Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6068—Other bacterial proteins, e.g. OMP

Definitions

• the invention relates to the fields of microbiology and vaccine technology, and concerns the development of a vaccine capable of conferring immunity to infection by group B Streptococcus.
• Streptococcus genus Bacteria of the Streptococcus genus have been implicated as causal agents of disease in humans and animals.
• the Streptococci have been divided into immunological groups based upon the presence of specific carbohydrate antigens on their cell surfaces. At present, groups A through O are recognized (Davis, B.D. et al, In: Microbiology, 3rd. Edition, page 609,
• Streptococci are among the most common and impor ⁇ tant bacteria causing human disease. Although Streptococci of the B group are associated with animal disease (such as mastitis in cattle), Streptococcus agalactiae (a group B Streptococci) has emerged as the most common cause of human neonatal sepsis in the United States and is thought to be responsible for over 6000 deaths annually (Hill, H.R. et al, Sexually Transmitted Diseases, McGraw Hill, pp. 397-407).
• Group B Streptococcus is also an important pathogen in late-onset meningitis in infants, in postpartum endometritis, and in infections in immunocompromised adults (Patterson, M.J. et al., Bact. Rev. 40:114-192 (1976)). Although the organism is sensitive to antibiotics, the high attack rate and rapid onset of sepsis in neonates and meningitis in infants results in both high morbidity (50%) and mortality (20%) (Baker, C.J. et al., New Eng. J. Med. (Editorial) 314(26): 1702-1704 (1986);
• Group B Streptococcus is a common component of normal human vaginal and colonic flora. While the most common route of neonatal infection is intrapartum from vaginal colonization, nosocomial spread in newborn nurseries has also been described (Patterson, M.J. et al., Bact. Rev. 40:114-
• Intrapartum chemoprophylaxis has not gained wide acceptance for the following reasons: (1) It has not been possible to identify maternal colonization by group B Streptococcus in a fast, reliable and cost-effective manner; (2) About 40% of neonatal cases occur in low-risk settings; (3) It has not been considered practical to screen and/or treat all mothers or infants who are potentially at risk; and (4) antibiotic prophylaxis has not appeared to be feasible in preventing late-onset meningitis (7200 cases per year in the United States) or postpartum endometritis (45,000 cases annually) (Baker, C.J. et al., New Eng. J. Med. (Editorial) 314: 1102-1104 (1986)).
• Plasmids pJMSl and pJMS23 are derivatives of plasmid pUX12 which contain DNA capable of encoding antigenic Streptococci proteins that may be used in accordance with the present invention. Plasmid pUX12 is a derivative of plasmid pUC12. Plasmids pJMSl and pJMS23 were deposited on September 15, 1989, at the American Type Culture Collection, Rockville, MD. and given the designations ATCC 40659 and ATCC 40660, respectively. Summary of the Invention
• Streptococcus agalactiae is the most common cause of neonatal sepsis in the United States and is responsible for between 6,000 and 10,000 deaths per year. While the type-specific polysaccharide capsule of group B Streptococcus is immunogenic and carries important protective antigens, clinical trials of a polysaccharide vaccine have shown a poor response rate (Baker, C.J. et al., New Engl. J. Med. 379: 1180 (1980); Hans, R.A, et al, New Eng. J. Med. (Editorial) 379(18): 1219-1220 (1988)).
• the present invention concerns the development of a conjugate vaccine to group B Streptococcus, (i.e. Streptococcus agalactiae) that utilizes to a protective protein antigen expressed from a gene cloned from group B Streptococcus.
• group B Streptococcus i.e. Streptococcus agalactiae
• This novel conjugate vaccine has the advantages both of eliciting T-cell dependent protection via the adjuvant action of the carrier protein and also providing additional protective epitopes that are present on the cloned group B Streptococcus protein (Insel, R.A, et al, New Eng. J. Med.
• the invention provides a conjugate vaccine capable of conferring host immunity to an infection by group B Streptococcus which comprises (a) a polysaccharide conjugated to (b) a protein; wherein both the polysaccharide and the protein are characteristic molecules of the group B Streptococcus, and wherein the protein is a derivative of the C protein alpha antigen that retains the ability to elicit protective antibodies against the group B Streptococcus.
• the invention also concerns a method for preventing or attenuating an infection caused by a group B Streptococcus which comprises administering to an individual, suspected of being at risk for such an infection, an effective amount of the conjugate vaccine of the invention, such that it provides host immunity against the infection.
• the invention further concerns a method for preventing or attenuating infection caused by a group B Streptococcus which comprises administering to a pregnant female an effective amount of a conjugate vaccine of the invention, such that it provides immunity to the infection to an unborn offspring of the female.
• the invention also provides a method for preventing or attenuating an infection caused by a group B Streptococcus which comprises administering to an individual suspected of being at risk for such an infection an effective amount of an antisera elicited from the exposure of a second individual to a conjugate vaccine of the invention, such that is provides host immunity to the infection.
• Figure 1 shows the modifications of pUC12 to create the plasmid pUX12.
• Figure 2 shows the restriction and transcriptional map of the plasmid pUX12.
• Figure 3 shows the modifications which were made to pUX12 in order to produce the + 1 reading frame plasmid pUX12 + 1 (A), and which produce the -1 reading frame plasmid pUX12-l (C).
• B shows a construction which is additionally capable of resulting in a -1 reading frame plasmid.
• Figure 4 shows the result of mouse protection studies employing rabbit antisera against SI and S23. Protection was observed in mice inoculated with anti-Si antisera (p ⁇ 0.002) or with anti-S23 antisera (p ⁇ 0.022). Due to the sample size used, this difference in the observed statistical significicance between the SI and S23 experiments is not significant. In the Figure, the mice surviving per total tested is reported as a fraction above each bar.
• Figure 5 shows the sequencing strategy and restriction endonuclease map of bca.
• the partial restriction endonuclease map encompasses the region of pJMS23 from an Nde I site to a Sry I site located at nucleotide 3594 for which the nucleotide sequence of bca and flanking region was determined.
• the open reading frame is illustrated by an open box.
• Transposon Tn5seql mutations serve to prime nucleotide sequencing in both directions from each of the insertions.
• the regions of sequence obtained from oligonucleotide primers (open arrows) and the nested deletions (closed arrows) are also shown.
• Restriction endonuclease cleavage sites are abbreviated as follows: A, Alu I; B, Bsm I; F, Fok I; H, Hindi; N, Nde I; S, Sty I. bp, base pairs.
• Figure 6 shows the nucleotide [SEQ ID NO: 14] and deduced amino acid sequences [SEQ ID NO: 15] of bca and the flanking regions.
• the DNA strand is shown 5' to 3', and nucleotides are listed on the upper line beginning 78 base pairs upstream from the open reading frame.
• the deduced amino acid sequence for the open reading frame is below the nucleic acid sequence.
• the G + C content of 40% and the codon usage are similar to other streptococcal genes (Hollingshead, S.K. et al, J. Biol Chem. 267: 1677-1686 (1986)).
• Highlighted features include the -10 (TATAAT) promoter consensus site, ribosomal binding site (RBS), signal sequence, repeat region 1, the C terminus, with the termination codon (TAA) at position 3161, and two regions of dyad symmetry that are potential transcriptional terminators.
• Figure 7 is represented by two panels (A and B) that show homologies to the putative signal sequences and C-terminal membrane anchor of the C protein alpha antigen, rewspectively.
• Panel 7A the N terminus of the C protein alpha antigen on the top line (sequence 1) [SEQ ID NO: 16] and is compared with the following Gram-positive signal sequences (accession codes are listed for each of the sequence numbers): sequence 2 [SEQ ID NO: 17], the C protein beta antigen (S 15330; STRBAGBA) and four M proteins of group A Streptococcus; sequence 3 [SEQ ID NO: 18], ennX (STRENNX); sequence 4 [SEQ ID NO: 19], emm24 (STREMM24); sequence 5 [SEQ ID NO: 20], M l (S00767); sequence 6 [SEQ ID NO: 21], S01260.
• Lysine (K) and arginine (R) residues preceding the underlined hydrophobic stretch are in boldface type, as are se ⁇ ne (S) and threonine (T) residues preceding the probable signal cleavage sites.
• the probable cleavage site for the alpha signal is following the valine at position 41; however, alternative cleavage sites exist at positions 53-56.
• Panel B The C terminus of the C protein alpha antigen is shown on the top line (sequence 1) [SEQ ID NO: 22] and compared with the following Gram-positive membrane anchor peptides: sequence 2 [SEQ ID NO:
• M24, emml2, emm49, and ennX are all M proteins; arp4 is a binding protein of group A Streptococcus. S00128, STRPROTG, spg, and A26314 are IgG binding proteins of group G Streptococcus. Sequence 8 [SEQ ID NO: 29] illustrates the membrane anchor for the beta antigen, which lacks the PPFFXXAA [SEQ ID NO: 1] motif. Highlighed areas include lysine residues
• FIG. 8 shows a comparison of the cloned and native gene products of bca.
• Surface proteins of the A909 strain of group B Streptococcus (type la/C) and C protein alpha antigen clone pJMS23-l were analyzed by SDS/PAGE and Western blotting and were probed with the alpha antigen- specific monoclonal antibody 4G8. Arrowheads illustrate an example of the difference between proteins. Molecular mass markers (in kDa) are shown on the right.
• Figure 9 shows a schematic of the open reading frame of bca.
• Maternal immunoprophylaxis with a vaccine to group B Streptococcus has been proposed as a potential route for protecting against infection both in the mother and in the young infant through the peripartum transfer of antibodies (Baker, C.J. et al, New Eng. J. Med. (Editorial) 314(26): 1702- 1704 (1986); Baker, C.J. et al, New Eng. J. Med. 379: 1180 (1988); Baker, C.J. et al, J. Infect. Dis. 7:458 (1985)).
• susceptibility to infection correlates with the absence of type-specific antibody (Kasper, D.L., et al, J. Clin. Invest.
• T-cell independent functions of the host's immune system are often required for mounting an antibody response to polysaccharide antigens.
• the lack of a T-cell independent response to polysaccharide antigens may be responsible for the low levels of antibody against group B Streptococcus present in mothers whose children subsequently develop an infection with group B Streptococcus.
• children prior to 18 or 24 months of age have a poorly developed immune response to T-cell independent antigens.
• Wilkinson defined a fifth serotype. Ic, by the identification of a protein antigen (originally called the Ibc protein) present on all strains of serotype lb and some strains with the type la capsule (Wilkinson, H.W, et al, J. Bacteriol 97:629-634 (1969), Wilkinson, H.W, et al, Infec. and Immun. 4:596-604 (1971)). This protein was later found to vary in prevalence between the different serotypes of group B Streptococcus but was absent in serotype la (Johnson, D.R, et al, J. Clin. Microbiol. 79:506-510 (1984)).
• Controversy has existed regarding the structural arrangement of the type-specific and group B streptococcal polysaccharides on the cell surface, on the immunologically important determinants with in the type-specific polysaccharide, and on the mechanisms of capsule determined virulence of group B Streptococcus (Kasper, D.L. et al, J. Infec. Dis. 753:407-415 (1986)).
• group B Streptococcus Korean, D.L. et al, J. Infec. Dis. 753:407-415 (1986)
• Rubens et al. used transposon mutagenesis to create an isogeneic strain of type III group B Streptococcus that is unencapsulated (Rubens, C.E, et al, Proc. Natl. Acad. Sci.
• CAMP factor is an extracellular protein of group B Streptococcus with a molecule weight of 23,500 daltons that in the presence of staphylococcal beta-toxin (a sphingomyelinase) leads to the lysis of erythrocyte membranes.
• staphylococcal beta-toxin a sphingomyelinase
• the C protein(s) are a group of a cell surface associated protein antigens of group B Streptococcus that were originally extracted from group
• Cleat et al. attempted to clone the C proteins by using two preparations of antisera to group B Streptococcus obtained from Bevanger ( and ⁇ ) to screen a library of group B Streptococcus DNA in E. coli (Bevanger, L. et al, Acta Path. Microbiol. Immunol. Scand. Sect. B. 93: 113-119 (1985), Cleat, P.H, et al, Infec. and Immun. 55(5): 1151-1155 (1987), which references are incorporated herein by reference). These investigators described two clones that produce proteins that bind to anti strep tococcal antibodies.
• the present invention surmounts the above-discussed deficiencies of prior vaccines to group B Streptococcus through the development of a conjugate vaccine in which the capsular polysaccharides are covalently linked to a protein backbone.
• This approach supports the development of a T-cell dependent antibody response to the capsular polysaccharide antigens and circumvents the T-cell independent requirements for antibody production (Baker, C.J, et al, Rev. of Infec. Dis. 7:458-467 (1985), Kasper, D.L. et al,
• an antigenic molecule such as the capsular polysaccharides of group B Streptococcus (discussed above) is covalently linked to a "carrier” protein or polypeptide.
• the linkage serves to increase the antigenicity of the conjugated molecule.
• the protein backbones for conjugate vaccines such as the Hemophilus influenzae vaccine have utilized proteins that do not share antigenic properties with the target organism from which the bacterial capsular polysaccharides were obtained (Ward, J. et al, In: Vaccines, Plotkin, S.A, et al, eds,
• conjugate vaccine of the present invention employs im- munogenic proteins of group B Streptococcus as the backbone for a conjugate vaccine. Such an approach is believed to lead to more effective vaccines
• the conjugate, protein-polysaccharide vaccine of the present invention is the first to specifically characterize group B Streptococcus proteins that may be used in a conjugate vaccine. Any protein which is characteristic of group B
• Streptococcus may be employed as the protein in the conjugate vaccines of the present invention. It is, however, prefered to employ a C protein of a group
• plasmids pJMSl and pJMS23 contain DNA which encode Streptococcus C protein.
• the most preferred C proteins are those obtained upon the expression of such
• the present invention concerns the cloning and expression of genes which encode the protective group B Streptococcus protein antigens.
• proteins are preferably used as the protein backbone to which the one or more of the polysaccharides of the group B Streptococcus can be conjugated in order to form a conjugate vaccine against these bacteria.
• one or more proteins as described herein may be conjugated to the structure of a polysaccharide of the group B Streptococcus.
• the present invention thus concerns the cloning of the C proteins of group B Streptococcus, their role in virulence and immunity, and their ability to serve as an immunogen for a conjugate vaccine against group B Streptococcus.
• the present invention acomplishes the cloning of the C proteins (and of any other proteins which are involved in the virulence of the group B Streptococcus, or which affect host immunity to the group B Streptococcus) through the use of a novel plasmid vector.
• a novel plasmid vector For this purpose, it is desirable to employ a cloning vector that could be rapidly screened for expression of proteins which bind to naturally elicited antibodies to group B Streptococcus. Since such antibodies are heterologous polyclonal antibodies and not monoclonal antibodies, it was necessary that a vector be employed which could be easily screened through many positive clones to identify genes of interest.
• the present invention provides a plasmid vector which was developed for screening cloned bacterial chromosomal DNA for the expression of proteins involved in virulence and/or immunity.
• the present invention thus further concerns the development and use of an efficient cloning vector that can be rapidly screened for expression of proteins which bind to naturally elicited antibodies to group B Streptococcus.
• the vector was prepared by modifying the commonly used plasmid cloning vector, pUC12 (Messing, J, et al, Gene 79:269-276 (1982); Norrander, J, et al, Gene 26: 101-106 (1983); Vieira, J, et al, Gene 79:259-268 (1982); which references are incorporated herein by reference).
• the invention concerns the vector described below, and its functional equivalents. Using this system, plasmid clones can be easily manipulated, mapped with restriction endonucleases and their DNA inserts sequences, probes prepared and gene products studied without the necessity for subcloning.
• pUC12 is a 2.73 kilobase (kb) high copy number plasmid that carries a ColEl origin of replication, ampicillin resistance and a polylinker in the lacZ gene
• Step 1 In order to provide a site for the insertion of foreign DNA with a high efficiency and to minimize the possibility for self-ligation of the plasmid, inverted, non-cohesive BstXl ends were added to the polylinker.
• pUC12 was first cut with BamHI (Step 1) and the plasmid was mixed with two synthetic oligonucleotide adaptors that are partially complementary: a 15-mer (GATCCATTGTGCTGG) [SEQ ID NO: 3] and an 11-mer (GTAACACGACC) [SEQ ID NO: 4] (Step 2).
• the adaptors When the adaptors are ligated into pUC12, two new Bstl sites are created but the original BamHI sites are also restored (Step 3).
• the plasmid was then treated with polynucleotide kinase and ligated to form a closed circular plasmid (Step 4).
• a second modification in the polylinker was done to allow for the purification of the linear plasmid for cloning without contamination from partially cut plasmid that can self-ligate.
• a blunt end, 365 base pair (bp) was used to allow for the purification of the linear plasmid for cloning without contamination from partially cut plasmid that can self-ligate.
• FnuD2 fragment was obtained from the plasmid pCDM.
• This cassette or "stuffer” fragment which does not contain a BstXl site, was blunt end ligated to two synthetic oligonucleotides that are partially complementary: a 12-mer (ACACGAGATTTC) [SEQ ID NO: 5] and an 8-mer (CTCTAAAG) (Step 6).
• the resulting fragment with adaptors has 4 bp overhangs (ACAC) that are complementary to the ends of the modified pUC12 plasmid shown in Step 5.
• the modified pUC12 plasmid was ligated to the pCDM insert with adaptors; the resulting construct, named pUX12, is shown in Figure 2.
• the pUX12 plasmid can be recreated from plasmids pJMSl or pJMS23 by excision of the introduced Streptococcus DNA sequences. Alternatively, it may be formed by recombinant methods (or by homologous recombination), using plasmid pUC12.
• pUX12 is to be used as an expression vector, it is preferable to further modified the polylinker such that it will contain all three potential reading frames for the ]ac promoter. These changes allow for the correct translational reading frame for cloned gene fragments with a frequency of one in six. For example, a cloned fragment can insert in the vector in one of two orientations and one of three reading frames.
• the pUX12 plasmid was cut with the restriction enzyme EcoRI which cleaves at a unique site in the polylinker. The single stranded 5' sticky ends were filled in using the 5'-3' polymerase activity of T4 DNA polymerase, and the two blunt ends ligated. This resulted in the loss of the EcoRI site, and the creation of a new Xmnl site ( Figure 3A). This construction was confirmed by demonstrating the loss of the EcoRI site and confirming the presence of a new
• the pUX12 vector was cut with the restriction enzyme Sacl which cuts at a unique site in the polylinker of pUX12.
• Sacl The single stranded 3' sticky ends were cut back to blunt ends using the 3 '-5' exonuclease activity of T4 polymerase, and the resulting blunt ends ligated.
• the resulting sequence should eliminate the Sacl site while resulting in a new FnuD2 site ( Figure 3B).
• restriction mapping of the pUX12-l plasmids showed that while the Sacl site was absent, there was no FnuD2 site present.
• the Smal/Xmal sites on the polylinker were no longer present.
• pUX12 vectors in the cloning of antigenic proteins of group B Streptococcus are discussed in detail in the Examples below.
• DNA derived from group B Streptococcus, or complementary to such DNA is introduced into the pUX12, pUX12+ 1 or pUX12-l vectors and transformed into Escherichia coli.
• the cloned DNA is expressed in E. coli and the cellular lysate is tested to determine whether it contains any protein capable of binding to antisera to group B Streptococcus.
• the lac promoter could be replaced by another promoter
• the origin of replication could be modified to produce a lower copy number vector
• the drug resistance marker could be changed.
• Any vector capable of providing the desired genetic information to the desired host cell may be used to provide genetic sequences encoding the alpha antigen derivatives of the invention to a host cell.
• such vectors include linear DNA, cosmids, transposons, and phage.
• the host cell is not limited to E. coli. Any bacterial or yeast (such as S. cerevisiae) host that is capable of expressing the derivatives of the invention may be used as an appropriate host.
• Any bacterial or yeast (such as S. cerevisiae) host that is capable of expressing the derivatives of the invention may be used as an appropriate host.
• B. subtilis and the group B Streptococcus may be used as hosts. Methods for cloning and into such hosts are known.
• the present invention concerns a vaccine comprising a polysaccharide (such as the capsular polysaccharide) which is characteristic of the group B Streptococcus conjugated to a protein which is also characteristic of the group B Streptococcus.
• a polysaccharide such as the capsular polysaccharide
• the "polysaccharide” and “protein” of such a conjugated vaccine may be identical to a molecule which is characteristic of the group B Streptococcus, or they may be functional derivatives of such molecules.
• a group B Streptococcus polysaccharide is any group B-specific or type-specific polysaccharide.
• such polysaccharide is one which, when introduced into a mammal
• any protein which when introduced into a mammal (either animal or human) either elicits antibodies which are capable of reacting a protein expressed by group B Streptococcus, or which increases the immunogenicity of a polysaccharide to elicit antibodies to a polysaccharide of the group B Streptococcus may be employed.
• the preferred proteins of the present invention include the C proteins of the group B Streptococcus, or their equivalents.
• Examples of functional derivatives of the peptide antigens include fragments of a natural protein, such as N-terminal fragment, C-terminal fragment or internal sequence fragments of the group B Streptococcus C protein alpha antigen that retain their ability to elicit protective antibodies against the group B Streptococcus.
• the term functional derivatives is also intended to include variants of a natural protein (such as proteins having changes in amino acid sequence but which retain the ability to elicit an immunogenic, virulence or antigenic property as exhibited by the natural mole ⁇ cule), for example, the variants of the alpha antigen recited below that possess fewer of the internal repeats than does the native alpha antigen, and/or an altered flanking sequence.
• the peptide antigen that is conjugated to the polysaccharide in the vaccine of the invention may be a peptide encoding the native amino acid sequence of the alpha antigen, as encoded on plasmid pJMS23 (with or without the signal peptide sequence) or it may be a functional derivative of the native sequence.
• the native group B Streptococcus C protein alpha antigen as encoded on pJMS23 contains an open reading frame of 3060 nucleotides and encodes a precursor protein of 108,705 daltons. Cleavage of the putative signal sequence of 41 amino aicds yields a mature protein of 104, 106 daltons.
• the 20,417 dalton N-terminal region of the alpha antigen shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein.
• Each repeating unit (denoted herein as "R") is identical and consists of 82 amino acids with a molecular mass of 8665 daltons, which is encoded by 246 nucleotides.
• the size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins naturally expressed by the group B Streptococcus.
• the C-terminal region of the alpha antigen contains a membrane anchor deomain motif that is hared by a number of Gram-positive surface proteins.
• the large region of identical repeating units in this gene (termed the bca gene, for group B Streptococcus, C protein, alpha antigen) defines protective eoptopes and may be used to generate diversity of alpha antigen functional derivatives that are useful in the vaccines of the invention.
• the sequence of such a functional alpha antigen derivative contains 1-9 copies of the 82 amino acid repeat (246 nucleotides) that begin at amino acid 679 of the DNA sequence of Figure 6, (as used herein, the partial repeat designed as repeat 9' therein is also useful in this regard).
• the functional derivative may lack the 185 amino acid 5' flanking sequence (555 nucleotides) that is found in the native protein prior to the repeating sequence or it may retain this sequence and/or the derivative may lack the 48 amino acid (246 nucleotides) C-terminal anchor sequence or it may retain this sequence.
• the functional derivative may be the N-terminal fragment that precedes the start of the alpha antigen repeating unit(s) or the functional derivative may be only the C-terminal fragment that follows the end of the alph antigen repeating unit(s) or the function derivative may be a hybrid of the N-terminal fragment and C-terminal fragment with no copies of the "R" units as defined below.
• the amino terminal sequence of the native alpha antigen may or may not contain the signal sequence. Either of the alpha antigen's amino terminal sequence or carboxy terminal sequence may be used in the conjugate vaccines of the invention, with or without one or more copies of the sequence that is repeated in the core of the native alpha antigen protein.
• R represents one copy of the 82 amino acid repeat that beings at amino acid 679 of the alpha antigen DNA sequence of Figure 6,
• R x represents "X” number of tandem copies of this repeat, tandemly joined at the carboxyl end of one R unit to the amino terminal end of the adjoining R unit,
• N represents the 5' amino acid flanking sequence that is found in the sequence shown on figure 6, with or without the signal sequence; when the signal sequence is lacking, "N” is a 185 amino acid 5' flanking sequence that is found in the native protein as shown on Figure 6; when the signal sequence is present, “N” is a 226 amino acid 5' flanking sequence as shown in Figure 6.
• C represents the 48 amino acid C-terminal anchor sequence as shown on Figure 6.
• R units including, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 R units, may be constructed in a similar manner.
• fragments of R, N, or C may be used if such fragments enhance the functional ability of the derivative to elicit protective antibodies against the group B Streptococcus, or if such fragment provides another desired property to the construct, such as a secretion signal or membrane localization signal.
• Alpha antigens from other strains of the group B Streptococcus may be prepared and used in a similar manner as a slight variability in the sequence of the protein, such as in the N terminus or C terminus or R repeat would not alter the biological properties and their functional ability to elicit protective antibodies.
• a group B Streptococcus alpha antigen isolated from a different strain of the group B Streotococcus and having the same repeat unit but a different N-terminal amino acid sequence is intended to be within the scope of the invention.
• the peptides of the invention are conjugated to a group B Streptococcus carbohydrate moiety by any means that retains the ability of these proteins to induce protective antibodies against the group B Streptococcus.
• Heterogeneity in the vaccine may be provided by mixing specific conjugated species.
• the vaccine preparation may contain one or more copies of one of the peptide forms conjugated to the carbohydrate, or the vaccine preparation may be prepared to contain more than one form of the above functional derivatives and/or the native sequence, each conjugated to a polysaccharide used therein.
• Conjugates providing a peptide can be mixed with conjugates providing any other peptide (such as a second example from group numbers 1-43) to arrive at a "compound" conjugate vaccine.
• a multivalent vaccine may also be prepared by mixing the group B-specific conjugates as prepared above with other proteins, such as diphtheria toxin or tetanus toxin, and/or other polysaccharides, using techniques known in the art.
• Heterogeneity in the vaccine may also be provided by utilizing group
• B Streptococcal preparations from group B Streptococcal hosts that have been transformed with the recombinant constructs of the invention such that the streptoccal host expresses the alph antigen protein or functional derivative thereof.
• homologous recombination between the genetic sequences encoding the repeating R units will result in spontaneous mutation of the host, such that a population of hosts is easily generated and such hosts express a wide range of antigenic alpha antigen functional derivatives useful in the vaccines of the invention.
• spontaneous mutation usually results in the deletion of R units, or portions thereof, although mutation of other regions of the alpha antigen may also occur.
• a polysaccharide or protein is "characteristic" of a bacteria if it is substantially similar in structure or sequence to a molecule naturally associateed with the bacteria.
• the term is intended to include both molecules which are specific to the organism, as well as molecules which, though present on other organisms, are involved in the virulence or antigenicity of the bacteria in a human or animal host.
• the vaccine of the present invention may confer resistance to group B Streptococcus by either passive immunization or active immunization.
• the vaccine is provided to a host (i.e. a human or mammal) volunteer, and the elicited antisera is recovered and directly provided to a recipient suspected of having an infection caused by a group B Streptococcus.
• antibodies, or fragments of antibodies, with toxin labels provides an additional method for treating group B Streptococcus infections when this type of passive immunization is conducted.
• antibodies, or fragments of antibodies which are capable of recognizing the group B Streptococcus antigens are labeled with toxin molecules prior to their administration to the patient.
• toxin molecules When such a toxin derivatized molecule binds to a group B Streptococcus cell, the toxin moiety will cause the death of the cell.
• the vaccine is provided to a female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of antisera which serve to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta).
• the present invention thus concerns and provides a means for preventing or attenuating infection by group B Streptococcus, or by organisms which have antigens that can be recognized and bound by antisera to the polysaccharide and/or protein of the conjugated vaccine.
• a vaccine is said to prevent or attenuate a disease if its administration to an individual results either in the total or partial attenuation (i.e. suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease.
• the administration of the vaccine may be for either a "prophylactic" or "therapeutic" purpose.
• the compound(s) are provided in advance of any symptom of group B Streptococcus infection.
• the prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent infection.
• the compound(s) is provided upon the detection of a symptom of actual infection.
• the therapeutic administration of the compound(s) serves to attenuate any actual infection.
• the anti-inflammatory agents of the present invention may, thus, be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.
• a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
• Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
• An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
• the vaccine of the present invention when it is provided to an individual, it may be in a composition which may contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition.
• Adjuvants are substances that can be used to specifically augment a specific immune response. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized. Adjuvants can be loosely divided into several groups based upon their composition.
• These groups include oil adjuvants (for example, Freund's complete and incomplete), mineral salts (for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucella.
• saponins such as, for example, Quil A. (Superfos A/S, Denmark). Examples of materials suitable for use in vaccine compositions are provided in Remington 's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, PA, pp. 1324-1341 (1980), which reference is incorporated herein by reference).
• compositions of the present invention can be administered parenterally by injection, rapid infusion, nasopharyngeal absorption . (intranasopharangeally), dermoabsorption, or orally.
• the compositions may alternatively be administered intramuscularly, or intravenously.
• Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
• Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form.
• Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water.
• inert diluents such as purified water.
• such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.
• compositions of the invention are used more than once to increase the levels and diversities of expression of the immunoglobulin repertoire expressed by the immunized animal. Typically, if multiple immunizations are given, they will be given one to two months apart. According to the present invention, an "effective amount" of a therapeutic composition is one which is sufficient to achieve a desired biological effect. Generally, the dosage needed to provide an effective amount of the composition will vary depending upon such factors as the animal's or human's age, condition, sex, and extent of disease, if any, and other variables which can be adjusted by one of ordinary skill in the art.
• the antigenic preparations of the invention can be administered by either single or multiple dosages of an effective amount.
• Effective amounts of the compositions of the invention can vary from 0.01-1 ,000 ⁇ g/ml per dose, more preferably 0.1-500 ⁇ g/ml per dose, and most preferably 10-300 ⁇ g/ml per dose.
• DNA with signal stranded ends can be modified to create blunt ends.
• the blunt ends of the DNA fragments were mixed with the two synthetic oligonucleotide adaptors. These are the same 12-mer and 8-mer used in preparing the stuffer fragment.
• the modified DNA fragments were separated from the unincorporated synthetic oligonucleotides on a potassium acetate gradient. These fragments were then ligated into the linear pUX12 family of plasmids and used to transform E. coli.
• pUX12 has a ⁇ -lactamase gene and carriers resistance to ampicillin (amp R ).
• amp R resistance to ampicillin
• tetracycline resistance gene (tet R ) from the plasmid pBR322 was cloned into the polylinker of pUX12 with the adaptors described above.
• a group of test ligations were run to establish the optimal concentration of oligonucleotide adaptor to fragment ends, and the ratio of modified insert to linear pUX12 plasmid for ligation and transformation.
• tet* gene as a marker, we were able to determine cloning parameters so that greater than 99% of the transformants selected on ampicillin containing plates also carried the ter marker.
• the frequency of self-ligation is very low in this system and it is not necessary to screen for the presence of an insert in the polylinker prior to screening a library in pUX12.
• the Apal site is within the structural gene near the N-terminal and the Hindlll site lies just outside of the C-terminal of the tox gene. This 1.2 kb restriction fragment was separated from the remaining 4.1 kb of the pABC402 vector using low melting point agarose.
• the tox fragment was treated with T4 DNA polymerase.
• the exonuclease activity of the polymerase cut back the
• extracts from these clones were screened using Western blots probed with antisera to diphtheria toxin (Blake, M.S., et al, Anal Biochem. 736:175-179 (1984), Murphy, J.R., et al, Curr. Topics Microbiol. and Immun. 778:235-251 (1985)).
• Reactive toxin related proteins were only detected from clones that contained the structural gene in the correct orientation and reading frame.
• This plasmid is called pUDTAH-1; the DNA sequence of the polylinker and beginning of the tox structural gene is shown in Table 1.
• the depisted sequence is the DNA sequence of the beginning of the tox ' ' structural gene in pUDTAH-1.
• ATG is the start signal for the transcript (lacT)
• GAT begins the modified polylinker of pUX12 and GCC starts the correct translational reading frame for the tax' gene.
• chromosomal DNA was isolated from the A909 strain of group B Streptococcus (Lancefield, R.C., et al, J. Exp. Med. 742:165-179 (1975)) by the method of Hull et al. (Hull, R.A., et al, Infect, and Immun. 33:933- 938 (1981)) as modified by Rubens et al (Rubens, C.E., et al, Proc. Natl. Acad. Sci USA 84:1208-1212 (1987) both of which references are incorporated herein by reference).
• mutanolysin was used to convert the group B Streptococcus strain A909 (Ia/c) strain into protoplasts.
• the resulting surface extract was found to contain numerous proteins that immunoreact with protective antisera raised to the intact bacteria.
• An insoluble protein fraction was partially purified using conventional column chromatography. Two fractions, including one which was highly concentrated for a single 14 kilodalton (kd) species, were used to immunize rabbits. Antisera raised against these partially purified group B Streptococcus proteins were found to be able to confer passive protection in a mouse virulence assay against a heterologous capsule type of group B Streptococcus which carries the C proteins.
• Group B Streptococcus DNA was purified by centrifugation in a buoyant-density cesium chloride (CsCl) gradient, and the chromosomal DNA was dialyzed exhaustively against TAE buffer, pH 8.0 (Maniatis, T. et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982).
• the A909 strain of group B Streptococcus has a type 1 capsule, expresses the C proteins and has been used previously in studies of the C proteins (Valtonen, M.V., et al, Microb. Path. 7:191-204 (1986)). It is also the strain of group B Streptococcus that was used in preparing the protective antisera for screening.
• the yield of Group B Streptococcus chromosomal DNA averages 3 to 5 mg for each 500 ml of an overnight culture of group B Streptococcus.
• the purified DNA was digested separately with 24 commonly used restriction endonucleases and the resulting fragments were run on a 1.0% agarose gel.
• Ethidium bromide (EtBr) staining of the gel showed that all of the restriction enzymes yielded a distribution of discrete fragment sizes of group B Streptococcus DNA. This suggests that group B Streptococcus DNA is not modified for any of the restriction enzymes tested.
• Group B Streptococcus chromosomal DNA was partially digested individually with Alul, Fu ⁇ D2, Haelll and Rsal.
• the pUX12 plasmid containing all three translational reading frames was digested with BstXl and the stuffer fragment was removed using a low melting point agarose gel.
• the group B Streptococcus library was prepared by mixing 10 ng of the adapted group B Streptococcus fragments with 100 ng of the linear pUX12 vector in 100 ⁇ l of ligation buffer to which 0.1 % T4 DNA Iigase was added. The ligation reaction was maintained overnight at 14°C and then used to transform the MCI 061 strain of E. coli on plates containing ampicillin (Ausubel, F.M., et al, Current Topics in Molecular Biology (1987)).
• the library of group B Streptococcus chromosomal DNA in the pUX12 vector was screened with the above-discussed protective anti-C proteins antisera.
• the group B Streptococcus library had an average fragment size of 1.4 kb.
• Transformants were screened as described above, and then subcloned and rescreened with the antisera three times. Of 20,000 clones screened, there were 35 independently isolated clones that reacted with the protective antisera. The clones were denominated S1-S35, and the plasmids containing the clones were denominated pJMSl-pJMS35. The clones ranged in size from 0.9 to 13.7 kb and have an average size of 4.5 kb.
• Plasmid DNA was isolated from the clones by minipreps and the inserts surveyed with four restriction endonucleases. Fourteen of the clones can be divided into three groups based on sharing identical insert sizes and common restriction endonuclease mapping patterns within each group. Clones SI and S23, discussed below, were found to be members of different groups.
• Extracts of the clones were prepared, run on Western blots and probed with the antisera used in screening the library.
• Six size classes of protein antigens were identified (A-F).
• A-F The initial classification was done only to get a rough survey of the potential number of genes involved.
• SI was found to be 3.5 kd in size, and to belong to antigen protein pattern A.
• S23 was found to be 13.7 kd in size, and to belong to antigen protein pattern D.
• the typing antisera, and ⁇ were used to screen the cloned gene products on Western blots (Bevanger, L., etal, Acta Path. Microbiol. Scand. Sect. B. 87:51-54 (1979); Bevanger, L., et al, Acta. Path. Microbiol Scand. Sect. B. 89:205-209 (1981); Bevanger, L., et al, Acta. Path. Microbiol
• C proteins The most important and characteristic property of the C proteins is their ability to elicit protective antibodies against group B Streptococcus strains that express C proteins.
• Several approaches could be used to prepare antisera against the cloned gene products. For example, lysates of the E. coli clones could be directly injected into rabbits in order to determine if the lysates contain proteins capable of eliciting antibodies to any of the E. coli or group B Streptococcus proteins introduced. The resulting antisera can be preab- sorbed with a lysate of E. coli prior to testing the antisera to reduce the number of cross-reacting antibodies.
• Such a lysate can be used to reduce the number of cross-reacting antibodies in both colony blots used for screening the clones for expression and in Western blots used to study both cellular extracts of group B Streptococcus and partially purified group B Streptococcus proteins.
• the control rabbits are injected with E. coli that carries pUX12 without an insert in the polylinker.
• the antisera is preadsorbed with an E. coli lysate and screened first on Western blots against extracts of the clones in the library. Therefore, it is possible to determine if there are cross-reacting epitopes between the clones and to confirm that these antisera are directed against the cloned proteins identified during the preliminary round of screening.
• the preadsorbed antisera may be tested in the mouse protection model.
• the mice are injected intraperitoneally with rabbit antisera (Lancefield, R.C., et al, J. Exp. Med. 142: 165-119 (1975)).
• rabbit antisera Lirange, R.C., et al, J. Exp. Med. 142: 165-119 (1975)
• the following day they are again injected intraperitoneally with an LDgo of viable group B Streptococcus that are known to carry C proteins.
• the endpoint is the death of the mice over a 48 hour period.
• Escherichia coli cells containing pJMSl and pJMS23 were grown, and used to prepare cellular extracts.
• Streptococcus surface extract which ranged in MW from > 180 kd to 40 kd.
• a monoclonal antibody derived from the A909 extract showed this same repeating pattern of immunoreactivity. This indicates that a single epitope was recognized in different molecular weight proteins and suggests a regularly repeating structure.
• the proteins recognized by the SI antiserum were susceptible to pepsin and trypsin degradation whereas those recognized by the S23 antiserum were susceptible to pepsin but not to trypsin. This experiment shows that these proteins partially purified from group B Streptococcus and expressed from the group B Streptococcus cloned genes represent the alpha and beta antigens of the C protein of group B Streptococcus.
• the 35 potential C protein clones described above may be evaluated both genetically and immunologically to determine the number of genes that are present.
• the isolation of these clones permits the genes which confer protective immunity to group B Streptococcus infection may be identified. It is likely that the protective antisera used to obtain the initial clones also detected proteins other than the C proteins. The use of such other proteins in a therapy against Streptococcus B infection is also contemplated by the present invention. Since a major goal of the present invention is the isolation and identification of the proteins involved in immunity, antisera prepared against the proteins expressed by these clones may be studied in the mouse protection model. Those genes that express proteins that are protective are preferred proteins for a conjugate vaccine.
• a single colony of each of the clones is placed in a well of microtiter dish containing LB broth and grown at 37°C overnight.
• Control colonies include the host E. coli strain and the E. coli strain containing pUX12.
• the overnight cultures are transferred onto a nitrocellulose filter on an agar plate containing the same culture medium. These plates are grown up over 8 hours at 37°C and the nitrocellulose filter containing the freshly grown colonies is prepared to be screened for DNA-DNA hybridization.
• the probes are prepared from the group B Streptococcus DNA inserts in the pUX12 library. Mini-preps are used to obtain plasmid DNA from the clones.
• the polylinker in pUX12 has both a BamHI and BstXl site on either side of the insert; therefore, the group B Streptococcus insert is excised from the plasmid using either BamHI or BstXl.
• the chromosomal DNA of group B Streptococcus contains few BamHI sites and many of the inserts are removed from the vector in one fragment as the result of digestion with BamHI.
• Low melting point agarose is used to separate the plasmid vector from the inserts.
• the inserts will be cut from the agarose gel and directly labelled by random prime labelling.
• the labelled inserts are then used to probe the colony blots. This results in the identification of clones that share DNA sequences.
• the 35 clones are placed into groups that share DNA sequences. These groups are mapped with multiple restriction endonucleases to determine the relationship of each clone to the others within that region of the DNA. Since the host plasmid, pUX12, contains many unique restriction endonucleases sites that are present only in the polylinker, much of the restriction mapping can be done utilizing the plasmid mini-prep DNA without needing to purify the inserts separately. By combining the colony blot data with detailed restriction mapping it is possible to get a reasonable assessment of the number of genetic loci involved.
• antisera is preferably prepared against the cloned gene products, and utilized in the mouse protection model to determine the ability of these antisera to protect against infection with group B Streptococcus (Lancefield, R.C., et al, J. Exp. Med. 142: 165-119 (1975), Valtonen, M.V., et al, Microb. Path. 7: 191-204 (1986)).
• a clone whose expressed protein is able to elicit protective antibodies is a preferred candidate for use in a conjugate vaccine.
• Clones whose expressed protein fails to elicit protective antibodies may be further analyzed to determine whether they are also candidates for a vaccine. Since the C proteins are membrane associated, a failure of protein expressed by a clone to elicit protective antibodies may reflect the fact that the protein may not be stable in E. coli, and in a high copy number vector. This problem has occurred in cloning other membrane proteins from both group A and group B Streptococcus (Kehoe, M. et al , Kehoe, M., et al, Infect, and Immun. 43:804-810 (1984), Schneewind, O., et al, Infect, and Immun. 56:2174-2179
• coli host pcnB, that restricts the copy number of pBR322 derived plasmids like pUX12 (Lopilato J., et al, Mol. Gen. Genet. 205:285-290 (1986) which reference is incorporated herein by reference).
• a failure of a clone to express protein which elicits protective antibod ⁇ ies may also indicate that the expressed protein lacks an epitope which is important for protection. This could be the case if the entire gene was not cloned or could not be expressed in E. coli. It might also be problem if there is post-transcriptional processing of the C proteins in group B Streptococcus but not for the cloned C protein genes in E. coli. It might be necessary either to subclone out the complete gene and/or transfer it into an alternate host background where it can be expressed.
• a failure of a clone to express protein which elicits protective antibod- ies may also indicate that antibodies elicited from antigens produced in
• Escherichia coli may differ from those elicited from an animal by the native C proteins on group B Streptococcus.
• the lysed bacterial extracts used to immunize the rabbits contain a number of E. coli protein antigens. Therefore, it may be necessary to obtain antisera for testing in the animal model from partially purified gene products instead of from the entire organism.
• a fine structure genetic map of C protein gene clones described above may be prepared and their DNA sequence(s) determined. Such mapping is preferably accomplished utilizing genomic Southern blots. By determining the DNA sequences of the
• C protein genes one can determine the structure of the genes including their ribosomal binding sites, potential promoters, signal sequences, and any unusual repetitive sequences.
• the DNA sequences are preferably compared to a library of known DNA sequences to see if there is homology with other genes that have been characterized.
• C proteins can be determined from DNA sequences of their genes. It is often possible to make predictions about the structure, function and cellular location of a protein from the analysis of its protein sequence.
• Genomic Southern blots are, thus, preferably used to determine if any of the genes are linked.
• group B Streptococcus chromoso ⁇ mal DNA is digested individually with several different restriction endonucleases that identify sequences containing six or more base pairs. The purpose is to obtain larger segments of chromosomal DNA that may carry more than one gene.
• the individual endonuclease digestions are then run out on an agarose gel and transferred onto nitrocellulose.
• the Southern blots are then probed with the labelled inserts derived from the above-described library.
• the identification of the above-described clones permits their DNA sequences to be determined. If the clones are on the pUX12 plasmid, it is possible to use double stranded DNA sequencing with reverse transcriptase to sequence from oligonucleotide primers prepared to the polylinker. This technique was used earlier in characterizing the pUX12 plasmid and is a rapid way to sequence multiple additional oligonucleotide primers to sequence a gene that is larger than 600 base pairs. Therefore, the DNA sequencing for the C protein genes is preferably performed by subcloning into an M 13, single stranded DNA sequencing system (Ausubel, F.M., et al, Current Topics in Molecular Biology (1987)).
• the DNA sequences of the C proteins are preferably compared with a library of known DNA sequences.
• the amino acid sequences derived from the DNA sequences are compared with a library of known amino acid sequences.
• chromosomal DNA from clinical and laboratory isolates of the various serotypes of group B Streptococcus are probed on genomic Southern blots with the C protein genes.
• comparison of the phenotypic expression as determined by precipitin techniques with genetic composition as shown by DNA-DNA hybridization is preformed in order to provide information regarding the regulation of expression of the C protein genes.
• the probes of the C protein genes are used to screen chromosomal DNA from other types of
• Streptococcus and other bacterial pathogens.
• Probes are prepared and labelled from the C protein genes of isolates of group B Streptococcus which includes most of the original typing strains used by Lancefield (Lancefield, R.C., et al, J. Exp. Med. 142: 165-119 (1975)). Colony blots of the 24 clinical and laboratory isolates of group B
• Streptococcus are screened using the microtiter technique described above. The ability of the various strains to hybridize to the C protein genes is then compared with the phenotypic characteristics of these organisms in binding to typing antisera directed against the C proteins. In this manner, it is possible to determine what strains carry any or all of the C protein genes, and whether some strains carry silent or cryptic copies of these genes.
• Those strains that hybridize to the C protein gene probes on colony blots are then screened using genomic Southern blots to determine the size, structure and location of their C protein genes.
• Chromosomal DNA isolated from the strains of group B Streptococcus that show binding on the colony blots is digested with restriction endonucleases, run on an agarose gel and blotted onto nitrocellulose. These Southern blots are probed with probes of the C protein genes. In this manner, it is possible to determine if there are differences in the location and size of these genes in the different serotypes of group B Streptococcus and to compare clinical (i.e. potentially virulent) isolates with laboratory strains (and with those which colonize clinically but are not associated with infection).
• the C protein gene probes are also preferably used to screen other streptococcal strains and a variety of pathogenic bacteria. Streptococcal strains are known to share other proteins associated with virulence including the M and G proteins (Fahnestock, S.R., et al, J. Bact. 767(3):870-880 (1986), Heath, D.G. , et al, Infec. and Immun. 55: 1233-1238 (1987), Scott, J.R., et al, Infec. and Immun. 52:609-612 (1986), Walker, J.A., et al, Infec. and Immun. 55: 1184-1189 (1987) which references are incorporated herein by reference).
• the strains to be tested are first screened using colony blots to determine whether they have any homologous sequences with the C protein genes probes. Genomic Southern blots are then prepared with the chromosomal DNA of the bacterial strains that test positive on the colony blots. These blots are then probed with the C protein genes to localize and define the areas of homology, such as a region of a C protein which serves as a membrane anchor, binds to the Fc region of immunoglobulins, or shares regions of homology with other genes with similar functions in other bacteria.
• tranposon mutagenesis with the self-conjugative transposon tn916 may be employed.
• site-directed mutagenesis may be used.
• Streptococcus and create isogeneic strains for studying virulence (Lopilato, J., et al, Mol Gen. Genet. 205:285-290 (1986) which reference is incorporated herein by reference).
• Transposon insertional mutagenesis is a commonly used technique for constructing isogeneic strains that differ in the expression of antigens associated with virulence, and its use in group B Streptococcus is well described (Caparon, M.G., et al, Proc. Natl. Acad. Sci. USA 84:8677-8681 (1987), Rubens, C.E. , et al, Proc. Natl. Acad. Sci USA 84:1208-1212 (1987), Wanger, A.R, Res. Vet. Sci. 38:202-208 (1985), Weiser, J.N., Trans Assoc. Amer. Phys.
• Tn916 strains are selected for the acquisition of an antibiotic resistance marker, and screened on colony blots for the absence of expression of the C proteins as detected by the specific antisera prepared as described above. Isolates that do not appear to express the C proteins can be further mapped using genomic Southern blots to localize the insertion within the C protein genes.
• the original Tn916 strain carried tet R ; however, an erythromycin resistance marker has recently been cloned into Tn916 (Rubens, C.E., et al, Plasmid 20: 137-142 (1988)). It is necessary to show that, following mutagenesis with Tn916, only one copy of the transposon is carried by the mutant strain and that the transposon is localized within the
• site-directed mutagenesis may be employed (for example to produce conditional mutants). Site-directed mutagenesis may thus be used for the genetic analysis of group B Streptococcus proteins.
• One problem that has delayed the development of these techniques in group B Streptococcus is the difficulty encountered in transforming group B Streptococcus. Electroporation has proven valuable in introducing DNA into bacteria that are otherwise difficult to transform (Shigekawa, K., etal, BioTech. 6:742-751 (1988) which reference is incorporated herein by reference).
• Conditions for transforming group B Streptococcus utilizing electroporation may be utilized to surmount this obstacle. It is thus possible to do site directed mutagenesis, to evaluate complementation, and to introduce C protein genes into group B Streptococcus strains that do not express the C proteins. Any of several approaches may be utilized to insert native or mutated C protein genes into strains of group B Streptococcus. For example, a drug resistance marker may be inserted within the C protein gene clones in pUX12. A drug resistance marker that can be expressed in group B Streptococcus, but that is not normally present, is preferred.
• This modified pUX12 protein clone is transformed into group B Streptococcus using electroporation (Shigekawa, K., etal, BioTech 6:142-151 (1988) which reference is incorporated herein by reference). Since the pUX12 plasmid cannot replicate in group B Streptococcus, those strains that acquire the drug resistance phenotype would likely do so by homologous recombination between the C protein gene on the host GB chromosome and the mutated C protein carried on the pUX12 plasmid. The mutants are screened as described above.
• modified C protein genes could be introduced into the group B Streptococcus chromosome by inserting the genes into the self- conjugating transposon Tn916 and introducing the modified transposons via mating from Streptococcus faecalis .
• This technique was used to successfully modify Tn916 with an erythromycin gene and insert this gene into the chromosome of group B Streptococcus (Rubens, C.E., et al, Plasmid 20: 131- 142 (1988)). It is necessary to show that, following mutagenesis with Tn916, only one copy of the transposon is carried by the mutant strain and that the transposon is localized within the C protein gene.
• a second important test of virulence is the ability of a gene to restore virulence through reversion of allelic replacement in a mutant strain.
• Isogeneic strains of group B Streptococcus in which the C protein genes are individually mutated may be created using either transposon mutagenesis or site-directed mutagenesis. Such strains are preferably characterized on genomic Southern blots to determine that only a single insertion is present on the chromosome. The location of these insertions may be ascertained using the fine structure genetic mapping techniques discussed above. The isogeneic strains are then tested for virulence in the neonatal rat model (Zeligs, B.J., et al, Infec. and Immun. 37:255-263 (1982)).
• Transposon mutagenesis permits the identification of genes involved in regulating the expression of the C proteins. For example, strains carrying the wild type C protein genes which are found to no longer express C proteins following transposon mutagenesis and in which transposon is not located within the C protein structural gene, carry mutations in sequences involved in the regulation of expression of the C protein genes. This approach was used successfully in characterizing the mry locus in group A Streptococcus that is involved in regulation of the M protein (Caparon, M.G., et al, Proc. Natl.
• Such methods may also be used to produce strains which overexpresses the C proteins, or which produce C proteins of altered virulence or immunity.
• C protein is an outer membrane protein.
• C proteins can also be isolated from the supernatants of cultures of group B Streptococcus, indicating that these proteins may be either secreted by group B Streptococcus or lost at a high rate from the cell surface.
• DNA and protein sequences derived from the C protein genes are valuable in determining the structure and function of the C proteins.
• One potential virulence determinant commonly described for the C proteins is the ability to bind to the immunoglobulin, IgA (Ferrieri, P., et al, Rev. Inf. Dis. 70(2): 1004-1071 (1988), Russell-Jones, G.J., et al, J. Exp. Med. 760: 1467-
• Immuno-electron microscopy has been utilized to localize cell surface determinants that are detected by specific antibody.
• Antisera raised against the C protein clones of group B Streptococcus is incubated with group B Streptococcus strains that carry the C proteins.
• Ferritin-conjugated goat anti- rabbit IgG is used to detect the antigen on the cell surface as previously described (Rubens, C.E., et al, Proc. Natl. Acad. Sci. USA 84:1208-1212 (1987), Wagner B., et al, J. Gen. Microbiol 778:95-105 (1980)).
• a simple determination of the ability of C proteins to bind to immunoglobulins can be assessed using Western blots.
• Cellular extracts of both the E. coli clones containing the C protein genes and of group B Streptococcus strains that carry the C proteins can be run on SDS-PAGE and blotted onto nitrocellulose.
• Controls include extracts of E coli carrying the wild type pUX12 plasmid, strains of group B Streptococcus that do not carry the C protein genes, and isogeneic group B Streptococcus strains in which the C protein genes have been inactivated.
• the Western blots can be probed individually with labelled immunoglobulins, e.g., IgG, IgM, IgA, and their components, e.g. , the Fc or F(ab) 2 fragments (Heath, D.G., et al, Infect, and Immun. 55: 1233-1238 (1987), Russell-Jones, G.J., et al, J. Exp. Med. 760:1467-1475 (1984)).
• the immunoglobulins are preferably iodinated using either iodogen or chloramine T.
• the above-described protective C protein antigens of group B Streptococcus were tested for their potential in a conjugate vaccine.
• cellular extracts of E. coli containing pJMSl or pJMS23 were prepared as decribed above, and used to immunize rabbits.
• the resulting antisera was tested in the mouse lethality model for its ability to protect mice from infection by the group B Streptococcus strain H36B.
• Strain H36B carries the C protein of group B Streptococcus.
• Streptococcus agalactine group B Streptococcus (GBS)
• GBS group B Streptococcus
• the C protein beta antigen that specifically binds to human serum IgA has been cloned (Michel, J.L., et al, Infect. Immun. 59:2023-2028 (1991); Cleat, P.H., et al, Infect. Immun. 55: 1151-1155 (1987)) and sequenced (Heden, L.-O., et al, Eur. J. Immunol. 27: 1481-1490 (1991); Jerlstrom,
• Opsonophagocytic killing in the presence of alpha antigen-specific monoclonal antibody (4G8) correlated directly with increasing molecular mass of the alpha antigen and with the quantity of alpha antigen expressed on the bacterial cell surface (Madoff, L.C., et al, Infect. Immun. 59:2638-2644 (1991)).
• GBS strains expressing the alpha antigen were resistant to killing by polymorphonuclear leukocytes in the absence of specific antibody; however, this resistance was not dependent on the size of the alpha antigen.
• the completed nucleotide sequence of bca and flanking regions reported here provides information regarding the size, structure, and composition of the alpha antigen gene.
• An interesting feature of both the native and cloned gene products of the alpha antigen is that they exhibit protein heterogeneity by expressing a regularly repeating ladder of proteins differing by approximately 8000 Da (Madoff, L.C., et al, Infect. Immun. 59:2638-2644 (1991); Michel, J.L. , et al, Infect. Immun. 59:2023-2028 (1991)). Since the protective monoclonal antibody 4G8 binds to the repeat region, this region defines a protective epitope (Madoff, L.C. , Infect. Immun.
• the nucleotide sequence of bca contains nine identical 246-nucleotide tandem repeating units.
• the estimated size of the peptide encoded by each of these repeats is 8665 Da and correlates with the intervals found in the heterogeneous laddering of the alpha antigen.
• the amino acid sequence derived from the DNA sequence revealed both significant homologies and important differences between the alpha antigen and other streptococcal proteins (Heden, L.-O., et al, Eur. J. Immunol. 27: 1481-1490 (1991); Jerlstrom, P.G., et al, Mol.
• GBS strain A909 type IsJC
• E. coli strains MC1061 and DK1 Ausubel, F.M., et al, Current Protocols in Molecular Biology, Wiley, New York (1990)
• DH5 ⁇ a derivative of DH1; GIBCO/BRL
• NK-8032 E. coli plasmids and clones pUC12, pUX12, and pJMS23
• the plasmid pG ⁇ M-7Zf(-) was purchased from Promega, Madison, WI, USA. Additional subclones of pJMS23 (pJMS23-l, -7, -9, and -10) are described below. Growth media for GBS and E. coli and antibiotics for selection have been described (Michel, J.L., et al, Infect. Immun. 59:2023-2028 (1991)).
• Transposon mutagenesis utilized lambda- Tn5seql (Nag, D.K., et al, Gene 64: 135-145 (1988)).
• Nucleotide sequencing of double-stranded DNA used plasmids containing transposon Tn5seql insertions using primers of Sp6 or T7 promoters for bidirectional sequencing, synthetic oligonucleotide primers, and nested deletions using Erase-a-Base (Promega, Madison WI, USA; Henikoff, S., Gene 28:351 (1984)). A total of 12 primers were prepared to obtain the sequence in both directions for the areas of the gene flanking the repeat region. Sequencing of the region of repetitive DNA was completed with exonuclease Ill-generated nested deletions.
• Subclones pJMS23-l, pJMS23-7, and pJMS23-10 were prepared for transposon mutagenesis to target smaller regions within bca (Michel, J.L. et al, Infect. Immun. 59:2023-2028 (1991)).
• Subclone pJMS23-l contains a 5.9-kilobase Hindlll fragment in pUX12;
• pJMS23-7 contains 2.8-kilobase Alu
• pJMS23-10 is a BsaBl/Sma I double restriction endonuclease digestion of pJMS23-7 that yielded a 2.3 kilobase insert containing the repeat region.
• Alu I fragment from pJMS23-l was ligated into the Sma I site on pGEM-7Zf(-) to create pJMS23-9.
• Nested deletions were constructed in the forward direction from the Hindlll and Nsi I sites and in the reverse direction from EcoRI and Sph I sites. The sizes of the subclones, mutants, and deletions used for sequencing were confirmed by restriction endonuclease mapping and/or PCR with primers to the pUC12 polylinker and to Tn5seql (Sp6 and T7).
• the complete nucleotide sequence of the bca locus and derived amino acid sequence for a single, large open reading frame are shown in Figure 6.
• the structural gene consists of 3063 nucleotides, encodes 1020 amino acids, and has a calculated molecular mass of 108,705 Da.
• TATAAT prokaryotic promoter consensus sequence upstream (at -10) from the initiating codon
• Doi, R.H., et al, Microbiol. Rev. 50:221-243 (1986) There are no clear homologies in the -35 region assuming a spacing of 5-19 bases upstream from the -10 region (Hawley, D.K. , et al, Nucleic Acids Res.
• the derived amino acid sequence of the mature peptide of bca predicts a pK a of 4.49, which is close to the experimentally measured values for both the native and the cloned C protein alpha antigen.
• the alpha antigen contains no cysteine and only a single methionine at the initiation codon.
• the alpha antigen is rich in proline (11 in the mature protein) but does not show the XPZ motif identified in the C protein beta antigen of GBS (Heden, L.-O., et al, Eur. J. Immunol. 27: 1481-1490 (1991); Jerlstrom, P.G. et al, Mol. Microbiol. 5:843-849 (1991)) or the proline repeat motifs described in M protein of group A streptococci (Fischetti, V.A., et al, Mol Microbiol
• alpha antigen may use a signal sequence to be exported from the cytoplasm.
• a BLAST search identified five Gram-positive surface proteins with homology to the first 41 amino acids of the alpha antigen ( Figure
• the mature protein would contain 979 amino acids with a molecular mass of 104,106 Da. This suggests that the signal sequence is encoded by 123 nucleotides, making up 4% of the gene, and has a molecular mass of 4616 Da. Further support for a signal sequence of this size comes from Western blots comparing the sizes of the native and cloned alpha antigens probed with the monoclonal antibody 4G8. As shown in Figure 8, each of the steps of the alpha antigen protein ladder from clone pJMS23 is slightly larger than that of the native protein from GBS
• Repeating Unit Region of bca Beginning at amino acid 679 of the DNA sequence, there are nine large tandem repeating units with identical nucleic acid and amino acid structures that encompass 74% of the gene. The size and repetitive nature of this region of bca are illustrated in Figure 9. Each repeating unit consists of 246 nucleotides encoding 82 amino acids with a calculated molecular mass of 8665 Da. The entire repeat region contains 749 amino acids and consists of the nine identical repeating units and a partial repeating unit designated 9'. The calculated molecular mass of this region is 79,053 Da.
• the determination of the beginning and end of the repeat is somewhat arbitrary. Here, the determination starts from the N terminus, beginning with the first codon that was in the open reading frame.
• the repeating units could also be defined as beginning out of frame or starting at the C- terminal side.
• BLAST computer searches for nucleic acid and derived amino acid homologies showed to significant matches for the repeat units. Therefore, these repeating units appear to be unique to the alpha antigen and are different in size and structure from those described for other streptococcal proteins (Heden, L.-O., et al.Eur. J. Immunol. 27: 1481-1490 (1991); Jerlstrom, P.G., et al, Mol.
• a BLAST search for amino acid homologies identified a class of Gram-positive surface proteins with a common membrane anchor motif (Figure 7B), including the M proteins of group A Streptococcus and IgG binding proteins from both group A and group G Streptococcus (Wren, B.W., Mol. Microbiol. 5:797-803 (1991)).
• the amino acid composition at the C terminus is characteristic of the peptide membrane anchor, including a hydrophilic stretch with lysine before the LPXTGE [SEQ ID NO: 2] motif ( Figure 7B) (Fischetti, V.A. et al, Mol. Microbiol. 4: 1603-
• PPFFXXAA [SEQ ID NO: 1], where X designates a hydrophobic amino acid.
• Figure 9 illustrates four distinct regions within the open reading frame of bca as determined from the nucleotide and derived amino acid sequences.
• a hydrophobicity plot of the amino acid sequence shows that the putative signal sequence has a short, hydrophilic N terminus, followed by a hydrophobic stretch, and ending in a hydrophilic region, whereas the C- peptide membrane anchor has a hydrophobic wall-spanning domain and a small hydrophilic tail (Engelman, D.M., et al, Annu. Rev. Biophys. Biophys. Chem. 75:321-353 (1986); Kyte, J., et al, J. Mol Biol 757: 105-132 (1982)).
• the native alpha antigen demonstrates a ladder of polypep tides at regularly repeating intervals that is also seen with the cloned gene product
• the alpha antigen sequence identified a region of large, identical, tandem repeats composing 74% of the gene and demonstrating no homology to previously described protein or nucleic acids sequences.
• a number of virulence-associated proteins contain multiple repetitive elements.
• the M protein of group A Streptococcus which is antiphagocytic, carries protective epitopes and displays variability in antigen size and presentation, contains two extended tandem repeat regions and one nontandem repeat region occupying nearly two-thirds of the gene (Fischetti, V.A., et al, Rev. Infect. Dis. S356-S359 (1988); Hollingshead, S.K., et al, J. Biol. Chem.
• Pneumococcal surface protein A contains a region containing up to 10 repetitive segments of 20 amino acids each (Yother, J. et al, J.
• Immunodominant epitopes associated with repetitive sequences have been identified in a number of other pathogens including Rickettsia rickettsii, Trichomonas vaginatis, and Clostridium difficile (Dai ley, D.C., et al, Infect. Immun. 59:2083-2088 (1991); Anderson, B.E., et al, Infect.
• strain A909 contains only one copy of bca it was proposed that these fragments may be responsible for the protein heterogeneity.
• the nucleotide sequence confirms the repetitive nature of the gene but does not identify the mechanism of protein laddering.
• tandem repeating units could provide convenient fixed recombination sites for deletion or duplication of the repeat region. Deletion would reduce the size of the gene and might occur during DNA replication by unequal crossover or mispaired template slippage, which would occur in frame (Harayama, S., et al, J. Bacteriol 173:7540-7548 (1991)). Duplication of DNA could be a mechanism to amplify mutations within a repeat and create antigenic diversity. However, we have no evidence that the variation in the protein size of the alpha antigen is accompanied by antigenic diversity and the expression of different protective or opsonic epitopes.
• the nine complete tandem repeats in the alpha antigen from A909 are identical at the nucleic acid level, which demonstrates a highly conserved structure. This suggests that the duplication causing the repeats is a recent event, that there are properties internal to the repeats that maintain their integrity, or that their structure is essential for the gene.
• Southern blots of genomic DNA from alpha antigen-bearing strains of GBS probed with alpha antigen-specific DNA show variability in gene size among strains. To look at the mechanism of genotypic diversity among strains, it will be necessary to clone and sequence bca from other phenotypic variants and to determine the phylogenetic relationships among C protein-bearing strains of GBS (Michel,
• Each repeating unit is identical and consists of 82 amino acids with a molecular mass of 8665 Da, which is encoded by 246 nucleotides.
• the size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins expressed by GBS.
• the C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of Gram-positive surface proteins.
• the large region of identical repeating units in bca defines protective epitopes and its structure may be manipulated for the construction of protective vaccines that are directed to the phenotypic and genotypic diversity of the alpha antigen.
• protective C protein alpha antigen functional derivatives such as a protein moiety of N, C, N-C, R., R 2 , R 3 , R 4 , R 5 , R 6 ,
• R 7 , R 8 , R,, N-R negligence N-R-, N-R 3 , N-R 4 , N-R 5 , N-R,, N-R 7 , N-R 8 , N-R,, R,-C, R 2 -C, R 3 -C, R 4 -C, R 5 -C, Re-C, R 7 -C, R 8 -C, R ⁇ C, N-R.-C, N-R 2 -C, N-R 3 -C, N-R 4 -C, N-R 5 -C, N-R 6 -C, N-R 7 -C, N-R g -C, or N-Rg-C) may be prepared by recombinant means using recombinant methods similar to those described above for cloning and expressing the native group B Streptococcus alpha antigen and beta antigen in hosts such as E.
• the recombinantly expressed, above-described protective C protein alpha antigen functional derivatives may be purified, above-described protective C protein alpha antigen functional derivatives (such as a protein moiety of N, C, N-C, Rlie R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , ⁇ , N-R réelle N-R 2 , N-R 3 , N-R 4 , N-R s , N-R 6 , N- R 7 , N-R 8 , N-R ⁇ , R r C, R 2 -C, R 3 -C, R 4 -C, R 5 -C, R ⁇ -C, R 7 -C, R 8 -C, R ⁇ -C, N- R r C, N-R 2 -C, N-R 3 -C, N-R 4 -C, N-R 5 -C, N-R 6 -C, N-R 7 -C, N-R
• Each peptide species may be tested alone, or in combination with other peptides.
• cellular extracts of E. coli containing recombinant plasmids are prepared as described above, and used to immunize rabbits.
• the resulting antisera are tested in the mouse lethality model for their ability to protect mice from infection by the group B Streptococcus strain H36B.
• Strain H36B carries the C protein of group B Streptococcus.
• the ability of the antisera to protect the mice against infection by Streptococcus strain 515 (which does not carry the C protein) is determined.
• a similar assay may be used to assess the conjugated form wherein the peptide is conjugated to a group B Streptococcus polysaccharide using the above described techniques known in the art.
• this is a group B Streptococcus capsid polysaccharide.
• the conjugates are used to immunize rabbits.
• the resulting antisera are tested in the mouse lethality model for their ability to protect mice from infection by the group B Streptococcus strain
• H36B Strain H36B carries the C protein of group B Streptococcus. As a control, the ability of the antisera to protect the mice against infection by Streptococcus strain 515 (which does not carry the C protein) is determined.
• ADDRESSEE Sterne, Kessler, Goldstein & Fox
• NAME Cimbala, Michele A.
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:2 :
• GGT AAT CTT AAT ATT TTT GAA GAG TCA ATA GTT GCT GCA TCT ACA ATT 255 Gly Asn Leu Asn He Phe Glu Glu Ser He Val Ala Ala Ser Thr He 45 50 55

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