Group b streptococcus vaccine

GROUP B STREPTOCOCO VACCINE

p00002Technical field

p00003This invention relates to Streptococcus agalactiae (GBS) polysaccharides and their use in immunization.

p00004Prior art

p00005Once thought to infect only cows, the highly positive bacterium Streptococcus agalactiae (or “group B streptococcus”, abbreviated “GBS”, (Ref. 1), is now known to cause serious disease, bacteremia and meningitis, in immunocompromised individuals and in neonates There are two types of neonatal infection The first (early onset, usually after 5 days of birth) is manifested by bacteremia and pneumonia. It contracts vertically when a baby passes through the birth canal. GBS colonizes the vagina of approximately 25% of young women, and approximately 1% of babies born through vaginal birth in colonized mothers will be infected. Mortality is between 50-70%. The second is a meningitis that occurs 10 to 60 days after birth. If pregnant women are vaccinated with a type III capsule so that babies are passively immunized, the incidence of late onset meningitis is reduced but not completely eliminated.

p00006The "B" in "GBS" refers to the Lancefield classification, which is based on the antigenicity of a carbohydrate that is soluble in dilute acid and is called carbohydrate C. Lancefield identified 13 types of carbohydrate C, designated A through O, that could be identified serologically. The organisms that most commonly infect humans are found in groups A, B, D and G. In group B, the strains can be divided into at least 9 serotypes (Ia, Ib, Ia / c, II, III, IV, V, VI, VII and VIII) based on the structure of their polysaccharide capsule. In the past, serotypes Ia, Ib, II and III were equally prevalent in normal vaginal delivery and early onset sepsis in newborns. However, GBS type V has appeared as an important cause of GBS infection in the United States of America, and type VI and VIII strains have become prevalent among Japanese women.

p00007The genome sequence of a strain 2603 V / R serotype V (Ref. 2) has been published and several polypeptides have been identified for use in vaccine antigens (Ref. 3). However, vaccines currently in clinical trials are based on polysaccharide antigens. They suffer from serotype specificity and poor immunogenicity, so there is a need for effective vaccines against S. agalactiae infection.

p00008It is an object of the invention to provide more and better GBS vaccines.

p00009Disclosure of the invention

p00010The inventors have realized that saccharide-based vaccines can be improved by using them in combination with polypeptide antigens, and vice versa, so that the polypeptide and saccharide can individually contribute to the immune response in a receptor. The combination is particularly advantageous when the saccharide and the polypeptide are of different GBS serotypes.

p00011The combined antigens may be present as a simple combination where the separated saccharide and polypeptide antigens are administered together, or they may be present as a conjugate combination, where the saccharide and polypeptide antigens bind covalently to each other.

p00012Thus, the invention provides an immunogenic composition comprising (i) one or more GBS polypeptide antigens and (ii) one or more GBS saccharide antigens. The polypeptide and the polysaccharide may be advantageously covalently linked together to form a conjugate.

p00013Among them, the combined polypeptide and saccharide antigens preferably cover two or more GBS serotypes (eg, 2, 3, 4, 5, 6, 7, 8 or more serotypes). The serotypes of the polypeptide and saccharide antigens may or may not overlap. For example, the polypeptide can protect against serogroup II or V, while the saccharide protects against serogroups Ia, Ib or II. Preferred combinations protect against the following groups of serotypes: (1) serotypes Ia and Ib, (2) serotypes Ia and II, (3) serotypes Ia and III, (4) serotypes Ia and IV, (5) serotypes Ia and V, (6) serotypes Ia and VI, (7 ) serotypes Ia and VII, (8) serotypes Ia and VIII, (9) serotypes Ib and II, (10) serotypes Ia and III, (11) serotypes Ia and IV, (12) serotypes Ia and V, (13) serotypes Ia and VI, (14) serotypes Ia and VII, (15) serotypes Ia and VIII, (16) serotypes II and III, (17) serotypes II and IV, (18) serotypes II and V, (19) serotypes II and VI, (20) serotypes II and VII, (21) serotypes II and VIII, (22) serotypes III and IV, (23) serotypes III and V, (24) serotypes III and VI, (25) serotypes III and VII, (26) serotypes III and VIII, (27 ) serotypes IV and V, (28) serotypes IV and VI, (29) serotypes IV and VII, (30) serotypes IV and VIII, (31) serotypes V and VI, (32) serotypes V and VII, (33) serotypes V and VIII, (34) serotypes VI and VII, (35) serotypes VI and VIII and (36) serotypes VII and VIII.

p00014Even more preferably, the combinations protect against the following groups of serotypes: (1) serotypes Ia and II, (2) serotypes Ia and V, (3) serotypes Ib and II, (4) serotypes Ib and V, (5) serotypes III and II and (6) serotypes III and

p00017V. More preferably, the combinations protect against serotypes III and V.

p00018Protection against serotypes II and V is preferably provided by polypeptide antigens. The protection against serotypes Ia, Ib and / or II may be of polypeptide or saccharide antigens.

p00019Preferably, the immunogenic composition comprises one or more serogroup V antigens or fragments thereof selected from the antigen group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690 and GBS 691. Preferably, the composition comprises a composition of at least two of these GBS antigens or fragments thereof .

p00020In one embodiment, the immunogenic composition comprises a GBS saccharide antigen and at least two GBS polypeptide antigens or fragments thereof, wherein said GBS saccharide antigen comprises a saccharide selected from GBS serotype Ia, Ib and III, and wherein said antigens GBS polypeptide comprise a combination of at least two polypeptides or fragments thereof selected from the antigen group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690 and GBS 691.

p00021Preferably, the combination comprises GBS 80 or a fragment thereof. In one embodiment, GBS polypeptide antigens comprise a combination of two GBS antigens or fragments thereof selected from the antigen group consisting of (1) GBS 80 and GBS 91, (2) GBS 80 and GBS 104, (3) GBS 80 and GBS 147, (4) GBS 80 and GBS 173, (5) GBS 80 and GBS 276, (6) GBS 80 and GBS 305, (7) GBS 80 and GBS 313, (8) GBS 80 and GBS 322, (9) GBS 80 and GBS 328, (10) GBS 80 and GBS 330, (11) GBS 80 and GBS 338, (12) GBS 80 and GBS 358,

p00022(13) GBS 80 and GBS 361, (14) GBS 80 and GBS 404,, (15) GBS 80 and GBS 656, (16) GBS 80 and GBS 690 and (17) GBS 80 and GBS 691.

p00023Even more preferably, the combination is selected from the antigen group consisting of (1) GBS 80 and GBS 338, (2) GBS 80 and GBS 361, (3) GBS 80 and GBS 305, (4) GBS 80 and GBS 328, (5) GBS 80 and GBS 690, (6) GBS 80 and GBS 691 and (7) GBS 80 and GBS 147. Even more preferably, the combination comprises GBS 80 and GBS

p00024691.

p00025In one embodiment, the composition comprises a combination of at least three GBS polypeptide antigens. Preferably, this combination comprises GBS 80 and GBS 691.

p00026Preferably, the immunogenic combination further comprises a GBS polypeptide or a fragment thereof of serogroup II.

p00027Polypeptide antigen

p00028The polypeptide is preferably: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ IDs 2-10966 with an even number of Ref. 3; (b) a polypeptide comprising an amino acid sequence having a sequential identity with an amino acid sequence of (a); or (c) a polypeptide comprising a fragment of an amino acid sequence of (a).

p00029In (a), the preferred SEQ IDs are those encoding GBS1 to GBS689 (see Table IV of reference 3).

p00030In (b), the degree of sequential identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (eg, 60%, 70%, 80%, 90%, 95%, 99% or higher). The polypeptides in (b) include homologs, orthologs, allelic variants and functional mutants of (a). Typically, 50% identity or more between two proteins is considered an indication of functional equivalence. The identity between proteins is preferably determined by the homology search with the Smith-Waterman algorithm as implemented by the MPSRCH (Oxford Molecular) program, using a search for related gaps with open hole penalty parameters = 12 and hole extension penalty = 1.

p00031In (c), the length of the fragment may vary depending on the amino acid sequence (a) in question, but the fragment preferably has 7 consecutive amino acids of the sequences of (a) for example 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more. Preferably, the fragment comprises one or more epitopes of the sequence. Other preferred fragments are the N-terminal signal peptides of SEQ ID s 110966 of Ref. 3, SEQ IDs 1-10966 of Ref. 3 without its N-terminal signal peptides and SEQ IDs 1-10966 of Ref. 3 where up to 10 amino acid residues are eliminated (ie, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues ) from terminal N and / or terminal C, for example, the amino acid residue of terminal N can be removed.

p00032Polypeptides, of course, can be prepared by various methods (for example, recombinant expression, GBS purification, chemical synthesis, etc.) and in various ways (for example, natives, fusions, glycosylated, non-glycosylated, etc.). They are preferably prepared in substantially pure form (that is,

p00035substantially free of other streptococcus or host cell proteins) or in substantially isolated form.

p00036Preferred polypeptide antigens are: GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690 and GBS 691, including polypeptides having amino acid sequences with sequential identity thereto, etc.

p00037The nucleotide and amino acid sequences of GBS 80 in Ref. 3 are SEQ ID 8779 and SEQ ID 8780. These sequences are set forth below as SEQ ID NOS 1 and 2:

SEQ ID NO. 1

p00039SEQ ID NO. two

p00040The nucleotide and amino acid sequences of GBS 91 in Ref. 3 are SEQ ID 8937 and SEQ ID 8938. These sequences are set forth below as SEQ ID NOS 3 and 4:

SEQ ID NO. 3

SEQ ID NO. 4

p00045The nucleotide and amino acid sequences of GBS 104 in Ref. 3 are SEQ ID 8777 and SEQ ID 8778. These sequences are set forth below as SEQ ID NOS 5 and 6:

SEQ ID NO. 5

SEQ ID NO. 6

p00048The nucleotide and amino acid sequences of GBS 147 in Ref. 3 are SEQ ID 8525 and SEQ ID 8526. These sequences are set forth below as SEQ ID NOS 7 and 8:

SEQ ID NO. 7

SEQ ID NO. 8

p00053The nucleotide and amino acid sequences of GBS 173 in Ref. 3 are SEQ ID 8787 and SEQ ID 8788. 45 These sequences are set forth below as SEQ ID NOS 9 and 10:

SEQ ID NO. 9

SEQ ID NO. 10

p00058The nucleotide and amino acid sequences of GBS 276 in Ref. 3 are SEQ ID 8941 and SEQ ID 8942. These sequences are set forth below as SEQ ID NOS 11 and 12:

p00059SEQ ID NO. eleven

SEQ ID NO. 12

p00061The nucleotide and amino acid sequences of GBS 305 in Ref. 3 are SEQ ID 207 and SEQ ID 208. These sequences are set forth below as SEQ ID NOS 13 and 14:

SEQ ID NO. 13

SEQ ID NO. 14

p00066The nucleotide and amino acid sequences of GBS 313 in Ref. 3 are SEQ ID 4089 and SEQ ID 4090.25 These sequences are set forth below as SEQ ID NOS 15 and 16:

p00067SEQ ID NO. fifteen

SEQ ID NO. 16

p00069The nucleotide and amino acid sequences of GBS 322 in Ref. 3 are SEQ ID 8539 and SEQ ID 8540. 45 These sequences are set forth below as SEQ ID NOS 17 and 18:

SEQ ID NO. 17

SEQ ID NO. 18

p00074The nucleotide and amino acid sequences of GBS 328 in Ref. 3 are SEQ ID 6015 and SEQ ID 6016. These sequences are set forth below as SEQ ID NOS 19 and 20:

SEQ ID NO. 19

p0007635 SEQ ID NO. twenty

p00077The nucleotide and amino acid sequences of GBS 330 in Ref. 3 are SEQ ID 8791 and SEQ ID 8792. These sequences are set forth below as SEQ ID NOS 21 and 22:

p00078SEQ ID NO. twenty-one

SEQ ID NO. 22

p00082The nucleotide and amino acid sequences of GBS 338 in Ref. 3 are SEQ ID 8637 and SEQ ID 8638. These sequences are set forth below as SEQ ID NOS 23 and 24:

SEQ ID NO. 2. 3

SEQ ID NO. 24

p00085The nucleotide and amino acid sequences of GBS 358 in Ref. 3 are SEQ ID 3183 and SEQ ID 3184. These sequences are set forth below as SEQ ID NOS 25 and 26:

SEQ ID NO. 25

p00087The nucleotide and amino acid sequences of GBS 361 in Ref. 3 are SEQ ID 8769 and SEQ ID 8770. These sequences are set forth below as SEQ ID NOS 27 and 28:

35 SEQ ID NO. 27

SEQ ID NO. 28

p00090The nucleotide and amino acid sequences of GBS 404 in Ref. 3 are SEQ ID 8799 and SEQ ID 8800. These sequences are set forth below as SEQ ID NOS 29 and 30:

SEQ ID NO. 29

SEQ ID NO. 30

p00095The nucleotide and amino acid sequences of GBS 656 in Ref. 3 are SEQ ID 9323 and SEQ ID 9324. These sequences are set forth below as SEQ ID NOS 31 and 32:

SEQ ID NO. 31

SEQ ID NO. 32

p00098The nucleotide and amino acid sequences of GBS 690 in Ref. 3 are SEQ ID 9965 and SEQ ID 9966. These sequences are set forth below as SEQ ID NOS 33 and 34:

SEQ ID NO. 33

SEQ ID NO. 3. 4

p00101The nucleotide and amino acid sequences of GBS 691 in Ref. 3 are SEQ ID 3691 and SEQ ID 3692. 55 These sequences are set forth below as SEQ ID NOS 35 and 36:

SEQ ID NO. 35

SEQ ID NO. 36

p00106Other preferred polypeptide antigens include: GBS4 (SEQ ID 2 of Ref. 3); GBS22 (SEQ ID 8584 of Ref. 3); and GBS85 (SEQ ID 216 of Ref. 3), including polypeptides having amino acid sequences with sequential identity thereto, etc.

p00107The polypeptide is preferably not a C protein (alpha or beta or epsilon) or an R (Rib) protein.

p00108The nucleotide and amino acid sequences of GBS 4 in Ref. 3 are SEQ ID 1 and SEQ ID 2. These 15 sequences are set forth below as SEQ ID NOS 37 and 38:

SEQ ID NO. 37

25 SEQ ID NO. 38

p00111The nucleotide and amino acid sequences of GBS 22 in Ref. 3 are SEQ ID 8583 and SEQ ID 8584. These sequences are set forth below as SEQ ID NOS 39 and 40:

SEQ ID NO. 39

SEQ ID NO. 40

p00114The nucleotide and amino acid sequences of GBS 85 in Ref. 3 are SEQ ID 215 and SEQ ID 216. These sequences are set forth below as SEQ ID NOS 41 and 42:

SEQ ID NO. 41

SEQ ID NO. 42

p00119GBS polypeptides of the invention may be present in the composition as separate individual polypeptides. However, it is preferred that two or more (that is, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ) of the antigens are expressed as a single polypeptide chain (a "hybrid" polypeptide). Hybrid polypeptides offer two main advantages: first, a polypeptide that can be unstable

p00120or expressing oneself poorly alone can be helped by adding a suitable hybrid partner that overcomes the problem; second, commercial manufacturing is simplified since only one expression and purification need to be employed in order to produce two polypeptides that are antigenically useful.

p00121The hybrid polypeptide may comprise two or more polypeptide sequences of the first antigen group. Accordingly, the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequence is selected from a GBS antigen or a fragment thereof. Preferably, the first and second amino acid sequence in the hybrid polypeptide comprise different epitopes.

p00122The hybrid polypeptide may comprise one or more polypeptide sequences of different GBS serotypes. Accordingly, the invention includes a composition comprising a first amino acid sequence and a second amino acid sequence, said first amino acid sequence and said second amino acid sequence selected from a GBS serotype selected from the group consisting of serotypes Ia, Ib, Ia / c, II, III, IV, V, VI; VII and VIII. The first and second amino acid sequence may be of the same GBS serotype or they may be of different GBS serotypes. Preferably, the first and second amino acid sequence are selected from a GBS serotype selected from the group consisting of serotypes II and V. More preferably, at least one of the first and second amino acid sequence is GBS serotype V. preferably, the first and Second amino acid sequence in the hybrid polypeptide comprise different epitopes.

p00123In one embodiment, the hybrid polypeptide comprises one or more GBS serotype V antigens. preferably, the hybrid polypeptide comprises a first amino acid sequence and a second amino acid sequence, said first amino acid sequence and said second amino acid sequence comprising a GBS antigen or a fragment thereof selected from the group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690 and GBS 691. Preferably, the GBS antigen or fragment thereof is selected from the group consisting of GBS 80 and GBS 691. Preferably, the first and second amino acid sequence in the hybrid polypeptide comprise different epitopes.

p00124Hybrids consisting of amino acid sequences of two, three, four, five, six, seven, eight, new or ten GBS antigens are preferred. In particular, hybrids consisting of amino acid sequences of two, three, four or five GBS antigens are preferred.

p00125Different hybrid polypeptides can be mixed together in a single formulation. In such combinations, a GBS antigen may be present in more than one hybrid polypeptide and / or as a non-hybrid polypeptide. However, it is preferred that an antigen be present either as a hybrid or as a non-hybrid, but not as both.

p00126Preferably, the GBS antigen in one of the hybrid polypeptides is GBS 80 or a fragment thereof. Accordingly, examples of hybrids of two antigens for use in the invention may comprise: (1) GBS 80 and GBS 91, (2) GBS 80 and GBS 104, (3) GBS 80 and GBS 147, (4) GBS 80 and GBS 173, (5) GBS 80 and GBS 276,

p00127(6) GBS 80 and GBS 305, (7) GBS 80 and GBS 313, (8) GBS 80 and GBS 322, (9) GBS 80 and GBS 328, (10) GBS 80 and GBS 330, (11) GBS 80 and GBS 338, (12) GBS 80 and GBS 358, (13) GBS 80 and GBS 361, (14) GBS 80 and GBS 404, (15) GBS 80 and GBS 656, (16) GBS 80 and GBS 690 and ( 17) GBS 80 and GBS 691. Preferably, a hybrid of two antigens for use in the invention comprises GBS 80 and GBS 691.

p00128Hybrid polypeptides may be represented by the formula NH2-A - {- ZL-} nB-COOH, where: X is an amino acid sequence of a GBS antigen or a fragment thereof; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional terminal C amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

p00129If a fraction - X - has a leader peptide sequence in its wild type form, it can be included or omitted in the hybrid protein. In some embodiments, the leader peptides will be removed except for the fraction -X located in the N terminal of the hybrid protein, that is, the leader peptide of X1 will be maintained, but the leader peptides of X2 ... Xn will be omitted. This is equivalent to eliminating all the leader peptides and using the leader peptide of X1 as the –A- fraction.

p00132For each case n of {-XL-}, the linker amino acid sequence -L-may be present or absent. For example, when –n = 2 the hybrid can be NH2-X1-L1-X2-L2-COOH, NH2-X1-X2-COOH, NH2-X1-L1-X2-COOH, NH2-X1-X2-L2- COOH, etc. The linking amino acid sequence or sequences -L- will typically be short (for example, 20 or less amino acids, that is, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1). Examples include short peptide sequences that facilitate cloning, polyglycine linkers (i.e., comprising Glin where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (that is, Hisn where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable amino acid linker sequences will be apparent to those skilled in the art. A useful linker is GSGGGG (SEQ ID 1), with the Gli-Ser dipeptide formed from a BamHI restriction site, thus aiding cloning and manipulation, and the tetrapeptide (Gli) 4 being a typical polyglycine linker.

p00133-A-is an optional N-terminal amino acid sequence. This will be typically short (for example, 40 or less amino acids, that is, 39, 38, 37, 36, 35, 34, 33, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leading sequences to direct protein traffic, or short peptide sequences that facilitate cloning or purification (eg, histidine tags, this is Hisn where n = 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If X1 lacks its own N-terminal methionine, -A- is preferably an oligopeptide (for example, with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) that provides a terminal A methionine.

p00134-B-is an optional C-terminal amino acid sequence. This will be typically short (for example, 40 or less amino acids, that is, 39, 38, 37, 36, 35, 34, 33, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences that facilitate cloning or purification (for example, comprising histidine tags, that is Hisn where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences that improve protein stability. Other suitable terminal C amino acid sequences will be apparent to those skilled in the art.

p00135More preferably, n is 2 or 3.

p00136Saccharide antigen

p00137The saccharide antigen is generally the capsular polysaccharide of a GBS or a derivative thereof. Suitable derivatives include oligosaccharide (for example, from 3 to 150, preferably from 8 to 100 monosaccharide units), polysaccharide fragments (eg, refs. 12 to 16), de-acetylated saccharides (Ref. 16), N saccharides -acrylates (16), saccharides with terminal aldehyde groups, etc.

p00138The saccharide is preferably conjugated with a transporter molecule to improve immunogenicity (for example, see refs. 4 to 23 etc.). In some embodiments of the invention the GBS saccharide is conjugated to a GBS protein as defined above, thereby giving a polypeptide / saccharide combination of the invention in a single molecule. In other embodiments, the GBS saccharide is conjugated to a non-GBS protein, in which case the conjugate will be combined with a separate GBS protein to give a polypeptide / saccharide combination of the invention.

p00139Non-GBS transporter polypeptides include tetanus toxoid, the outer membrane protein of

p00140N. meningitidis (24), synthetic peptides (25, 26), heat shock proteins (27, 28), pertussis proteins (29, 30), H. influenzae D protein (31), cytokines (32), lymphokines ( 32), hormones (32) growth factors (32), C. difficile toxin A or B (33), iron absorption proteins (34), etc. Preferred carrier proteins are diphtheria toxoid CRM197 (35) and tetanus toxoid.

p00141The saccharide and the polypeptide covalently bind. This may involve a direct covalent bond between the saccharide and the polypeptide, or an indirect linkage can be used by means of a linker or spacer (for example, by means of a B-propionamide linker (16), etc.). Any suitable conjugation chemistry (eg reductive amination (21), etc.) may be used. The connection is preferably made by means of a terminal saccharide in the polysaccharide.

p00142A single transport molecule can carry saccharide antigens of a single type (for example, saccharide derived from a single GBS serotype) or can transport multiple different antigens (for example, saccharides derived from multiple GBS serotypes, all conjugated with the same transporter).

p00143The saccharides can, of course, be prepared in various ways (for example, GBS saccharide purification, chemical synthesis, etc.), of various sizes (e.g., full length, fragmented, etc.) or can be derivatized to bind to conveyors Preferably they will be prepared in substantially pure form (that is, substantially free of other streptococcal saccharides) or in substantially isolated form. Processes for preparing GBS capsular polysaccharides are well known in the art (for example, refs. 36 to 39) and processes for preparing polysaccharide oligosaccharides are also known (for example, hydrolysis, sonication, enzymatic treatment, treatment with a base followed nitrosation, etc. (12 to 16)).

p00146As an alternative to using a saccharide antigen in unconjugated combinations, a peptide mimetic of the GBS capsular polysaccharide (for example, 40) can be used. Suitable peptides can be selected by technique such as phage expression using protective anti-saccharide antibodies. As a further alternative, an anti-idiotopic antibody can be used instead of a saccharide antigen (for example, ref. 41).

p00147Awareness / reinforcement programs

p00148The polypeptide / saccharide combinations of the invention can be given as single doses or as part of a sensitization / boost program. In a sensitization / reinforcement program, the combinations can be used as the sensitization dose, the booster dose or dose, or both.

p00149If a combination is used for sensitization and reinforcement, it is preferred to use the same combination at both times. If a combination is used for only one of sensitization and reinforcement, it is preferred that the other dose uses the polypeptide or saccharide on which the combination is based. Thus the invention provides a sensitization-enhancement program where (i) one of the saccharide and polypeptide antigens is used to sensitize an immune response and a combination is used to enhance the response, or (ii) combined saccharide antigens and Polypeptide are used to sensitize an immune response but only one is used to strengthen the response.

p00150Various rhythms for sensitization and reinforcement are suitable for use with the invention. In one embodiment, a sensitization dose is given to a child and a teenager (13-18 years old) or young adult (19-25 years old) is given reinforcement. In another embodiment, a dose of sensitization is given to a teenager or young adult and reinforcement is given during pregnancy. In another embodiment, a dose of sensitization is given to a woman who tries to become pregnant and is given a boost during pregnancy.

p00151Immunogenic pharmaceutical compositions

p00152Polypeptide / saccharide combinations are formulated as immunogenic compositions, and more preferably as compositions suitable for use as a vaccine in humans (eg, children or adults). The vaccines of the invention can be prophylactic (that is, to prevent infection) or therapeutic (that is, to treat the disease after infection), but will typically be prophylactic. Accordingly, the invention includes a method for therapeutic or prophylactic treatment of GBS infection in an animal susceptible to GBS infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention.

p00153The composition of the invention is preferably sterile.

p00154The composition of the invention is preferably pyrogen free.

p00155The composition of the invention generally has a pH of between 6.0 and 7.0, more preferably between 6.3 and 6.9, for example, 6.6 ± 0.2. The composition is preferably buffered at this pH.

p00156Other components suitable for human administration are disclosed in reference 42.

p00157The vaccines of the invention can be administered together with other immunoregulatory agents. In particular, the compositions will normally include an adjuvant. More preferred adjuvants include, but are not limited to, one or more of the following set forth below:

p00158A. Compositions containing minerals

p00159Compositions containing minerals suitable for use as adjuvants in the invention include mineral salts, such as aluminum salts and calcium salts. The invention includes mineral salts such as hydroxides (for example, oxyhydroxides), phosphates (for example, hydroxyphosphates, orthophosphates), sulfates, etc., {for example, see chapters 8 and 9 of ref. 43}), or mixtures of different mineral compounds with the compound taking any suitable form (for example, gel, crystalline, amorphous, etc.), and adsorption being preferred. Mineral-containing compositions can also be formulated as a metal salt particle. See ref.

p0016044.

p00161B. Emulsions in oil

p00162Compositions of oil emulsions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80 and 0.5% Span 85, formulated in submicron particles using a microfluidizer). See ref. Four. Five.

p00163Complete Freund's Adjuvant (AFC) and Incomplete Freund's Adjuvant (AFI) can also be used

p00166as adjuvants in the invention.

p00167C. Saponin formulations

p00168Saponin formulations can also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots and even flowers of a wide variety of plant species. Saponins from the bark of the Quillaja saponaria Molina tree have been studied extensively as adjuvants. Saponin can also be obtained at the Smilax ornata (sarsaparilla), Gypsophilla paniculata (bridal veil) and Saponaria officianalis (soap root) market. Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.

p00169Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-LC) and Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-

p00170C. Preferably, saponin is QS21. A method for the production of QS21 is disclosed in U.S. Patent No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO 96/33739).

p00171Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulatory Complexes (ISCOMs). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP 0 109 942, WO 96/11711 and WO 96/33739. Optionally, ISCOMs may be free of additional detergent. See ref. 46.

p00172An analysis of the development of saponin-based adjuvants can be found in ref. 47

p00173C. Virosomes and Virus-like Particles (PTVs)

p00174Virosomes or virus-like particles (PTVs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins of a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any native viral genome. Viral proteins can be recombinantly produced or isolated from whole viruses. These viral proteins for use in virosomes or PTVs include proteins derived from influenza viruses (such as HA or NA), hepatitis B viruses (such as nucleus or capsid proteins), heptatis E virus, measles virus, Sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, Norwalk virus, human papillomavirus, HIV, RNA phage, Qβ-phage, fr-phage, phage AP205 and Ty (such as TY p1 retrotransposon protein). PTVs are further analyzed in WO 03/024480, WO 03/024481 and Refs. 48, 49, 50 and 51. Virosomes are further analyzed, for example, in Ref. 52.

p00175D. Bacterial and Microbial Derivatives

p00176Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as:

p00177(1) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS)

p00178Such derivatives include Monophosphoryl lipid A (MPL) and MPL 3-O-deacylate (3dMPL). 3dMPL is a mixture of monophosphoryl lipid A 3 De-O-acylated with 4, 5 or 6 acylated chains. A preferred "small particle" form of monophosphoryl lipid A 3 De-O-acylated disclosed in EP 0 689 454. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane. (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics, such as aminoalkyl glucosaminide phosphate derivatives, for example, RC-529. See Ref. 53.

p00179(2) Lipid A derivatives

p00180Lipid A derivatives include lipid A derivatives of Escherichia coli such as OM-174. OM-174 is described for example in Ref. 54 and 55.

p00181(3) Immunostimulatory oligonucleotides

p00182Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG unit (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double helix RNA or oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory.

p00183CpGs may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double helix or single helix. Optionally, guanosine can be replaced by a

p00186analogue such as 2'-deoxy-7-deazaguanosine. See ref. 56, WO 02/26757 and WO 99/62923 for examples as possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further analyzed in Refs. 57, 58, WO 98/40100, United States Patent No. 6,207,646, United States Patent No. 6,239,116 and United States Patent No. 6,429,199.

p00187The CpG sequence can be directed to TLR9, such as the GTCGTT or TTCGTT unit. See ref. 59. The CpG sequence may be specific to induce a Th1 immune response, such as CpG-A ODN or may be more specific to induce a B cell response, such as CpG-B ODN. CpG-A and CpG-B ODNs are analyzed in refs. 60, 61 and WO 01/95935. Preferably, CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5 'end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can bind to their 3 'ends to form "immunomers." See, for example, refs. 62, 63, 64 and WO 03/035836.

p00188(4) ADP-ribosilant toxins and detoxified derivatives thereof

p00189ADP bacterial ribosilant toxins and their purified derivatives can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (thermolabile enterotoxin "TL" from E. coli), cholera ("CT") or whooping cough ("PT"). The use of purified ribosilating ADP toxins as mucosal adjuvants is described in WO 95/17211 and as parenteral adjuvants in WO 98/42375. Preferably, the adjuvant is a purified TL mutant such as LT-K63 and LT-R72 and LT-G192. The use of ribosilating ADP toxins and purified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in references 65,66, 67, 68, 69, 70, 71 and 72, each specifically incorporated herein by reference in its entirety. The numerical reference for amino acid substitutions is preferably based on the alignments of subunits A and B of the ribosilating ADP toxins set forth in Domenighini et al., Mol. Microbiol (1995) 15 (6): 1165-1167.

p00190E. Human immunomodulators

p00191Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (for example IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc. .), interferons (for example interferon-γ), macrophage colony stimulating factor and tumor necrosis factor.

p00192F. Bioadhesives and mucoadhesives

p00193In the invention, bioadhesive and mucoadhesive adjuvants can also be used. Suitable bioadhesives include esterified hyaluronic acid microspheres (Ref. 73) or mucoadhesives such as crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethyl cellulose. Chitosan and its derivatives can also be used as adjuvants in the invention. For example, ref. 74.

p00194G. Microparticles

p00195In the invention microparticles can also be used as adjuvants. Microparticles (i.e. a particle with a diameter of ∼100 nm to ∼150 µm, more preferably with a diameter of ∼200 nm to ∼30 µm and more preferably with a diameter of ∼500 nm to ∼10 µm) formed with materials that are biodegradable and non-toxic (for example a poly (α-hydroxy acid), a poly (hydroxybutyric acid), a polyortoester, a polyanhydride, a polycaprolactone, etc.), with poly (lactide-co-glycolic acid), optionally treated to have a negatively charged surface (for example, with SDS) or a positively charged surface (for example, with a cationic detergent, such as CTAB)

p00196H. Liposomes

p00197Examples of formulations suitable with liposomes for use as adjuvants are described in U.S. Patent No. 6,090,406, U.S. Patent No. 5,916,588 and EP 0 626,169.

p00198I. Formulations of polyoxyethylene ether and polyoxyethylene ester

p00199Adjuvants suitable for use in the invention include polyoxyethylene ether and polyoxyethylene ester. Ref. 75. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (Ref. 765) as well as polyoxyethylene alkyl ether or ester surfactants in combination with at least one other non-ionic surfactant such as octoxynol (Ref. 77 ). Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.

p00200J. Polyphosphazene (PCPP)

p00203PCPP formulations are described, for example, in Ref. 78 and 79.

p00204K. Muramil peptide

p00205Examples of muramyl peptides suitable for use as adjuvants in the invention include Nacethyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP) and Nacethylmuramil-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 ', 2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy) -ethylamine MTP-PE).

p00206L. Imidazoquinolone Compounds

p00207Examples of imidazoquinolone suitable for use as adjuvants in the invention including Imiquamod and its counterparts, further described in Ref. 80 and 81. The invention may also comprise combinations of aspects of one or more adjuvants identified above. For example, the following adjuvant compositions can be used in the invention:

<dl><dt>(1) </dt><dd>a saponin and an oil-in-water emulsion (ref. 82); </dd></dl>

<dl><dt>(2) </dt><dd>a saponin (for example, QS21) + a non-toxic LPS derivatives (for example, 3dMPL) (see WO 94/00153); </dd></dl>

<dl><dt>(3) </dt><dd>a saponin (for example, QS21) + a non-toxic LPS derivatives (for example, 3dMPL) + a cholesterol; </dd></dl>

<dl><dt>(4) </dt><dd>a saponin (for example, QS21) + 3dMPL + IL-12 (optionally + a sterol) (Ref. 83); </dd></dl>

<dl><dt>(5) </dt><dd>SAF, which contains 10% Squalene, 0.4% Tween 80, 5% L121 pluronic block polymer, and thr-MPD, either microfluidized in a submicron emulsion or vortexed to generate a larger particle emulsion. </dd></dl>

<dl><dt>(6) </dt><dd>Adjuvant system Ribi ™ (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80 and one or more bacterial cell wall components of the group consisting of monophosphorylipid A (MPL), trehalum dimicolate (TDM) and cell wall skeleton (EPC), preferably MPL + EPC (Detox ™); and</dd></dl>

<dl><dt>(7) </dt><dd>one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL). </dd></dl>

p00208Aluminum salts and MF59 are preferred adjuvants for parenteral immunization. Mutant bacterial toxins are preferred mucosal adjuvants.

p00209The composition may include an antibiotic.

p00210The GBS polypeptides and saccharides in the compositions of the invention will be present in "immunologically effective amounts", that is, the administration of the amount to an individual, either in a single dose or as part of a series, is effective for treatment. or disease prevention. This amount varies depending on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the evaluation of the medical situation by the treating doctor and other relevant factors. The quantity is expected to correspond to a relatively wide range that can be determined through routine tests.

p00211Typically, the compositions of the invention are prepared as injectables. The direct administration of the compositions will generally be parenteral (for example, by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or administered to the interstitial space of a tissue) or of the mucosa (for example, oral or intranasal [85, 86] The compositions can also be administered to an injury. The invention provides syringes containing a composition of the invention.

p00212Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated may be animals; in particular, human subjects can be treated. Vaccines are particularly useful for vaccinating children and adolescents, and more particularly women.

p00213As well as GBS polypeptides and saccharides, the composition of the invention may also comprise antigens. For example, the composition may comprise one or more of the following antigens: Helicobacter pylori antigens such as CagA [87 to 90], VacA [91, 92], NAP [93, 94, 95], HopX (for example, 96], HopY [for example, 96] and / or urease - a saccharide antigen of N. meningitidis serogroup A, C, W135 and / or Y, such as the oligosaccharide disclosed in ref. 97 from serogroup C [see also ref. 98] or the oligosaccharide of ref. 99. -a saccharide antigen of Streptococcus pneumoniae [eg 100, 101, 102]. -a hepatitis A virus antigen, such as inactivated virus [eg 103, 104]. -a hepatitis B virus antigen, such as surface and / or core antigens [eg 104, 105]. -A Bordetella pertussis antigen, such as holotoxin pertussis (PT) and filamentous hemagglutinin (FH) of B. pertussis, optionally also in combination with pertactin and / or agglutinogens 2 and 3 [eg, refs. 106 and 107]. -a diphtheria antigen, such as a diphtheria toxoid [eg, chapter 3 of ref. 108], for example the mutant

p00216CRM197 [for example 109]. -a tetanus antigen, such as a tetanus toxoid [eg, chapter 4 of ref. 128]. -a saccharide antigen from Haemophilus influenzae B [eg 98]. -a hepatitis C virus antigen [for example 110]. - an antigen of N. gonorrhoeae [for example 111, 112, 113, 114]. -a Chlamydia pneumoniae antigen [eg refs. 115 to 121]. -a Chlamydia trachomatis antigen [for example 122]. -a Porphyromonas gingivalis antigen [for example 123]. -polio antigens [eg 124, 125] such as OPV or preferably IPV. rabies antigens [for example 126] such as lyophilized inactivated virus [for example 127, RabAvert ™]. - measles, mumps and / or rubella antigens [eg, chapters 9, 10 and 11 of ref. 128]. - influenza antigen (s) [eg chapter 19 of ref. 128], such as hemagglutinin and / or surface proteins of neuraminidase. -a Moraxella catarrhalis antigen [for example. 129]. -a Streptococcus pyogenes antigen (group A streptococcus) [for example 3, 130, 131]. -a Staphylococcus aureus antigen [for example 132]. -a Bacillus anthracis antigen [for example 133, 134, 135]. -a virus antigen in the family flaviviridae (genus flavivirus), such as yellow fever virus, virus Japanese encephalitis, four serotypes of Dengue virus, tick-borne encephalitis virus, Nile virus Western. -a pestivirus antigen, such as classical swine fever virus, bovine viral diarrhea virus and / or disease virus from the border. -a parvovirus antigen, for example of parvovirus B19. -a prion protein (for example, the prion protein CJD) - an amyloid protein, such as a beta peptide [136] -a cancer antigen, such as those listed in Table 1 of ref. 137 or in tables 3 and 4 of ref. 138.

p00217The composition may comprise one or more of these additional antigens.

p00218Toxic protein antigens can be purified where necessary (for example, purification of pertussis toxin by chemical and / or genetic means [107]).

p00219When a diphtheria antigen is included in the composition it is also preferred to include tetanus antigen and pertussis antigens. Similarly, when a tetanus antigen is included it is also preferred to include diphtheria and pertussis antigens. Similarly, when a pertussis antigen is included it is also preferred to include diphtheria and tetanus antigens. DTP combinations are therefore preferred. Saccharide antigens are preferably in the form of conjugates. The carrier proteins for the conjugates are the same as those described above for conjugation of saccharide GBS, with CRM197 being preferred.

p00220The antigens in the composition will typically be present in a concentration of at least 1 µg / ml each. In general, the concentration of any given antigen will be sufficient to obtain an immune response against that antigen.

p00221As an alternative to the use of protein antigens in the composition of the invention, nucleic acid encoding the antigen can be used. The protein components of the compositions of the invention can thus be replaced by nucleic acid (preferably DNA, for example, in the form of a plasmid) encoding the protein.

p00222Methods to treat patients

p00223The invention provides polypeptide / saccharide combinations of the invention for use as medicaments. The medicament is preferably capable of eliciting an immune response in an animal (that is, it is an immunogenic composition) and is more preferably a vaccine.

p00224The invention also provides a method for eliciting an immune response in a patient, comprising administering to a patient a composition of the invention. The immune response is preferably protective against streptococcal disease, and may comprise a humoral immune response and / or a cellular immune response.

p00225The invention also provides the use of polypeptide / saccharide combination of the invention in the manufacture of a medicament to obtain an immune response in a patient. The medicament is preferably an immunogenic composition (for example, a vaccine). The medicament is preferably for the prevention and / or treatment of a disease caused by GBS (for example, meningitis, sepsis, chorioamnionitis).

p00226The invention also provides a kit comprising a first component comprising the

p00229immunogenic compositions of the invention. The kit may also include a second component comprising one or more of the following: instructions, syringe or other delivery device, adjuvant or pharmaceutically acceptable formulation solution.

p00230The invention also provides a pre-filled administration device with the immunogenic compositions of the invention.

p00231The invention also provides a method for eliciting an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention. The immune response is preferably protective and preferably involves antibodies and cell-mediated immunity. The method can cause a booster response.

p00232Fabrication process

p00233The invention provides a process for the preparation of a composition of the invention, which comprises the step of mixing (i) one or more GBS polypeptide antigens with (ii) one or more GBS saccharide antigens.

p00234The process may comprise the step of covalently binding the GBS polypeptide with the GBS saccharide in order to form a conjugate.

p00235Definitions

p00236The terms "comprising" mean "including" as well as "consisting", for example, a composition "comprising" X may consist exclusively n X or may include something additional, for example X +

p00237AND.

p00238The term "approximately" in relation to a numerical value x means, for example, x ± 10%.

p00239The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may not be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

p00240Modes for carrying out the invention

p00241GBS serotype III grows in Todd-Hewitt broth as described in reference 36 and its capsular polysaccharide is purified. The polysaccharide is depolymerized, classified and purified as described in reference 14 to give oligosaccharide antigen. Similar procedures are used to prepare capsular polysaccharides of other GBS serotypes.

p00242The oligosaccharide is mixed with or covalently conjugated (directly or through a linker) with purified serotype V protein. Preferably, the protein comprises a GBS antigen or a fragment thereof selected from the group consisting of GBS 80, GBS 91, GBS 104, GBS 147, GBS 173, GBS 276, GBS 305, GBS 313, GBS 322, GBS 328, GBS 330, GBS 338, GBS 358, GBS 361, GBS 404, GBS 656, GBS 690 and GBS 691.

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SEQUENCE LIST

p00346<110> Chiron Corporation 25

p00347<120> Group B Streptococcus Vaccine

p00348<130> 002441.00084

p00349<140> PCT / US03 / 29167

p00350<141> 2003-09-15

p00351<160> 42

p0035235 <170> PatentIn version 3.1

p00353<210> 1

p00354<211> 1662

p00355<212> DNA

p00356<213> group B streptococcus

p00357<400> 1

65 <210> 2

p00360<211> 554

p00363<212> PRT

p00364<213> group B streptococcus

p00365<220>

p00366<221> MISC_FEATURE

p00367<223> Strain 80

p00368<400> 2

p00372<210> 3 55 <211> 1629

p00373<212> DNA

p00374<213> group B streptococcus

p00375<400> 3

p00380<211> 543

p00381<212> PRT

p00382<213> group B streptococcus

p00383<400> 4

<dl><dt><210> 5 </dt><dd /></dl>

<dl><dt><211> 2670 </dt><dd /></dl>

<dl><dt>15 </dt><dd><212> DNA </dd></dl>

<dl><dt><213> group B streptococcus </dt><dd /></dl>

<dl><dt><400> 5 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

p00390<210> 6

p00391<211> 890

p00392<212> PRT 55 <213> group B streptococcus

p00393<400> 6

<dl><dt>45 </dt><dd><210> 7 <211> 3699 <212> DNA <213> group B streptococcus <400> 7 </dd></dl>

<dl><dt>55 </dt><dd /></dl>

p00405<210> 8

p00406<211> 1233 45 <212> PRT

p00407<213> group B streptococcus

p00408<400> 8

p00415<210> 9

p00416<211> 2040

p00417<212> DNA

p00418<213> group B streptococcus 65 <400> 9

<dl><dt><210> 10 </dt><dd /></dl>

<dl><dt><211> 680 </dt><dd /></dl>

<dl><dt><212> PRT </dt><dd /></dl>

<dl><dt>15 </dt><dd><213> group B streptococcus </dd></dl>

<dl><dt><400> 10 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

<dl><dt><210> 11 </dt><dd /></dl>

<dl><dt>5 </dt><dd> <211> 3402 </dd></dl>

<dl><dt><212> DNA </dt><dd /></dl>

<dl><dt><213> group B streptococcus </dt><dd /></dl>

<dl><dt><400> 11’ . </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

<dl><dt><210> 12 </dt><dd /></dl>

<dl><dt><211> 1134 </dt><dd /></dl>

<dl><dt>5 </dt><dd><212> PRT </dd></dl>

<dl><dt><213> group B streptococcus </dt><dd /></dl>

<dl><dt><400> 12 </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

p00434<210> 13

p00435<211> 1365

p00436<212> DNA

p00437<213> group B streptococcus

p00438<400> 13

p0043945 <210> 14

p00440<211> 455

p00441<212> PRT 55 <213> group B streptococcus

p00442<400> 14

p00443<210> 15

p00444<211> 1020

p00445<212> DNA

p00446<213> group B streptococcus

p00447<400> 15

p00448<210> 16

p00449<211> 340

p00450<212> PRT

p00451<213> group B streptococcus

p0045245 <400> 16

p00453<210> 17

p00454<211> 1376

p00455<212> DNA

p00456<213> group B streptococcus

p00457<400> 17

p00458<210> 18

p00459<211> 432

p00460<212> PRT

p00461<213> group B streptococcus

p00462<400> 18

p00463<210> 19

p00464<211> 2070

p00465<212> DNA

p00466<213> group B streptococcus

p00467<400> 19

p00468<210> 20

p00469<211> 690

p00470<212> PRT

p00471<213> group B streptococcus

p00472<400> 20

p00473<210> 21

p00474<211> 1500

p00475<212> DNA

p00476<213> group B streptococcus

p00477<400> 21

p00478<210> 22

p00479<211> 500

p00480<212> PRT

p00481<213> group B streptococcus

p00482<220>

p00483<221> MISC_FEATURE

p00484<222> (240)..(240)

p00485<223> X is Asn or His

p00486<400> 22

p00487<210> 23

p00488<211> 720

p00489<212> DNA

p00490<213> group B streptococcus

p00491<400> 23

p00492<210> 24

p00493<211> 240

p00494<212> PRT

p00495<213> group B streptococcus

p0049655 <400> 24

p00497<210> 25

p00498<211> 870

p00499<212> DNA

p00500<213> group B streptococcus

p00501<400> 25

<dl><dt>45 </dt><dd><210> 26 <211> 290 <212> PRT <213> group B streptococcus <400> 26 </dd></dl>

<dl><dt>55 </dt><dd /></dl>

p00502<210> 27

p00503<211> 2193

p00504<212> DNA

p00505<213> group B streptococcus

p00506<400> 27

<dl><dt><210> 28 </dt><dd /></dl>

<dl><dt><211> 731 </dt><dd /></dl>

<dl><dt>15 </dt><dd><212> PRT </dd></dl>

<dl><dt><213> group B streptococcus </dt><dd /></dl>

<dl><dt><400> 28 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

p00507<210> 29

p00508<211> 900

p00509<212> DNA

p00510<213> group B streptococcus

p00511<400> 29

p00512<210> 30

p00513<211> 300

p00514<212> PRT

p00515<213> group B streptococcus

p00516<210> 31

p00517<211> 783

p00518<212> DNA

p00519<213> group B streptococcus

p0052025 <400> 31

p00521<210> 32

p00522<211> 261

p00523<212> PRT

p00524<213> group B streptococcus

p00525<400> 32

p00526<210> 33

p00527<211> 1242

p00528<212> DNA

p00529<213> group B streptococcus

p00530<400> 33

55 <210> 34

p00531<211> 414

p00532<212> PRT

p00533<213> group B streptococcus

p00534<400> 34

p00535<210> 35

p00536<211> 930

p00537<212> DNA

p00538<213> group B streptococcus

p00539<400> 35

<dl><dt>45 </dt><dd><210> 36 <211> 310 <212> PRT <213> group B streptococcus <400> 36 </dd></dl>

<dl><dt>55 </dt><dd /></dl>

p00540<210> 37

p00541<211> 576

p00542<212> DNA

p00543<213> group B streptococcus

p0054455 <400> 37

p00545<210> 38

p00546<211> 192

p00547<212> PRT

p00548<213> group B streptococcus

p00549<400> 38

p00550<210> 39

p00551<211> 924

p00552<212> DNA

p00553<213> group B streptococcus

p00554<400> 39

p00555<210> 40

p00556<211> 307

p00557<212> PRT

p00558<213> group B streptococcus

p00559<220>

p00560<221> MISC_FEATURE

p00561<222> (281) .. (281) 45 <223> X is Lys or Glu

p00562<400> 40

<dl><dt><210> 41 </dt><dd /></dl>

<dl><dt><211> 1134 </dt><dd /></dl>

<dl><dt><212> DNA </dt><dd /></dl>

<dl><dt><213> group B streptococcus </dt><dd /></dl>

<dl><dt><400> 41 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

p00563<210> 42

p00564<211> 378 45 <212> PRT

p00565<213> group B streptococcus

p00566<400> 42