CA). The oligonucleotide was manually removed from the column by treatment with 1.5 ml of ammonium hydroxide for 2 hours with gentle mixing every 15 minutes. They were deprotected at 55 ° C for 16-18 hours. After deprotection, they were dried and used directly or on an oligonucleotide purification column (Applied Biosystems, Foster C).
, CA). Several different oligonucleotides were made to induce different changes in the DNA sequence in the region of interest. An example is as follows. The primers in this particular example are overlapping primers, both of which are amino acids 170 and 17 in the sequence shown in FIG. 1 (SEQ ID NO: 2).
5 contained a sequence that required changing lysine to alanine. This forward primer (designated Cβ613) has the sequence (SEQ ID NO: 6) 5′-GTT GA
A GC A ATG GCA GAG CAA GC G GGA ATC AC
A AAT GAA G-3 ′ and the reverse primer (Cβ642R
Is represented by the sequence (SEQ ID NO: 7) 5′-GAT TCC C GC TTG
CTC TGC CAT T GC TTC AAC TTG ACT TTT
With TTG-3 '(substitutions are underlined). The reaction conditions are as follows: 10 ng pNV222 template, 15 pmol of each primer, 1 m
M dNTPs, 1 × Vent polymerase buffer (20 mM Tris-HC)
1, pH 7.5; 10 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 2 mM M
gSO ₄ ; 0.1% (v / v) Triton® X-100; 0.1 m
g / ml bovine serum albumin (BSA)), 10 units of Vent polymerase, and H ₂ O to 100 μl. This reaction was combined with the PCR Gem10 wax beads in a Hot Start Protocol (Perkin Elder).
mer, Foster City, CA). This reaction system is called Pe
rkin Elmer® thermocycler (Perkin Elme)
r, Foster City, CA) under the following conditions: 5 at 94 ° C.
10 cycles of 30 minutes at 94 ° C., 2 minutes at 37 ° C., 10 minutes at 72 ° C .; 30 cycles of 30 seconds at 94 ° C., 2 minutes at 55 ° C., 10 minutes at 72 ° C .;
One cycle of 12 minutes at 2 ° C. The reaction is brought to 37 ° C. with 10 units of DpnI.
For 30 minutes to break the template DNA, then treat with PfuI polymerase at a temperature of 72 ° C. for 60 minutes to remove any remaining overhangs.
ng). The reaction was combined with 1 × Vent polymerase buffer and 0.3
1: 4.6 dilution in 8 mM dATP. Dilute the reaction at 25 ° C. for 24 hours.
Time-ligated and competent DH5α cells (Gibco / BRL, Gait
hersburg, MD). Select the colonies with 3 ml of LB
Grow in Kanamycin (50 mg / ml) for 16-18 hours at 37 ° C.
The DNA was applied to a QIAspin® column (Qiagen, Chatswood).
rth, CA). The clone was analyzed for the size of the insert on a 0.8% agarose gel and then sequenced. The selected colonies were then plated at 37 ° C. in 100 ml of LB + Kanamycin (50 mg / ml).
For 16-18 hours. The DNA is transferred to a Qiagen® chip 10
0 (Qiagen, Chatsworth, CA). Then
They were digested with NdeI and PstI and run on a 0.8% agarose gel to separate mutated regions. Isolating a 2300 bp fragment;
And Gene-Clean Spin Kit (registered trademark) (Bio 101
, Vista, CA). Expression vector pET 24a
(Novagen, Inc.) and pNV3 consisting of the native Cβ gene
The clone designated 4 was also digested with NdeI and PstI and
Run on an 8% agarose gel. A large band (6300 bp) containing the pET vector and the rest of the Cβ gene was isolated and Gene-Clean Sp
Gels were purified using in Kit® (Bio 101). These two fragments were ligated at 4 ° C. for 24 hours and transformed into competent BL21 (DE3) cells. Selected colonies were transformed with 3 ml of LB +
Grow in kanamycin (50 mg / ml) at 37 ° C. for 16-18 hours. D
NA was prepared using a QIAspin® column (Qiagen),
Clones were then analyzed on a 0.8% agarose gel for insert size.

A clone encoding a mutant Cβ protein was also constructed. Here, two glutaminyl residues were replaced with prolinyl residues and caused a deletion in the Cβ gene, which resulted in a six amino acid deletion in the region of interest (Table 1).
).

A clone expressing a Cβ protein lacking or reduced in IgA binding activity but still reacting with anti-βag antiserum was selected (Example 5) and 100 m
Grow in 1 LB + Kanamycin (50 mg / ml) at 37 ° C. for 16-18 hours. Plasmid DNA from these clones was prepared using a Qiagen® chip 100 (Qiagen) and the mutated Cβ gene was completely sequenced.

Example 5 Western Blot and ELI of IgA Binding by Cβ Mutants
SA analysis) To determine whether a mutation in the gene encoding the Cβ protein reduced or eliminated IgA binding while maintaining Cβ antigenicity, the protein encoded by the mutated gene And expressing SDS-PA
It was subjected to GE and Western blot analysis. Two Western blots were generated for each sample and reacted with either purified human IgA myeloma protein or hyperimmune rabbit anti-βag protein antiserum. A clone expressing a Cβ protein in which lysine was changed to alanine at amino acids 170 and 175 in the sequence shown in FIG. 1 (SEQ ID NO: 2) has little IgA binding activity, but this protein reacts with the anti-Cβ antiserum. The ability to do so remained high. IgA binding activity was also observed for clones expressing Cβ protein (where two glutaminyl residues were replaced with prolinyl residues) and Cβ with a six amino acid deletion.
Almost virtually eliminated in the protein encoding clones (Table 1), but both reactivity with anti-Cβ antiserum was maintained. Data for clones with six amino acid deletions show that the residues responsible for IgA binding activity of the Cβ protein are located within this region of the protein, and that other possible mutations within this region affect IgA binding activity. It was suggested to have an effect.

A competitive inhibition ELISA was used to more accurately determine the amount of antigenic / structural change of a sequence modification on the Cβ protein. 0.1M containing 0.02% azide
Microtiter plates (Nunc) were added by adding 0.1 ml of purified Cβ per well at a concentration of 2.0 μg / ml in carbonate buffer (pH 9.6).
-Immuno PlateIIF, Vanguard International
al, Neptune, NJ). The plate was incubated overnight at room temperature. The plate was washed with 0.9% NaCl, 0.05% Brij 3
Washed 5 times with 5, 10 mM sodium acetate (pH 7.0), 0.02% azide. Hyperimmune rabbit antiserum against Cβ protein was 0.5% Brij
Diluted with BPS containing 35, added to the plate, and incubated for 1 hour at room temperature. The plate was washed again as before and the secondary antibody alkaline phosphatase conjugated goat anti-rabbit IgG (Tago Inc., Burlingame, C
A) was diluted in PBS-Brij and added to the plate and incubated for 1 hour at room temperature. The plate was washed as before and p-nitrophenyl phosphate (Sigma 104®) in 0.1 M diethanolamine, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 0.02% azide, pH 9.8. Phosphate substrate) (1 mg / ml) was added. This plate
Incubate at 7 ° C. for 1 hour and measure the absorbance at 405 nm by Elida-
5 microtiter plate reader (Physica, New York, N
Y). Control wells lacked either the primary and / or secondary antibodies. This was done to obtain titers of rabbit anti-Cβ protein. This gave a half-maximum reading in the ELISA assay. This titer was used in the inhibition ELISA. Microtiter plates were sensitized and washed as before. Purified Cβ protein or a variant of Cβ protein was added and diluted in PBS-Brij. A dilution of the rabbit anti-Cβ protein that gave a half-maximum reading was then added. This mixture was then incubated for 1 hour at room temperature. The plate was washed again and the conjugated secondary antibody was added as described. The plate was then processed and read as described. The percentage of inhibition is calculated as follows: 1- (ELISA value with added protein) / (ELISA value without added protein). In this assay, the inhibition of wild-type Cβ protein from streptococci was compared to recombinant Cβ protein and a glutaminyl to prolinyl mutant,
E. FIG. E. coli (data not shown). This assay is sensitive enough to detect the absence of the transmembrane region in recombinant Cβ proteins. However, when the recombinant Cβ protein containing the wild-type sequence is compared to a substitution mutant lacking IgA binding activity, the difference in antigenicity is minimal. This suggests that such substitutional mutants retain most of the antigenic characteristics of the Cβ protein, but lack unwanted IgA binding activity.

Example 6 Initial Isolation of Recombinant Cβ Protein from E. coli Cell paste was added to an ice-cold cell disruption buffer (5 mM Tris (HCl), pH 7
. 6-7.8 (with 5 mM DTT), 0.2 mg / ml EDTA and P
EFABLOC PLUS (registered trademark) Protease Inhibitor
(Boehringer Mannheim, Indianapolis, IN
)). The cell suspension was passed twice through a Stansted® cell disrupter and then centrifuged at 12,000 g for 30 minutes. The supernatant was decanted and non-proteinaceous material was precipitated by adding ethanol / CaCl ₂ to a final concentration of 20% ethanol / 0.1 M CaCl ₂ .

After clarifying the protein by centrifugation at 10,000 g for 30 minutes,
Precipitate with final 80% ethanol, then 5% Zwittergent® 3,14, 25 mM Tris (HCl) and 5 mM DTT (pH
7.6-7.8).

Example 7 Initial Isolation of Cβ Protein from S. agalactiae Native Cβ protein was isolated from 5% Zwittergent® 3
, 14, and 25 mM Tris (HCl) (pH 7.6-7.8) by boiling the bacteria in Streptococcus agala.
ctiae. After boiling, the suspension is cooled in an ice bath to 0 ° C. to 10 ° C. and then a cooled (0 ° C. to 4 ° C.) solution of ethanol / CaCl ₂ is added to a final concentration of 2 ° C.
0% ethanol / 0.1 M CaCl ₂ was added. The solution was clarified by centrifugation, after which the protein was precipitated from the supernatant with 80% ethanol.
The protein was purified using 5% Zwittergent®, 25 mM Tri.
s (HCl), 5mM DTT and "PEFABLOC PLUS" Prot
Ease Inhibitor (Boehringer Mannheim C
corporation, Indianapolis, IN) (pH 7.6-7.
Redissolved in the buffer containing 8).

Example 8 Purification of Cβ Protein If necessary, the concentration of this protein in 5% Zwittergent® / Tris buffer was determined by using Pharmacia High Performance
Before applying to a Q.nce Q.RTM. column (1.6.times.20 cm).
Should be between 5 and 0.8 mg / ml. When eluting Cβ protein using a linear salt gradient, the gradient used was 25 mM Tris (HCl) /0.5%
0 in Zwittergent® 3,14 (pH 7.6-7.8)
０．１0.1 M NaCl. A step gradient at 0, 50, 75, and 90 mM NaCl in the Tris / Zwittergent® buffer can also be applied, and the Cβ protein can elute at 75 mM NaCl.
Alternatively, 50 mM NaCl / Tris (HCl) /0.5% Zwitte
After washing the column in Rgent® buffer, the Cβ protein was
60 mM NaCl / 0.6% CH in s (HCl) (pH 7.6-7.8)
Elution was performed using APS. The Cβ protein is eluted at low salt concentration with increasing total protein applied to the column.

SDS-PAGE was used to identify the Cβ fraction from the Q® column. These fractions are pooled and the protease inhibitor PEFABLOC
PLUS® (Boehringer Mannheim, Indi
anapolis, IN) was added. This protein pool was then tripled (ie, 2 parts diluent (v / v) (1 part protein pool) (15
% Zwittergent®, containing 25 mM Tris (HCl) and 5 mM DTT (pH 7.6-7.8)). The protein was then applied to a Pharmacia High Performance Heparin Sepharose® column (1.6 × 20 cm). The Cβ protein was subjected to a linear salt gradient (25 mM Tris (HCl) /0.5% Zwitt).
eluting with 0 to 0.1 M NaCl in ergent <(R)>. In general, Cβ proteins can be eluted from a heparin column utilizing the same buffers and gradient conditions used on the Q® column. FIG. 10 shows the elution profiles of the Q® column and the heparin column. Figures 3A and 3
B shows purification data and corresponding SDS-PAGE gel analysis.

When using a gel filtration column in the second chromatographic separation, the fraction from the Q® column containing the Cβ protein was precipitated by adding absolute ethanol to a final concentration of 80% ethanol. The precipitated protein was collected by centrifugation and a minimum volume of 25 mM HEPES, 1 M NaCl, 5 mM D
Redissolved in TT (pH 8.0) containing 10% Zwittergent®. The re-dissolved sample was applied to a gel filtration column (same buffer (Zwittergent® for recombinant protein without DTT and 0.5% Zwittergent for native protein purification).
(Registered trademark) was pre-equilibrated with).

Example 9 Protease Digestion of Cβ Protein and Isolation of Fragments Dulbecco's PBS (Life Technologies, Gaithers)
burg, MD) from 6.0 to 8.0 mg / ml.
10 mg / ml thermolysin stock solution in 0 mM CaCl ₂ (Boe
hringer Mannheim, Indianapolis, IN).
Digestion was performed by adding 025 ml. The reaction mixture was incubated overnight at 60 ° C. with constant stirring. The protease activity was then reduced to 10 mM
Stopped by adding EDTA (FIGS. 4A and 4B).

First, the peptide fragments T1 and T1 generated by thermolysin
2 (apparent molecular weights of 35 kD and 25 kD, respectively) were separated by electrophoresis, transferred to nitrocellulose, stained with Ponceau S, and then recovered by dissolving the membrane in acetonitrile (FIG. 5). . After dissolving the membrane, the peptide fragments were precipitated with ice-cold acetone and collected by centrifugation. After centrifugation, the peptide precipitate was dissolved in PBS and purified by Fusco et al., Adv.
Exp. Med. Biol 418: 841-845 (1997) and assayed for inhibition of bactericidal activity. Aliquots of the acetone-precipitated protein were also run on an SDS gel to confirm protein recovery.

To increase the amount of protein for subsequent digestion, the thermolysin fragment was precipitated with 80% final ethanol and collected by centrifugation. The pellet is mixed with 10 mM Tris (HCl) / 10 mM CaCl ₂ , 10%
Redissolve in 1-3 ml of glycerol (pH 7.2) and add 10 mM Tr
It was applied to a Sephacryl®-100 column (1.6 × 40 cm) equilibrated with is (HCl) / 10 mM CaCl ₂ (pH 7.2) (FIG. 6).

The isolated peptide fragment T1 was purified from Clostridium hysto
lyticum-derived endoprotease Arg-C (Boehringer
Mannheim, Indianapolis, IN). Arg-C digestion was assayed for inhibition of bactericidal activity. R1 and R2
The fragment represented by Polybuffer (registered trademark) 74 (P
(Pharmacia, Piscataway, NJ) on a Mono P® column (Figure 7).

Inhibition of bactericidal activity using unseparated thermolysin fragments T1 and T2 showed 50% inhibition at a protein concentration of 36 μg / ml. When the thermolysin fragments T1 and T2 were separated and recovered by electrophoresis and western transcription, 50% inhibition of bactericidal activity occurred at 15 μg / ml and 20 μg / ml for the T1 and T2 fragments, respectively. The T1 and T2 peptides were 100% at 18 μg / ml and 40 μg / ml, respectively.
Inhibition occurred. The yield of T1 peptide after Arg-C digestion was minimal. Ar
The protein concentration of the gC digested fragment was not determined. However, 50% inhibition of bactericidal activity was obtained with a 1:50 dilution of the sample. This activity was compared to the T1 peptide fraction separated by gel filtration. This T1 fraction contained 2.5 μg
/ Ml showed 50% inhibition.

N-terminal sequence data for the two major peptide fragments T1 and T2 (apparent molecular weights near 35 kD and 25 kD) from thermolysin digestion were obtained by SDS-PAGE on a tricine gel, which was obtained from 10 mM CA.
Transfer to a PVDF membrane in PS / 10% methanol and add 0% in 1% acetic acid.
. Stained with 1% Ponceau S, destained with 5% acetic acid, and washed thoroughly with water while vortexing. A piece of PVDF that has been sufficiently dried is cut out, and MA B
Sent to ioServices® (Rockville, MD) for sequencing.

The sequence analysis of the thermolysin digest showed that the N-terminus for T1 was VEQ
DQPAPIPENSE (SEQ ID NO: 52). For the T2 peptide, the N-terminus was LAANENNQQKIELTV (SEQ ID NO: 53). The T1 peptide fragment is closer to the C-terminus of the Cβ protein than the T2 peptide fragment (FIG. 8).

Example 10 Peptide Synthesis Chiron Mimotopes Multipin Non-Cleav
Able Peptide Kit (Chiron Mimotopes, Ra
Leigh, NC) using a Cβ mutant holoprotein (holoprotei).
The peptide of n) was synthesized. The computer program supplied with the kit helped to generate a contiguous 8-mer peptide with an overlapping sequence of 4 amino acid residues, and determined the approximate region of antibody binding. As recommended by the kit manual, FMOC protected amino acids were used for peptide synthesis and DIC / HOBt for amino acid activation. The side chain protecting groups were removed and the N-terminal peptide was acetylated. Preliminary ELISA results obtained using the 8-mer peptide showed an antibody binding region. A 20-mer peptide with overlapping sequences was synthesized and used to confirm initial results.

24 mer peptide sequence, PDVPKLPDVPKLPDVPKLPDAPKL
(SEQ ID NO: 47) was also synthesized by solid phase peptide synthesis, cleaved, and purified to give the peptide on a multiple mg scale (Protein / DNA Techno).
logy Center, Rockfeller University, NY
). This peptide was used in an ELISA inhibition assay.

For ELISA, mimotope pin was 2%
Blocked in BSA / PBS-0.1% Tween® (polyoxyethylene sorbitan) (PBST) for 30 minutes. After washing the pins with at least 5 changes of PBST, the pins are gently agitated for 1.5 hours in a 1: 20,000 dilution of polyclonal rabbit antibody 52.2 in 0.05% BSA / PBS-T. did. The pins are washed again with at least 5 changes of PBS-T, and then
Stirred in 1: 2000 dilution of alkaline phosphatase conjugated anti-rabbit antibody in 2% fetal calf serum / PBS-T.

[0142] The ELISA plates were placed in SIGMA 104 (diethanolamine buffer).
®) phosphate substrate (Sigma Chemical Company)
, St. (Louis, MO). The plate was developed at room temperature with constant stirring for 1 hour, then read at 405 nm on a plate reader.

ELISA results indicated that most antibody binding was directed to the repetitive sequence located near the C-terminus of this protein. This repeat sequence is PDVPKLPD
VPKLPD is present in the Cβ protein as VPKLPDAPKL (SEQ ID NO: 47). The 8-mer sequences PDVPKLPD (SEQ ID NO: 33), KLPDVPKL (SEQ ID NO: 34), VPKLPDVP (SEQ ID NO: 35), and KLPDAPKL (SEQ ID NO: 36) induced most antibody binding (FIG. 9A). To a lesser extent,
Some antibody binding activity is determined by the 8-mer sequence ETPDTPKI (SEQ ID NO: 38), R
Oriented to TVRLALG (SEQ ID NO: 39), and occasionally GGGTVRVF (SEQ ID NO: 40). The 20-mer peptides synthesized (in duplicate) to confirm antibody binding were: SPKTPEAPKIPEPPPKTPDVP (SEQ ID NO: 41), PEAPKIPEPPPKTPDVPKLLPD (SEQ ID NO: 42), K
IPEPPKTPDVPKLPDVPKL (SEQ ID NO: 43), PPKTPDVP
KLPDVPKLPDVP (SEQ ID NO: 44), PDVPKLPDVPKLPDV
PKLPD (SEQ ID NO: 45) and KLPDVPKLPDVPKLPDAPK
L (SEQ ID NO: 46) (FIG. 9B).

Example 11 Inhibition ELISA with Cβ Protein and Synthetic Peptide Polyclonal rabbit antibody 52.2 is in 0.05% BSA / PBS-T containing 0.1 mg / ml recombinant Cβ protein: 20,000 dilution 20 ml
Was incubated at room temperature for 30 minutes before the pins were assayed for antibody binding as described above. The result of the ELISA was the sequence PDVPKLPD (SEQ ID NO: 3).
3), KLPDVPKL (SEQ ID NO: 34), VPKLPDVP (SEQ ID NO: 35)
, And KLPDAPKL (SEQ ID NO: 36) showed inhibition of antibody binding to the pins. However, some antibody binding activity is due to the sequence RTVRLALG (SEQ ID NO: 39).
(Data not shown).

In an inhibition experiment using the synthetic peptide PDVPKLPDVPKLPDVPKLPDAPKL (SEQ ID NO: 47), the 8-merpin and 20-merpin were blocked in BSA. At the same time, the 52.2 anti-bag rabbit antibody was 0.05% BSA / 0
. 20 m 1: 20,000 dilution in 1% Tween®-PBS
1 for 30 minutes with (0.1 mg / ml) synthetic peptide (Protein / DNA).
Technology Center Rockefeller Universe
(Rsity, NY), and then assayed for pins for antibody binding as described above. One set of duplicated 20-mers
Served as a positive control for antibodies not exposed to the synthetic peptide.

The results of the ELISA were sequence PDVPKLPD (SEQ ID NO: 33), KLPDVP
KL (SEQ ID NO: 34), VPKLPDVP (SEQ ID NO: 35), and KLPDA
Inhibition of antibody binding to PKL (SEQ ID NO: 36) pins was demonstrated. Also, antibody binding was observed with the sequence ETPDTPKI (SEQ ID NO: 38) and somewhat less with RTVRLALG (SEQ ID NO: 39). Antibody binding to the 20-merpin was similarly inhibited. Positive control pins showed antibody binding for comparison (FIG. 10).

Example 12 Bactericidal Inhibition Assay to Establish That Specific Repetitive Amino Acid Sequences on Cβ Induce Antibody Binding Rabbits were vaccinated with a synthetic peptide conjugated to tetanus toxoid. Specific antibodies against the region on Cβ were generated. Array PDVPKLPDVPKL
A specific antibody directed against PDVPKLPDAPKL (SEQ ID NO: 48) was obtained and purified. This could be shown to kill GBS (Group B Streptococcus) type Ib. This killing was compared to the bactericidal activity of rabbit antisera against native Cβ holoprotein.

(Materials and Methods) (Conjugation of Peptide to Carrier Protein) Peptides were purchased from Rockfeller University Protein.
The ABI 430A peptide synthesizer (Applied Biosystems, Foster City,
CA) using NMP t-butoxycarbonyl chemistry. The peptide was deprotected by treatment with HF (0 ° C / 1 hour) in the presence of anisole and removed from the resin. Pre-purification of each peptide was performed using a C18 column (2.14 id × 30 cm) (Dynamax-Rainin, Woburn, Mass.). This peptide was analyzed by PTC amino acid analysis for Waters P.
Quantification was performed using the icotag system (Waters, Milford, MA). The synthetic peptide eluted from the C18 column as the major peak, consisting of more than 95% of the total elution profile. The amino acid composition of the purified peptide was in good agreement with this sequence. Perspective Biosys
Mass spectroscopy (MALDI-TOF) performed using the tem® DERP Mass Spectrometer showed that α-
It was run in a linear fashion using hydroxycinnamic acid (peptide mass indicated 2677 kD, which is in good agreement with the calculated molecular weight of about 2658 kD).

By dissolving about 26 mg of the synthetic peptide SPDVPKLPDVPKLPDVPKLPDAPKL (designated Ser-peptide) in 1.5 ml of deionized water and adding 60 μl of citraconic anhydride in 15 μl aliquots at room temperature intervals at 30 minute intervals. Citracone (Atassi, MZ and H
abeeb, A .; F. S. A. "Reaction of proteins
with citriconic anhydride "Methods in
Enzymology, Volume XXV, Enzyme Structure B
Section, Hirs, C.I. H. W. And Timasheff, S.M. N. Hen, New
York, Academic Press, pp. 546-533 (1972)).
The reaction mixture was maintained at a pH above 8 by titration with 5M NaOH.
The reaction lasted 1 hour after the final addition of citraconic anhydride. The solution is then
Thoroughly dialyzed against 0.2 M Na ₂ HPO ₄ (pH 8.5) using 100 MWCO dialysis tubing.

The serine residue on the peptide was then oxidized in the dark for 10 minutes at room temperature using 2.2 mg of sodium periodate (Geoghegan, KF and Strow, JG "Site"). -Directed conjugati
on of non-peptide groups to peptides
and proteins via period oxidatio
no of a 2 amino-alcohol-Application t
o modification at N-terminal serine "
Bioconjugate Chem. 3: 138-146 (1992)).
The reaction was quenched by adding ethylene glycol at room temperature with constant stirring, and then also dialyzed exhaustively against 0.2 M Na ₂ HPO _4, pH 8.5.

[0151] Purified tetanus toxoid monomer (Statens Serumminsti)
tut, Copenhagen S.M. , Denmark), Superdex
Chromatograph again on ® 200. This serine peptide was reductively aminated using 13 mg sodium cyanoborohydride to give
Bound to 15 mg tetanus toxoid. The reaction continued at 37 ° overnight. It was then extensively dialyzed against aqueous citric acid (pH 4.2) to remove protecting groups on the lysine residues of the peptide. Approximately 8 mg of product was recovered (weight determination including unbound peptide). Binding was monitored by HPLC using a Superdex® peptide column by injecting aliquots of peptide, tetanus toxoid, and a mixture of peptide and tetanus toxoid after coupling. In addition, conjugates that were dialyzed using 12,000-14,000 MWCO dialysis tubing to remove unbound peptides were assayed by ELISA. Microtiter plates were prepared using 1 μg of Ser-peptide conjugate for 4
Coated overnight at ° C. The polyclonal rabbit 52.2 primary antibody was used at 1: 2,000 in PBS-T, followed by the secondary antibody, alkaline phosphatase goat anti-rabbit conjugate (ICN, Costa Mesa, CA), in PBS-T. 1 in
: Used at 20,000 dilution. The ELISA plate was loaded with SIGMA 104 in 1 M diethanolamine buffer / 0.5 mM MgCl ₂ (pH 9.8).
Color was developed with Phospatase® substrate. This plate is loaded into a plate reader (Dynex Technologies, Inc., Chanti).
ly, VA) at 405 nm.

Ser-Peptide Antibodies New Zealand white rabbits were immunized on day 0 with a 100 μg dose of a mixture of both bound and unbound peptides in Freund's complete adjuvant. On days 21 and 42, rabbits were immunized again with a 100 μg dose in incomplete Freund's adjuvant. Rabbits were bled on day 51. In obtaining rabbit antiserum, ELISA was performed at 96 μl with 2 μg / ml Cβ protein.
SA plates were coated and then performed by diluting the Ser-peptide antibody using duplicate 2-fold dilutions starting at 1: 1000 until titers were obtained.

Further, to evaluate the specificity of the antibody binding, mimotopepin representing an 8-mer of the whole Cβ molecule was blocked in 2% BSA / PBS-T for 30 minutes. After washing, pins were washed with a 1: 20,000 dilution of ser-peptide antibody for 1
. Incubated for 5 hours. The pins were washed again and then incubated for 1.5 hours in a 1: 2,000 alkaline phosphatase anti-rabbit antibody. ELI
The SA plate was placed in a 1 M diethanolamine buffer / 0.5 mM MgCl ₂ (p
Color was developed with SIGMA 104® Phospatase substrate in H9.8). The plate was read at 405 nm on a plate reader.

(Purification of Ser-Peptide Rabbit Antibody) 30 mg of tetanus toxoid was added to 0.2 M NaHCO ₃ /0.5 M NaCl.
(PH 8.3) Dissolved in 7 ml and bound on a 5 ml HiTrap® NHS-activated affinity column (Pharmacia, Piscataway, NJ) by refluxing at room temperature for 4 hours using a peristaltic pump. After washing and quenching, the column is replaced with P as recommended by the manufacturer.
Equilibrated with BS, then 20 ml of the ser-peptide rabbit serum was passed through the column. The void fractions were pooled and assayed using mimotope pins displaying the N- and C-terminal sequences of Cβ. Immunoglobulin G
Further purification was performed using an opure® G column (Pierce, Rockford, IL). The fraction eluted from this column in low pH buffer is
ml HiTrap® desalting column (Pharmacia, Pisca)
(Taway, NJ) and desalted, then tested again for antibody binding specificity to mimotopepin, which displayed the N- and C-termini of Cβ.

Bactericidal Activity of Ser-Peptide Antibodies Antibodies and complement-mediated bactericidal activity were measured by Fusco et al.
activity elicited by the beta C pro
tein of Group B streptococci constra
"sted with capsular polysaccharides" A
dv. Exp. Med. And Biol. 418: 841-845 (1997)
). Comparisons were made using killings by: (1
A) an antibody against the native Cβ protein, (2) a Ser-peptide antibody purified against a tetanus toxoid column, and (3) a Ser-peptide antibody purified against a tetanus column and then a protein G column. Peptide PDVPKL
PDVPKLPDVPKLPDAPKL (SEQ ID NO: 48) was used to inhibit the bactericidal activity of these Ser-peptide antibodies.

GBS Colony Lift for Probe Ser-Peptide Antibody Binding GBS colonies were lifted from chocolate agar plates onto round nitrocellulose sheets, washed with PBS-Tween®, and then tetanus purified Ser. -Peptide antibody (1: 10,000 dilution and 1: 20,000 dilution) or tetanus and protein G purified Ser-peptide antibody (1: 300
0 dilution and 1: 6000 dilution) for 1.5 hours. After several washes in PBS-Tween <(R)>, the nitrocellulose sheet was treated with a goat anti-rabbit alkaline phosphatase conjugate for 1.5 times.
Incubate for hours, then substrate buffer (0.1 M Tris / 0.1 M
After washing in NaCl / 1 mM MgCl ₂ ), color was developed using NBT / BCIP substrate (Blake, MS, et al., “A rapid sensitiv”.
e method for detection of alkaline p
phosphatase conjugated anti-antibody
s on western blots "Anal. Biochem. 136:
175-179 (1984)).

(Results and Discussion) (Binding of Peptide to Carrier Protein) The lysine residues on this Ser-peptide are citracone to prevent these peptides from binding to tetanus toxoid and from binding to each other It is necessary. After dialysis, ELISA showed that the 52.2 antibody recognized the tetanus-Ser-peptide conjugate.

(Ser-peptide antibody) ELISA of this Ser-peptide antibody against the 8-mer mimotope sequence of Cβ
Shows that this antibody is only on the block of pins that represent the carboxy terminus of Cβ,
It was shown that the sequence PDVPKLPD (SEQ ID NO: 50) was recognized. Some sequences located towards the N-terminal part of Cβ also showed a positive signal in this ELISA. Antibodies raised against the tetanus conjugate cross-reacted with the N-terminal sequence of Cβ, and the reaction was eliminated by passing the antiserum through a tetanus affinity column.

(Purification of Ser-Peptide Antibody) Antibody binding to the N-terminal sequence was removed by once passing the antiserum through a tetanus column. The antibody specifically bound to the sequence PDVPKLPD (sequence 50). Further purification of the Ser-peptide antibody on a protein G column further purified the IgG and maintained affinity for the sequence PDVPKLPD (sequence 50).

Bactericidal Activity of Ser-Peptide Antibodies 50% of the bacteria survived for the 52.2 antibody against native Cβ:
It occurred with an antiserum dilution greater than 5500 (FIG. 12). Similarly, 50% survival occurred at a dilution of about 1: 8000 for Ser-peptide, whether or not it passed through a tetanus column (FIG. 13). Interestingly, antiserum purified against tetanus and protein G columns did not elicit bactericidal activity. This phenomenon could be attributed to structural changes due to the acidic conditions used to elute these proteins during purification on Protein G columns, which inhibited complement activation.

GBS Colony Lift Against Probe Ser-Peptide Antibody FIG. 14 shows Ser- on GBS Ib using only tetanus column and antiserum purified by protein G column followed by tetanus column. 3 shows peptide antibody binding. In each case, antibody binding occurred despite the lack of ability of Protein G purified antibodies to kill GBS for both preparations.

(Summary) A synthetic peptide sequence derived from Cβ protein was bound to tetanus toxoid.
This elicited an antibody response in rabbits vaccinated with this conjugate. After purification of the antiserum against a tetanus column or a tetanus column followed by a protein G affinity column, the antibody was raised against an ELISA against mimotope.
A specifically bound to the sequence PDVPKLPD (SEQ ID NO: 50) as measured by A.

The antisera and complement-mediated bactericidal activity of the antisera were measured and compared to antisera to Cβ haloprotein. Antisera against Cβ peptide purified against tetanus columns showed killing in a bactericidal assay similar to that with antisera against Cβ holoprotein. Antibodies are thought to infiltrate the peptidoglycan layer of this Gram-positive organism during cell division, allowing complement activity to result in cell lysis.

Example 13 Ser-Peptide Antibody Binding Introduction Many cell wall binding proteins of Gram positive bacteria have some common features. Its most prominent feature is the presence of the LPXTG sequence near the carboxy terminus of these proteins. This motif is well characterized, and it is through this sequence that these cell wall proteins are covalently linked to the cell wall. That is, an enzyme cleaves the sequence between its TGs, and then advances the protein to attach to the peptidoglycan peptide bridge via the carboxyl group on the threonine residue. Another common feature of these cell wall proteins of Gram-positive bacteria is that the region immediately before the amino terminus of the LPXTG sequence contains a number of prolyl residues. Prolyl residues are, due to their unique sequence,
Causes kink in protein. Therefore, this high prolyl region on these cell wall proteins has been postulated to be a region that weaves in and out of the peptidoglycan layer. However, there is no direct evidence for this theory. Anti-Se
It is in this region on the Cβ protein that the r-peptide antibody binds in the presence of complement and results in cell death. As mentioned earlier, the theory of how this rare activity can occur is that the antibody binds to this region of Cβ during cell division at the septum, where peptidoglycan is destroyed. Be rebuilt and rebuilt. To obtain further evidence for this theory, using confocal microscopy and FACScan analysis, where and when this Ser-
It was determined whether antibodies against the peptide bound to streptococcal organisms expressing the Cβ protein.

Materials and Methods Confocal Microscopy (CLSM) Analysis: CLSM was used to study the binding of rabbit antibodies (anti-Ser-peptide) to purified group B streptococci on tetanus columns. Bacterial cells of group B streptococcus strain H36B were grown in Todd-Hewitt broth and samples were taken at different stages of this growth. The stage of growth was determined by measuring the optical density of the sample at 600 nm. The cells were then fixed in 2% paraformaldehyde and aliquots were dried on microscope slides. The slide was coated with 5% normal goat serum. Samples were incubated with a 1: 500 dilution of rabbit antibody (anti-Ser-peptide) for 2 hours, washed, and incubated with FITC-conjugated goat antiserum to rabbit IgG (Molecular Probes, Eugene, OR). This sample was prepared using Vectaschild® mounting medium (Vector Laboratories,
Inc. (Burlingame, Calif.) And tested with a dual wavelength laser in a BioRad 1024 CLSM.

(FACScan analysis): FACScan analysis was performed using Becton-Dicke.
nson FACScan (Research Triangle Park,
NC) was performed on samples of bacterial cells obtained at different stages of growth. Bacterial cells of group B streptococcus strain H36B were grown in Todd-Hewitt broth and samples were taken at different stages of this growth. The stage of growth was determined by measuring the optical density of the sample at 600 nm. The bacterial cells were fixed in 2% paraformaldehyde, washed, and incubated with either anti-Ser-peptide antiserum or tetanus-adsorbed prebleed serum for 1 hour. Cells were washed three times and incubated for 1 hour with FITC-conjugated goat antiserum to rabbit IgG (Molecular Probes, Eugene, OR). Bacterial cells are washed three times with PBS, counterstained with ethidium bromide, and diluted 2 buffer (J & S Medical Ass.).
ociates, Farmingham, Mass.) to a volume of 1 ml. This data is stored in a Flo-Jo program (Tree Star Corp., Sa).
nCarlos, CA).

Results Confocal Microscopy (CLSM) Analysis: CLSM analysis showed that anti-Ser-peptide bound to small areas between streptococcal cells. The antibody bound more to actively dividing cells (ie, samples in logarithmic growth period) and, as the culture entered stationary phase, bound less, as further shown (see below). That). These data support the notion that the Ser-peptide antibody only accesses this region on the Cβ protein in regions where the peptidoglycan is in the fluid state (ie, the dividing septum). No bacteria were observed to be completely covered by the Ser-peptide antibody in any of the regions.

(FACScan analysis): Data for FACScan analysis can be shown in FIG. The x-axis is the time that has elapsed since the start of the culture, which took the bacterial sample. The y ¹ axis is the OD600 of the sample, indicating the number of bacteria in the sample.
The y ² axis is the amount of green fluorescence (ie, Ser-peptide antibody binding) compared to red fluorescence (ethidium bromide stains all bacteria). As can be appreciated, at the beginning of this culture, very little Ser-peptide antibody binds to the cells. This is the time to dilute the stationary phase cells from fresh culture into fresh culture medium. By 2 hours, the streptococci begin to divide and proliferate, as indicated by an increase in OD600, and at the same time, an increased amount of bound ser-peptide antibody is observed. Antibody binding peaks during the log phase (4-6
Coincides with the most active part of this culture). After 8 hours, the culture enters a stationary phase in which bacterial division slows and stops. Stationary phase sampled bacteria did not bind to the Ser-peptide antibody and resembled the organism initially found in this culture. These data show that the original theory is correct;
It is further confirmed that the epitope to which the peptide antibody binds is exposed only during active cell division and only in relatively small areas (ie, the septum of these dividing cells). Such septum divisions also expose the cell membrane of Gram-positive bacteria, allowing complement components activated by Ser-peptide antibodies to be unobstructed from accessing the bacterial cell membrane, resulting in cell lysis. Complete.

While the foregoing has described certain embodiments, it will be understood that the invention is not so limited. It will occur to those skilled in the art that various modifications may be made and are intended to be within the scope of the present invention (as defined by the appended claims). All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[Sequence list]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows wild-type Cβ1 (Jerlström, PG et al., Molec.
microbirol. 5: 843-849 (1991)). BglII and PstI shown in FIGS.
The site is identified.

FIGS. 2A and 2B show Q® columns (1.6 × 20 cm), respectively.
2 shows the protein elution profile under the following conditions for and heparin column (1.6 × 20 cm) chromatography: flow rate—5 ml / min; decay—
0.2; and a gradient of 0 to 0.1 M NaCl / 0.5% Zwitterge
nt® / 25 mM Tris (HCL), pH 7.6-7.8, 10 ml
/ Fraction.

FIGS. 2A and 2B show Q® columns (1.6 × 20 cm), respectively.
The elution profile is shown for the following conditions for and Heparin column (1.6 x 20 cm) chromatography: flow rate-5 ml / min; decay-0.2; and gradient-0-0.1 M NaCl / 0. 5% Zwittergent® / 25 mM Tris (HCL), pH 7.6-7.8, 10 ml / fraction.

FIGS. 3A and 3B show Cβ protein purification data and SD of corresponding preparations.
5 shows S-PAGE. FIG. 3A illustrates the purification of Cβ protein on a Q® column and a heparin column.

3A and 3B show Cβ protein purification data and SD of the corresponding preparation.
5 shows S-PAGE. FIG. 3B shows purification of Cβ protein on a Q® column and an S-300 gel filtration column.

FIGS. 4A and 4B illustrate thermolysin digestion of Cβ protein. FIG. 4A
Shows SDS-PAGE showing the digestion pattern of the resulting fragments with apparent molecular weights of 35 kD and 25 kD on a tricine gel.

4A and 4B illustrate thermolysin digestion of Cβ protein. FIG. 4B
Is a western transfer probed with antibody 52.2, indicating that the major fragment reacted with the antibody.

FIG. 5 shows separation and recovery of Cβ protein fragments by western transfer and subsequent lysis of nitrocellulose membrane. Aliquots were run on an SDS gel to confirm protein recovery.

FIG. 6 shows SDS-PAGE of thermolysin-digested Cβ protein separated by gel filtration on Sephacryl®-100.

FIG. 6 shows SDS-PAGE of thermolysin-digested Cβ protein separated by gel filtration on Sephacryl®-100.

FIG. 7 shows SDS-PAGE of the Arg-C fragment of the T1 peptide.

FIG. 1 shows the sequence of native Cβ protein from S. agalactiae. N-terminal sequence analysis showed the position where thermolysin-generating fragments T1 and T2 begin (the sequenced N-terminal section is underlined). Ar
The theoretical T1 fragment digestion by gC is indicated by a circle.

FIGS. 9A and 9B are graphical representations of antibodies that bind to 8-mer and 20-mer mimotopes, respectively. Duplicate experiments were performed with each mimotope, washing between each binding experiment.

FIGS. 9A and 9B are graphical representations of binding to 8-mer and 20-mer mimotopes, respectively. Duplicate experiments were performed with each mimotope, washing between each binding experiment (two bars). Some variability is due to incomplete cleaning.

FIG. 10 is a graphical representation of inhibition of antibody binding to a 20-mer mimotope by pre-exposing the antibody to a synthetic peptide corresponding to amino acids 880-906 of FIG. One set of data (open bars) for the 20 mer of the experiment in FIG. 9B is included as a positive control for antibodies not exposed to this synthetic peptide.

FIG. 11 is a graphical representation of bactericidal inhibition by Cβ protein and thermolysin digested fragments thereof.

FIG. 12 is a graphical representation of the survival of group B streptococcal Ib in a bactericidal assay using an antibody against the native Cβ protein.

FIG. 13 is a graphical representation of the survival of group B streptococcal Ib in a bactericidal assay using a Ser peptide antibody to Cβ protein.

FIG. 14 shows a Ser peptide antibody binding on a group B streptococcal Ib colony lift using tetanus column alone and antiserum purified by a protein G column followed by a tetanus column.

FIG. 15 is a graphical representation of the correlation between group B streptococcal Ib growth and Ser peptide antibody binding. Ser peptide antibody binding begins during the induced growth phase and peaks during the logarithmic growth phase. As bacteria reach stationary phase and divide, antibody binding decreases to zero.

FIG. 16 is a graphical representation of rabbit antibody 52.2 binding and inhibition of binding to the overlapping 8-mer mimotopepin (showing the sequence of the Arg-C digested R2 peptide fragment). Specifically, the bar graph has the highest antibody affinity (unadsorbed)
The sequence and the sequence with the ability (absorption) of the intact Cβ protein to inhibit the antibody 52.2 against the mimotope are shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 1/18 C07K 14/315 1/22 19/00 14/315 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12R 1:46) 5/10 C12N 15/00 ZNAA C12P 21/02 A // (C12N 15/09 5/00 A C12R 1: 46) (31) Priority claim number 60 / 154,017 (32) Priority date September 15, 1999 (September 15, 1999) (33) Priority claim country United States (US) (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, TZ, 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, CR, CU, CZ, DE, DK, DM, EE, ES, FI, 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, TZ, UA, UG, US, uz , VN, YU, ZA, ZW (72) Inventor Blake, Milan. United States Maryland 20759, Fulton, Beaufort Drive 8521 F-term (for reference) 4B024 AA01 BA80 CA04 DA02 DA05 DA11 EA04 GA11 HA01 HA03 4B064 AG01 CA01 CA19 CC24 CE08 CE11 CE12 DA01 4B065 AA01X AA49Y AA57X AA87X AB01 BA02 CA24 A45 A45 DD0 EE01 EE05 4H045 AA10 AA11 AA20 AA30 BA41 CA11 DA86 EA31 FA72 FA74 GA01 GA23 GA26

Claims (29)

[Claims] 1. A process for obtaining a substantially pure Cβ protein or a fragment and / or variant thereof, comprising the following steps: (a) obtaining the Cβ protein in a cell extract; (b) Subjecting the Cβ protein to ion exchange chromatography;
collecting the β protein-containing fraction; (c) pooling and diluting the Cβ protein-containing fraction; and (d) subjecting the diluted Cβ protein-containing fraction to ligand affinity chromatography, and Collecting the fractions, whereby a substantially pure Cβ protein or a fragment and / or variant thereof is obtained. 2. The process according to claim 1, wherein said ion exchange chromatography is performed utilizing an anion exchange medium containing trimethylaminomethyl groups. 3. The process of claim 1, wherein said ligand affinity chromatography is performed utilizing a ligand affinity medium comprising a heparin ligand. 4. The process of claim 1, wherein the Cβ protein or fragment and / or variant thereof is subjected to chromatography in a buffer containing about 5% zwitterionic detergent. 5. The method according to claim 1, wherein said pooled fractions from said ion exchange chromatography containing said Cβ protein or fragments and / or variants thereof are from about 10% to about 20% prior to ligand affinity chromatography.
2. The process of claim 1, wherein the process is diluted about 3-fold with a buffer containing about 10% zwitterionic surfactant. 6. The method according to claim 1, wherein the Cβ protein or a fragment and / or variant thereof is eluted from the medium by applying an eluent comprising a salt gradient during the ion exchange chromatography or ligand affinity chromatography. Item 2. The process according to Item 1. 7. The process of claim 6, wherein said eluent comprises about 0.5% zwitterionic surfactant. 8. The Cβ protein or fragment and / or variant thereof is obtained from a bacterial cell, wherein the bacterial cell has been transfected with a nucleotide sequence encoding the Cβ protein or a fragment and / or variant thereof. The process of claim 1, wherein the cells overexpress the Cβ protein or fragments and / or variants thereof. 9. The process of claim 8, wherein the Cβ protein and / or a fragment or variant thereof comprises: (a) destroying the cell; (b) about 20% ( v / v) ethanol / about the non-proteinaceous material by adding ethanol / CaCl ₂ to a concentration of 0.1 M CaCl ₂ step are precipitated from the cell; by removing the (c) non-proteinaceous agent that the precipitate (D) precipitating the protein from the solution by adding ethanol to a concentration of about 80% (v / v) and collecting the precipitated protein; and (e) about 1%. Resuspending the precipitated protein in a buffer solution containing from about 10% to about 10% of a zwitterionic detergent. 10. The process of claim 1, wherein the Cβ protein is obtained from a bacterial cell that naturally produces the Cβ protein. 11. The process of claim 10, wherein said Cβ protein
(A) said fine particles in a buffer containing from about 1% to about 10% of a zwitterionic surfactant;
Boiling the bacterial cells to obtain a solution; (b) cooling the solution in an ice bath; (c) ethanol / CaCl_TwoFrom the cells by adding a cold solution of
Precipitate the non-proteinaceous material and add about 20% ethanol / about 0.1 M CaCl _Two (D) removing the precipitated non-proteinaceous material to obtain a solution; and (e) adding ethanol to a concentration of about 80% (v / v).
Precipitating the protein from the solution and collecting the precipitated protein;
And (f) in a buffer solution containing about 1% to about 10% zwitterionic surfactant.
Resuspending the precipitated protein. 12. An isolated nucleic acid molecule encoding a protein fragment or peptide comprising a proline-rich region, wherein at least every third residue is proline and wherein said protein fragment or peptide An isolated nucleic acid molecule wherein the antibodies raised against are bactericidal against Gram-positive bacteria having complement alone. 13. The protein fragment or peptide of the formula-[P
_{-Y 1 -Y 2 -P-Y 1} -Y 2] r - or - [Y ₁ -Y include _{2 -P-Y 1 -Y 2 -P} ] successive having _r (repeat) the amino acid sequence, wherein The isolated nucleic acid molecule of claim 12, wherein Y ₁ represents either an acidic or basic residue, Y ₂ represents a neutral amino acid, and r is an integer from 1 to 5. . 14. The protein fragment or peptide of the formula-[P
_{-D-Y 3 -P-K-} L] r - or - [K-L-P- D-Y 3 -P] r - successive having include (repeated) amino acid, wherein Y ₃ is V or 13. The isolated nucleic acid molecule of claim 12, wherein A represents and r is an integer from 1 to 5. 15. The protein fragment or peptide of the formula-[S
_{-P-K-Y 4 -P-} E-A-P-Y 5 -V-P-E] r - successive having include (repeated) amino acid sequence, wherein Y ₄ represents T or A, Y ₅ represents H or R, and r is an integer from 1 to 5, an isolated nucleic acid molecule of claim 12. 16. An isolated nucleic acid molecule encoding a protein fragment or peptide having the formula XYZ, wherein X is the amino acid sequence of FIG. 1 (SEQ ID NO: 2).
) Represents at least 8 consecutive amino acid residues between amino acids 827 and 1028, wherein Y is hydrogen bonded to X or the N-terminal amino acid sequence of Figure 1 (SEQ ID NO: 2) or an N-terminal fragment thereof and / or Or a variant, and Z represents hydrogen bonded to X or the C-terminal amino acid sequence of FIG. 1 (SEQ ID NO: 2) or a C-terminal fragment and / or variant thereof, wherein the protein fragment or peptide Does not bind to the Fc region of human IgA immunoglobulin, provided that the protein is at least one of an N-terminal fragment or a C-terminal fragment of the amino acid sequence of FIG. 1 (SEQ ID NO: 2), provided that the protein fragment or The peptide is approximately 38 kD secreted by group B streptococcus strain HG806. Rather than Ripepuchido further proviso, FIG. 1 (SEQ ID NO: 2
3.) An isolated nucleic acid molecule wherein at least one of amino acids 1-164 of claim 1) is non-wild-type when present in Y. 17. The nucleic acid molecule of claim 16, wherein said Y does not include at least amino acids 1-176 of SEQ ID NO: 2. 18. The nucleic acid molecule of claim 16, wherein said Z comprises at least amino acid 901 of SEQ ID NO: 2. 19. The method according to claim 19, wherein said X is: 17. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule is selected from the group consisting of: 20. The nucleic acid molecule of claim 16, wherein X represents any 8-21 contiguous amino acid residues between amino acids 828 and 1027 of SEQ ID NO: 2. 21. The nucleic acid molecule of claim 16, wherein X represents an amino acid sequence between amino acids 828 and 1027 of the sequence shown in SEQ ID NO: 2. 22. A vector comprising the polynucleotide molecule according to claim 12 or 16. 23. A host cell transformed with the vector according to claim 22. 24. A protein fragment or peptide encoded by the nucleic acid molecule according to claim 12 or 16. 25. A protein fragment-polysaccharide conjugate or peptide-polysaccharide conjugate comprising the protein fragment or peptide of claim 24. 26. A vaccine comprising at least one protein fragment or peptide according to claim 24 together with a pharmaceutically acceptable carrier. 27. The protein fragment or peptide is conjugated to a polysaccharide selected from the group consisting of group B streptococcal capsular polysaccharides type Ia, II, III and V. 28. The vaccine according to 26. 28. A combination vaccine comprising at least two protein fragment-polysaccharide conjugates or peptide-polysaccharide conjugates according to claim 27. 29. A method for inducing an immune response in a mammal, comprising a pharmaceutically acceptable carrier and an amount of the protein fragment or peptide of claim 24 sufficient to induce an immune response in the mammal. Administering a vaccine comprising at least one of the following.