Patent Application
20090117113
This application incorporates by reference the contents of each of four CD-ROMs, each of which contains an identical 1.75 KB file labeled “sequence listing.txt” and containing the sequence listing for this application. The CD-ROMs were created on Oct. 11, 2005.
This invention is in the fields of immunology and vaccinology. In particular, it relates to antigens derived from Streptococcus pyogenes and their use in immunization.
Group A streptococcus (“GAS,” S. pyogenes) is a frequent human pathogen, estimated to be present in between 5-15% of normal individuals without signs of disease. An acute infection occurs, however, when host defenses are compromised, when the organism is able to exert its virulence, or when the organism is introduced to vulnerable tissues or hosts. Related diseases include puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotizing fasciitis, myositis, and streptococcal toxic shock syndrome.
GAS bacteria are gram positive, non-spore forming coccus-shaped bacteria which typically exist in chains or in pairs of cells. GAS bacteria are subdivided according to serotyping based on a large, highly variable cell surface antigen call the M protein. Lancefield, J. Exp. Med. 47, 9-10, 1928; Lancefield, J. Immunol. 89, 307-13, 1962. DNA sequencing of genes encoding M proteins has become the most common method of determining GASM types (emm sequence types). To date 124 different M types have been identified; 22 of these types were identified between 1995 and 1998 (Facklam et al., Clin. Infect. Dis. 34, 28-38, 2002). M1, M28, M12, M3, M11, and M6 are among the most prevalent GAS types worldwide. Li et al., Infect. Dis. 188, 1587-92, 2003; O'Brien et al., Clin. Infect. Dis. 35, 268-76, 2002.
Although S. pyogenes infections can be treated using antibiotics, there is a need in the art for prophylactic vaccines to prevent the onset of disease, as well as for additional therapies for treating S. pyogenes infections.
The invention provides compositions for preventing and/or treating S. pyogenes infection. These compositions comprise one or more active agents, which can be GAS antigens expressed on the surface of GAS bacteria, nucleic acid molecules encoding the GAS antigens, and/or antibodies which selectively bind to the GAS antigens.
“GAS antigens” according to the invention include (1) naturally occurring immunogenic proteins of a GAS bacterium, (2) immunogenic portions of such proteins, and (3) engineered proteins or portions of proteins with amino acid sequences which retain immunogenicity and which are at least 50% identical to the amino acid sequence of a naturally occurring GAS immunogenic protein or portion thereof, such as homologs, orthologs, allelic variants, and mutants. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). Typically, 50% identity or more between two polypeptide sequences is considered to be an indication of functional equivalence. Identity between polypeptides is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.
Amino acid sequences for examples of GAS proteins, as well as nucleotide sequences encoding the proteins, are identified in Table 1.
Preferably, a GAS antigen is shorter than a GAS protein by at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 76, 80, 85, 90, 95, 100, or more amino acids). More preferably a GAS antigen lacks a transmembrane domain. Even more preferably, a GAS antigen comprises a surface-exposed domain.
The invention also includes various polypeptide fragments (including immunogenic portions) of the identified GAS proteins. The length of a fragment may vary depending on the amino acid sequence of the particular GAS antigen. Typically, fragments of GAS proteins comprise at least 7 contiguous amino acids (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 178, 200, 203, 250 or more contiguous amino acids).
Preferably the fragment comprises one or more epitopes. The fragment may comprise at least one T-cell or, preferably, a B-cell epitope of the sequence. T- and B-cell epitopes can be identified empirically (e.g., using PEPSCAN (Geysen et al. (1984) PNAS USA 81:3998-4002; Carter (1994) Methods Mol. Biol. 36:207-223, or similar methods), or they can be predicted (e.g., using the Jameson-Wolf antigenic index (Jameson, B A et al. 1988, CABIOS 4(1):1818-186), matrix-based approaches (Raddrizzani and Hammer (2000) Brief Bioinform. 1(2):179-189), TEPITOPE (De Lalla et al. (199) J. Immunol. 163:1725-1729), neural networks (Brusic et al. (1998) Bioinformatics 14(2):121-130), OptiMer & EpiMer (Meister et al. (1995) Vaccine 13(6):581-591; Roberts et al. (1996) AIDS Res. Hum. Retroviruses 12(7):593-610), ADEPT (Maksyutov & Zagrebelnaya (1993) Comput. Appl. Biosci. 9(3):291-297), Tsites (Feller & de la Cruz (1991) Nature 349(6311):720-721), hydrophilicity (Hopp (1993) Peptide Research 6:183-190), antigenic index (Welling et al. (1985) FEBS Lett. 188:215-218) or the methods disclosed in Davenport et al. (1995) Immunogenetics 42:392-297, etc.
Other preferred fragments include (1) the N-terminal signal peptides of each identified GAS protein, (2) the identified GAS protein without its N-terminal signal peptide, (3) each identified GAS protein wherein up to 10 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the N-terminus and/or the C-terminus, and (4) GAS polypeptides without their N-terminal amino acid residue. Some fragments omit one or more domains of the protein (e.g., omission of a signal peptide, a cytoplasmic domain, a transmembrane domain, and/or an extracellular domain).
Some GAS antigens consist of immunogenic portions of GAS proteins, which can be surface exposed domains as disclosed herein. Other GAS antigens are “hybrid GAS antigens,” which comprise one or more immunogenic portions of a full-length GAS protein. Hybrid GAS antigens, which also can include full-length GAS antigens, are described in detail below. Other fusion proteins can comprise, for example, one or more additional antigens and/or a tag protein, such as polyhistidine (HIS) or glutathione-S-transferase (GST).
Preferably, a GAS antigen is expressed on the surface of a GAS bacterium, most preferably on the surface of more than one M type (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 M types), particularly M1, M3, M6, M11, M12, and/or M23 GAS types. GAS antigens also preferably are found on the surface of at least two different strains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more strains). Preferred GAS antigens are highly conserved among multiple M types and/or multiple strains within an M type. See Table 2, which lists full-length GAS proteins and M types on which the proteins are expressed. Columns 3-13 of Table 2 list M types (e.g., M1, M2, etc.). The presence these GAS proteins were detected on the surface of various strains of these M types as explained in Example 1; the number of strains tested within each type is shown in parentheses in columns 3-13. The final column lists the number of strains out of the total of 20 strains tested which express each of these GAS antigens.
As indicated in Table 2, some GAS antigens are expressed on the surface of a number of different M types as well as on the surface of multiple strains within some of these M types. In some embodiments, compositions of the invention comprise one or more GAS antigens which are expressed on the surface of an M1, M3, M6, M11, M12, and/or M23 type. Preferred GAS antigens of this type GAS 5, 99, 166, 96, 103, 188, 76, 108, 142, 190, 22, 56, 77, 67, 75, 93, 18, 23, 69, 206, 249, 123, 143, 68, 25, 30, 97, 105, 187, 195, 242, 81, 101, 6, 62, 49, 63, 85, 89, 100, 179, 205, 291, 98, 104, 36, 92, 158, 178, 218, 175, 78, 131, 29, 82, 91, 165, 327, 219, 60, 86, 380, 207, 271, 74, and 685 antigens. Even more preferably, a GAS antigen is exposed on at least 10 M types (e.g., GAS 5, 22, 40, 56, 67, 76, 77, 96, 99, 103, 108, 142, 166, 188, and 190).
GAS antigens of the invention also include surface-exposed domains of GAS proteins 4, 5, 15, 16, 23, 24, 25, 40, 49, 54, 57, 63, 64, 68, 72, 84, 86, 87, 89, 98, 102, 103, 108, 143, 149, 152, 157, 158, 163, 166, 168, 171, 177, 188, 190, 191, 192, 193, 194, 195, 198, 201, 224, 251, 259, 262, 264, 268, 277, 282, 299, 382, 405, 406, 425, 433, 460, 469, 493, 500, 545, 558, 587, 645, 650, 685, 362-1, spy0080a, spy0272, spy0461, spy0611, spy0717, spy0792, spy1029, spy1073, spy1260, spy1613, spy1835, spy2005, spy2093, spy2178, NT01SP0246, spy0047, spy0127, and spy0686 (see Table 7).
Other GAS antigens include surface-exposed domains of GAS proteins 5, 10, 23, 24, 49, 56, 63, 67, 72, 78, 81, 83, 84, 86, 89, 98, 100, 103, 157, 160, 177, 192, 194, 201, 205, 284, 286, 292, 382, 396, 405, 406, 500, spy0047, spy0053, spy0056, spy0063, spy0069, spy0098, spy0127, spy0274, spy0611, spy0666, spy0686, spy0688, spy0731, spy0913, spy1200, spy1281, spy1721, spy1750, spy1805, spy2070, spy2092, spy2178, and gi-21909751 (see Table 8).
Still others include surface-exposed domains of GAS proteins 16, 57, 68, 143, 158, 166, 171, 188, 190, 191, 192, 23, NT01SP0246, 49, 685, 63, 108, 84, 86, 89, 98, 103, 4, 149, 152, 157, 72, 405, 406, 299, 168, 251, 259, 262, 177, 264, 268, 277, 193, 194, 282, 195, 201, 40, 224, 163, 500, 198, 433, 54, 545, 469, 587, 645, 425, 493, 460, 558, 650, 5, 24, 25, 64, 87, 362-1, 382, 102, NT01SP0485, NT01SP0572, NT01SP0634, and NT01SP0877 (see Table 9).
Some surface-exposed domains are shown in SEQ ID NOS:591-649. Other surface-exposed GAS antigens comprise at least 7 contiguous amino acids selected from the group consisting of SEQ ID NOS:1-281 (i.e., 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 75, or 100 or more).
GAS antigens also include the surface-exposed domains of GAS proteins 35, 54, 70, 414, 421, 425, 426, 428, 433, 434, 437, 438, 439, 457, 461, 465, 469, 472, 473, 474, 475, 477, 478, 486, 492, 494, 495, 535, 538, 540, 543, 553, 560, 561, 564, 565, 574, 576, 577, 579, 586, 587, 591, 592, 607, 609, 625, 626, 636, 640, 643, 649, 653, 657, and 663. More preferred GAS antigens comprise a surface-exposed domain of 35, 414, 437, 438, 461, 465-2, 469, 472, 473, 475, 478, 495, 538, 553, 561, 577-2, 591, 593, 636, 643, 649, or 663. Even more preferred surface-exposed GAS antigens comprise a surface-exposed domain of GAS472, GAS473, or GAS553.
Other useful GAS antigens include GAS117, GAS130, GAS277, GAS236, GAS389, GAS504, GAS509, GAS366, GAS159, GAS217, GAS309, GAS372, GAS039, GAS042, GAS058, GAS290, GAS511, GAS533, GAS527, GAS294, GAS253, GAS529, GAS045, GAS095, GAS193, GAS137, GAS084, GAS384, GAS202, and GAS057 antigens, as well as M protein, GAS fibronectin-binding protein, GAS streptococcal heme-associated protein, and streptolysin S antigens.
Preferred groups of GAS antigens for use in vaccines of the present invention include:
(i) GAS4, GAS24, GAS54, GAS63, GAS64, GAS72, GAS86, GAS87, GAS102, GAS149, GAS152, GAS157, GAS163, GAS168, GAS171, GAS177, GAS191, GAS192, GAS194, GAS198, GAS201, GAS224, GAS251, GAS259, GAS262, GAS264, GAS268, GAS282, GAS299, GAS382, GAS405, GAS406, GAS425, GAS433, GAS460, GAS469, GAS493, GAS500, GAS545, GAS558, GAS587, GAS645, GAS650, GAS685, GAS362-1, spy611, spy717, spy792, spy1073, NT01SP0246, and NT01SP0102;
(ii) GAS64, GAS149, GAS158, GAS166, GAS191, GAS192, GAS193, SPY1664, and SPY0861;
(iii) GAS57, GAS64, GAS72, GAS84, GAS98, GAS108, GAS152, GAS157, GAS158, GAS166, GAS191, GAS192, GAS193, GAS268, NT01SP0246, NT01SP0908 (Spy1111), and NT01SP0182 (Spy0216);
(iv) GAS64, GAS158, GAS166, GAS191, GAS192, and GAS193; and
(v) GAS5, GAS6, GAS15, GAS16p2, GAS18, GAS22, GAS23, GAS25, GAS29, GAS30, GAS36, GAS40a-RR, GAS42, GAS45, GAS49, GAS56, GAS57, GAS60, GAS62, GAS63, GAS65, GAS67, GAS68, GAS69, GAS75, GAS76, GAS77, GAS81, GAS82, GAS84, GAS85, GAS86, GAS88, GAS89, GAS91, GAS92, GAS94, GAS95, GAS96, GAS97, GAS98, M30098, GAS99, GAS100, M3—0100, GAS101, M3—0102, GAS103, M3—0104, GAS105, SPs0106, GAS108, GAS117-40+A97, GAS130, GAS137, GAS142, GAS143, M6—0157, GAS158, M6—0159, GAS159a, M6—0160, GAS165, GAS166, GAS175, GAS178, GAS179-1, GAS187, GAS188, GAS190, GAS191, GAS193, GAS195, GAS205-1, GAS206, GAS208, GAS217, GAS218, GAS218-t, GAS219-1, GAS220, GAS242, GAS249, GAS277a, GAS290, GAS294-1, GAS327, GAS380, GAS384-RR, GAS504, GAS509, GAS511, GAS527, GAS529, GAS533, GAS680, 19224134, 19224135, 19224137, and 19224141 (see Table 16).
GAS 680 is annotated as a predicted CoA-binding protein and corresponds to M1 GenBank accession numbers GI:13621481 and GI:71909974, to M49 GenBank accession number GI:56808534, to M18 GenBank accession number GI:19747454, to M3 GenBank accession number GI: 28895062, and is also referred to as ‘Spy0186’ or ‘M5005_Spy—0160’ (M1), ‘SpyoM01000450’ (M49), ‘spyM18—0185’ (M18) and ‘SPs0150’ (M3).
GAS vaccines of the invention preferably include all or a surface portion of GAS57 and/or GAS40.
GAS40 Antigens
GAS40 antigens are particularly useful in compositions of the invention because GAS40 proteins are highly conserved both in many M types and in multiple strains of these M types (see
Amino acid sequences of a number of GAS40 proteins from various M strains are provided in SEQ ID NOS:17-43. The amino acid sequences of several GAS40 proteins also are contained in GenBank and have accession numbers GI:13621545 and
GI:15674449 (M1); accession number GI: 21909733 (M3), and accession number GI:19745402 (M18). GAS40 proteins also are known as “Spy0269” (M1), “SpyM3—0197” (M3), “SpyM18—0256” (M18) and “prgA.”
A GAS40 protein typically contains a leader peptide sequence (e.g., amino acids 1-26 of SEQ ID NO:17), a first coiled-coil region (e.g., amino acids 58-261 of SEQ ID NO:17), a second coiled coil region (e.g., amino acids 556-733 of SEQ ID NO:17), a leucine zipper region (e.g., amino acids 673-701 of SEQ ID NO:17) and a transmembrane region (e.g., amino acids 855-866 of SEQ ID NO:17).
Preferred fragments of a GAS40 protein lack one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the GAS40 protein. In one embodiment, the leader sequence is removed. In another embodiment, the transmembrane region is removed. Other fragments may omit one or more other domains of the GAS40 protein.
The coiled-coil regions of GAS40 are likely involved in the formation of oligomers such as dimers or trimers. Such oligomers could be homomers (containing two or more GAS40 proteins oligomerized together) or heteromers (containing one or more additional GAS proteins oligomerized with GAS40). Alternatively, two coiled-coil regions may interact together within the GAS40 protein to form oligomeric reactions between the first and second coiled-coil regions. Thus, in some embodiments the GAS40 antigen is in the form of an oligomer. Some oligomers comprise two more GAS40 antigens. Other oligomers comprise a GAS40 antigen oligomerized to a second GAS antigen.
Other useful GAS antigens include fusion proteins comprising GAS40 and GAS117. “40/117” is a GAS40 hybrid antigen in which the GAS 40 protein is placed to the N-terminus of the GAS117 protein and a HIS tag is added to the C terminus of the GAS117 protein (SEQ ID NO:234). “117/40” is a GAS40 hybrid antigen in which GAS117 is fused to GAS40 by the linker sequence YASGGGS (SEQ ID NO:278). Its amino acid sequence is shown in SEQ ID NO:233.
“GAS40a-HIS” is a GAS40 antigen with a HIS tag but without the leader and hydrophobic sequences (SEQ ID NO:235). A nucleotide sequence encoding GAS40a is shown in SEQ ID NO:892 (codon 824, AGA in the wild-type sequence, was mutagenized to CGT). “GAS40aRR” is similar to GAS40a except that two additional AGA codons (334 and 335) in the coding sequence were mutated to CGT.
Hybrid GAS Antigens
GAS antigens can be present in compositions of the invention as individual separate polypeptides. Alternatively, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) of any of the GAS antigens described above can be expressed as a single polypeptide chain, i.e., a “hybrid GAS antigen.” Hybrid GAS antigens offer two principal advantages. First, a polypeptide which may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner which overcomes the problem. Second, commercial manufacture is simplified because only one expression and purification produces two polypeptides, both of which are antigenically useful.
A hybrid GAS antigen can comprise two or more amino acid sequences for GAS40 antigens and/or one or more other GAS antigens of the invention. Hybrids can comprise amino acid sequences from two, three, four, five, six, seven, eight, nine, or ten or more GAS antigens. In compositions of the invention, a GAS antigen can be present in more than one hybrid GAS antigen and/or as a non hybrid GAS antigen.
A hybrid GAS antigen comprises at least two GAS antigens (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) expressed as a single polypeptide chain. Preferred hybrid GAS antigens comprise at least one surface-exposed and/or surface-associated GAS antigen. Hybrid GAS antigens offer two principal advantages. First, a polypeptide which may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner which overcomes the problem. Second, commercial manufacture is simplified because only one expression and purification produces two polypeptides, both of which are antigenically useful.
Hybrid GAS antigens can be represented by the formula:
NH2-A-(-X-L-)n-B—COOH
in which X is an amino acid sequence of a surface-exposed and/or surface-associated or secondary GAS antigen; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
If an —X— moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid antigen. In some embodiments, the leader peptides will be deleted except for that of the —X— moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of X1 will be retained, but the leader peptides of X2 . . . Xn will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of X1 as moiety -A-.
For each n instances of (—X-L-), linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH2—X1-L1-X2-L2-COOH, NH2—X1—X2—COOH, NH2—X1-L1-X2—COOH, NH2—X1—X2-L2-COOH, etc. Linker amino acid sequence(s)-L- will typically be short, e.g., 20 or fewer amino acids (i.e., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences which facilitate cloning, poly-glycine linkers (Gly, where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (Hisn where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG (SEQ ID NO:280), with the Gly-Ser dipeptide being formed from a BamHI restriction site, which aids cloning and manipulation, and the (Gly)4 tetrapeptide being a typical poly-glycine linker.
-A- is an optional N-terminal amino acid sequence. This will typically be short, e.g., 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35, 34, 33, 32, 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 leader sequences to direct protein trafficking or short peptide sequences which facilitate cloning or purification (e.g., a histidine tag His, 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-terminus methionine, -A- is preferably an oligopeptide (e.g., with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine.
—B— is an optional C-terminal amino acid sequence. This will typically be short, e.g., 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35, 34, 33, 32, 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 which facilitate cloning or purification (e.g., Hisn where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.
The individual GAS antigens within the hybrid (individual —X— moieties) may be from one or more strains or from one or more M types. Where n=2, for instance, X2 may be from the same strain or type as X1 or from a different strain or type. Where n=3, the strains might be (i) X1=X2=X3, (ii) X1=X2X3, (iii) X1
X2=X3, (iv) X1
X2/X3, or (v) X1=X3
X2, etc.
Identification of Surface-Exposed GAS Antigens
Surface-exposed and/or surface-associated GAS antigens (GAS “surfome”) can be identified using any one or combination of several proteomics approaches as outlined below. These proteomics strategies have great potential for shortening the time needed for vaccine discovery when compared with other strategies, such as reverse vaccinology. Surface-exposed and/or surface-associated GAS antigens identified by these methods can be used as active agents in compositions for preventing and for treating S. pyogenes infections.
One embodiment is described in Example 13. Briefly, the surface of whole GAS bacterial cells is digested in vivo under physiological conditions using reagents which cleave proteins. Typically the reagents are proteases (e.g., trypsin, protease K, papain), although any protein cleavage reagent can be used. These reagents include, for example, formic acid, hydroxylamine, BNPS-skatole (3-bromo-3-methyl-2-(o-nitrophenyl-sulfenyl)-indolenine), which cleaves at Trp residues), cyanogen bromide (which cleaves polypeptides on the carboxyl side of methionine residues), metal chelate reagents such as Fe-EDTA, and the like. Proteases can be either free or anchored, this latter condition favouring the identification of surface extruding regions. Combinations of more than one protein cleavage reagent can be used. The recovered peptides are then separated by liquid chromatography and identified by tandem mass spectrometry. The actual accessibility of identified proteins to the immune system can be assessed by fluorescence-activated cell sorting (FACS) analysis. This proteomic approach permits validation of software-based topology predictions and vice versa.
Another embodiment is described in Example 14. This approach involves overproduction of membrane-delimited from GAS bacteria after antibiotic treatment. See Hakenbeck et al., J. Bacteriol. 155, 1372-81, 1983, which is incorporated herein by reference. Either wild-type GAS bacteria or mutant GAS bacteria, for example those with “leaky” or destabilized peptidoglycan cell walls, can be used in this method. GAS bacteria naturally produce membrane-delimited structures which are released into the growth medium. When the bacteria are treated with an antibiotic that interferes with the synthesis of the cell-wall such as penicillins, cephalosporins, glycopeptides and cycloserine, production of these membrane-delimited structures increases. Vancomycin, a glycopeptide which inhibits both cell wall synthesis and the sortase, interferes with surface protein anchoring which is catalyzed by sortases and can be used to further increase the overproduction of membrane-delimited structures. The membrane-delimited structures contain GAS proteins which are potential vaccine candidates. The GAS proteins can be separated by electrophoresis and identified using mass spectrometry (e.g., MALDI-TOF). Alternatively, the GAS proteins can be digested with proteases, and the resulting fragments can be separated by liquid chromatography and identified using tandem mass spectrometry.
A third embodiment is described in Example 15. In this method, cell wall and/or membrane fractions are generated by chemical cell fractionation of bacterial cells using, for example, 6 M guanidinium, urea, or SDS. The cell wall is insoluble in these reagents. This property allows the isolation of the cell wall and identification of anchored cell wall proteins. GAS proteins in these fractions can be separated and identified as described above.
A fourth embodiment involves labeling cell surface GAS proteins (e.g., by biotinylation), lysing the cells, and isolating labeled proteins using affinity chromatography. The isolated proteins can be separated by electrophoresis and identified using mass spectrometry. Alternatively, the isolated proteins can be digested in solution, followed by isolation of labeled peptides by affinity chromatography, separation of the labeled peptides by liquid chromatography, and identification of the labeled peptides using tandem mass spectrometry. These methods selectively isolate the labeled peptides, therefore they allow identification of the truly exposed domains. In this case, the use of two affinity chromatography steps results in a reduction of complexity of the sample to be loaded on the chromatography column.
For all the above embodiments a mutant can be used which harbors a deleted gene for one of the more abundant known surface-exposed antigens, such as M protein and C5a peptidase. These mutants will increase the probability of spotting previously unidentified, less abundant surface proteins.
Analysis of the bacterial surfome provides powerful methods of identifying antigens useful in vaccines against S. pyogenes. For example, using these techniques, as described below, we identified a protein, Spy0416 (GAS57), which confers a remarkable protection in mice against the highly virulent M3 (MGAS315) strain. Spy0416 is a 1647 amino acid protein, carrying a C-terminal LPXTG-like motif, which shares 48% similarity with the C5a peptidase precursor. See SEQ ID NO:118. The protein has a Ca-dependent serine protease activity (Femandez-Espla, App. Env. Microbiol., 2000) which maps within the first 600 amino acids of the protein. Spy0416 has a homolog in Group B Streptococcus (GBS) (cspA) which was proposed to be involved in GBS virulence by potentially protecting the bacterium from opsonophagocytic killing (Harris et al., J. Clin. Invest. 111, 61-70, 2003). Lei and co-workers recently found that a 31 kDa N-terminal fragment of Spy0416 is released in the supernatant of GAS cultures and that the protein is well recognized by sera from GAS-infected patients (Lei et al, Inf. Immunol. 68, 6807-18, 2000). Based on the 5 available Streptococcus pyogenes genome sequences, the protein appears to be highly conserved (over 98%) and preliminary data on surface expression on a panel of 20 different GAS strains indicates that Spy0416 is a major component of over 70% of the circulating strains. It is, therefore, a preferred antigen for use in immunogenic compositions, either alone or in combination with one or more other GAS antigens.
The sequence listing provides coding sequences for the surface-exposed and/or surface-associated domains disclosed herein and their full-length proteins, as well as for the additional disclosed secondary GAS antigens. Any nucleotide sequence which encodes a particular antigen, however, can be used in a compositions of the invention, for example as a DNA vaccine, or to produce a GAS antigen recombinantly, as described below. The full genomic sequences of at least three GAS strains are publicly available and can be used to obtain coding sequences for GAS antigens. The genomic sequence of an M1 GAS strain is reported in Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98, 4658-63, 2002. The genomic sequence of an M3 GAS strain is reported in Beres et al., Proc. Natl. Acad. Sci. U.S.A. 99, 10078-83, 2002. The genomic sequence of an M18 GAS strain is reported in Smooet et al., Proc. Natl. Acad. Sci. U.S.A. 99, 4668-73, 2002.
The invention includes nucleic acid molecules which encode the identified GAS proteins and protein fragments. The invention also includes nucleic acid molecules comprising nucleotide sequences having at least 50% sequence identity to such molecules. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). Identity between nucleotide sequences is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.
The invention also provides nucleic acid molecules which can hybridize to these molecules. Hybridization reactions can be performed under conditions of different “stringency.” Conditions which increase stringency of a hybridization reaction are widely known and published in the art. See, e.g., page 7.52 of Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C., 55° C., and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, and 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or de-ionized water. Hybridization techniques and their optimization are well known in the art. See, e.g., Sambrook, 1989; Ausubel et al., eds., Short Protocols in Molecular Biology, 4th ed., 1999; U.S. Pat. No. 5,707,829; Ausubel et al., eds., Current Protocols in Molecular Biology, Supplement 30, 1987.
In some embodiments, nucleic acid molecules of the invention hybridize to a target under low stringency conditions; in other embodiments, nucleic acid molecules of the invention hybridize under intermediate stringency conditions; in preferred embodiments, nucleic acid molecules of the invention hybridize under high stringency conditions. An example of a low stringency hybridization condition is 50° C. and 10×SSC. An example of an intermediate stringency hybridization condition is 55° C. and 1×SSC. An example of a high stringency hybridization condition is 68° C. and 0.1×SSC.
Nucleic acid molecules comprising fragments of these sequences are also included in the invention. These comprise at least n consecutive nucleotides of these sequences and, depending on the particular sequence, n is 10 or more (e.g., 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more).
Nucleic acids (and polypeptides) of the invention may include sequences which:
(a) are identical (i.e., 100% identical) to the sequences disclosed in the sequence listing;
(b) share sequence identity with the sequences disclosed in the sequence listing;
(c) have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single nucleotide or amino acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b); and,
d) when aligned with a particular sequence from the sequence listing using a pairwise alignment algorithm, a moving window of x monomers (amino acids or nucleotides) moving from start (N-terminus or 5′) to end (C-terminus or 3′), such that for an alignment that extends to p monomers (where p>x) there are p−x+1 such windows, each window has at least x·y identical aligned monomers, where: x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and if x·y is not an integer then it is rounded up to the nearest integer. The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453], using default parameters (e.g., with Gap opening penalty=10.0, and with Gap extension penalty=0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [Rice et al. (2000) Trends Genet. 16:276-277].
The nucleic acids and polypeptides of the invention may additionally have further sequences to the N-terminus/5′ and/or C-terminus/3′ of these sequences (a) to (d).
Antibodies can be generated to bind specifically to a surface-exposed and/or surface-associated GAS antigen or to a secondary GAS or non-GAS polypeptide antigen disclosed herein. The term “antibody” includes intact immunoglobulin molecules, as well as fragments thereof which are capable of binding an antigen. These include hybrid (chimeric) antibody molecules (e.g., Winter et al., Nature 349, 293-99, 1991; U.S. Pat. No. 4,816,567); F(ab′)2 and F(ab) fragments and Fv molecules; non-covalent heterodimers (e.g., Inbar et al., Proc. Natl. Acad. Sci. U.S.A. 69, 2659-62, 1972; Ehrlich et al., Biochem 19, 4091-96, 1980); single-chain Fv molecules (sFv) (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A. 85, 5897-83, 1988); dimeric and trimeric antibody fragment constructs; minibodies (e.g., Pack et al., Biochem 31, 1579-84, 1992; Cumber et al., J. Immunology 149B, 120-26, 1992); humanized antibody molecules (e.g., Riechmann et al., Nature 332, 323-27, 1988; Verhoeyan et al., Science 239, 1534-36, 1988; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and any functional fragments obtained from such molecules, as well as antibodies obtained through non-conventional processes such as phage display. Preferably, the antibodies are monoclonal antibodies. Methods of obtaining monoclonal antibodies are well known in the art.
Typically, at least 6, 7, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids. Various immunoassays (e.g., Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art) can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen. A preparation of antibodies which specifically bind to a particular antigen typically provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, the antibodies do not detect other proteins in immunochemical assays and can immunoprecipitate the particular antigen from solution.
GAS antigens or non-GAS polypeptide antigens (described below) can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, an antigen can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.
Monoclonal antibodies which specifically bind to an antigen can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (Kohler et al., Nature 256, 495 497, 1985; Kozbor et al., J. Immunol. Methods 81, 3142, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026 2030, 1983; Cole et al., Mol. Cell. Biol. 62, 109 120, 1984).
In addition, techniques developed for the production of “chimeric antibodies,” the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et al., Nature 312, 604 608, 1984; Takeda et al., Nature 314, 452 454, 1985). Monoclonal and other antibodies also can be “humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
Alternatively, humanized antibodies can be produced using recombinant methods, as described below. Antibodies which specifically bind to a particular antigen can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.
Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to a particular antigen. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120 23, 1991).
Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).
Antibodies which specifically bind to a particular antigen also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833 3837, 1989; Winter et al., Nature 349, 293 299, 1991).
Chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the “diabodies” described in WO 94/13804, also can be prepared.
Antibodies can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which the relevant antigen is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
Recombinant Production of Polypeptides
Any nucleotide sequence which encodes a particular antigen can be used to produce that antigen recombinantly. If desired, an antibody can be produced recombinantly once its amino acid sequence is known.
Examples of sequences which can be used to produce GAS antigens of the invention are identified in Table 1. Nucleic acid molecules encoding surface-exposed and/or surface-associated or secondary GAS antigens can be isolated from the appropriate S. pyogenes bacterium using standard nucleic acid purification techniques or can be synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating nucleic acids are routine and are known in the art. Any such technique for obtaining nucleic acid molecules can be used to obtain a nucleic acid molecule which encodes a particular antigen. Sequences encoding a particular antigen or antibody can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser. 215 223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225 232, 1980).
cDNA molecules can be made with standard molecular biology techniques, using mRNA as a template. cDNA molecules can thereafter be replicated using molecular biology techniques well known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either genomic DNA or cDNA as a template.
If desired, nucleotide sequences can be engineered using methods generally known in the art to alter antigen-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
Sequence modifications, such as the addition of a purification tag sequence or codon optimization, can be used to facilitate expression. For example, the N-terminal leader sequence may be replaced with a sequence encoding for a tag protein such as polyhistidine (“HIS”) or glutathione S-transferase (“GST”). Such tag proteins may be used to facilitate purification, detection, and stability of the expressed protein. Codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half life which is longer than that of a transcript generated from the naturally occurring sequence. These methods are well known in the art and are further described in WO05/032582.
Expression Vectors
A nucleic acid molecule which encodes an antigen or antibody can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
Host Cells
The heterologous host can be prokaryotic or eukaryotic. E. coli is a preferred host cell, but other suitable hosts include Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis), yeasts, etc.
A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post translational processing which cleaves a “prepro” form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post translational activities are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of a foreign protein. See WO 01/98340.
Expression constructs can be introduced into host cells using well-established techniques which include, but are not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, “gene gun” methods, and DEAE- or calcium phosphate-mediated transfection.
Host cells transformed with expression vectors can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell can be secreted or contained intracellularly depending on the nucleotide sequence and/or the expression vector used. Those of skill in the art understand that expression vectors can be designed to contain signal sequences which direct secretion of soluble antigens through a prokaryotic or eukaryotic cell membrane.
Purification
Antigens used in the invention can be isolated from the appropriate Streptococcus pyogenes bacterium or from a host cell engineered to produce GAS or non-GAS antigens. A purified polypeptide antigen is separated from other components in the cell, such as proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified polypeptide antigens is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. Where appropriate, polypeptide antigens can be solubilized, for example, with urea.
Chemical Synthesis
GAS antigens, as well as other antigens used in compositions of the invention, can be synthesized, for example, using solid phase techniques. See, e.g., Merrifield, J. Am. Chem. Soc. 85, 2149 54, 1963; Roberge et al., Science 269, 202 04, 1995. Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of a surface-exposed and/or surface-associated GAS antigen can be separately synthesized and combined using chemical methods to produce a full-length molecule.
Nucleic acid molecules which encode antibodies or polypeptide antigens can be synthesized by conventional methodology, such as the phosphate triester method (Hunkapiller, M. et al. (1984), Nature 310: 105-111) or by the chemical synthesis of nucleic acids (Grantham, R. et al. (1981), Nucleic Acids Res. 9: r43-r74).
The invention also provides compositions for use as medicaments (e.g., as immunogenic compositions or vaccines) or as diagnostic reagents for detecting a GAS infection in a host subject. It also provides the use of the compositions in the manufacture of: (i) a medicament for treating or preventing infection due to GAS bacteria; (ii) a diagnostic reagent for detecting the presence of GAS bacteria or of antibodies raised against GAS bacteria; and/or (iii) a reagent which can raise antibodies against GAS bacteria.
For example, GAS antigens or nucleic acids encoding the antigens can be used in the manufacture of a diagnostic reagent for detecting the presence of a GAS infection or for detecting antibodies raised against GAS bacteria, or in the manufacture of a reagent which can raise antibodies against GAS bacteria. Nucleic acids encoding GAS antigens can be detected by contacting a nucleic acid probe with a biological sample under hybridizing conditions to form duplexes and detecting the duplexes as is known in the art. A GAS antigen can be detected using antibodies which specifically bind to the GAS antigen. Similarly, antibodies to GAS antigens can be used to detect GAS antigens by contacting a biological sample under conditions suitable for the formation of antibody-antigen complexes and detecting any complexes formed. The invention also provides kits comprising reagents suitable for use these methods.
Compositions of the invention are useful for preventing and/or treating S. pyogenes infection. Compositions containing GAS antigens are preferably immunogenic compositions, and are more preferably vaccine compositions. The pH of such compositions preferably is between 6 and 8, preferably about 7. The pH can be maintained by the use of a buffer. The composition can be sterile and/or pyrogen free. The composition can be isotonic with respect to humans.
Vaccines according to the invention may be used either prophylactically or therapeutically, but will typically be prophylactic. Accordingly, the invention includes a method for the therapeutic or prophylactic treatment of a Streptococcus pyogenes infection. The animal is preferably a mammal, most preferably a human. The methods involve administering to the animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention.
Some compositions of the invention comprise at least two surface-exposed GAS antigens as described above. Other compositions of the invention comprise at least one nucleic acid molecule which encodes two surface-exposed GAS antigens. Still other compositions of the invention comprise at least two antibodies, each of which specifically binds to one of two surface-exposed GAS antigens. Preferred compositions of the invention comprise at least one of the surface-exposed GAS antigens is a GAS40 antigen and the other antigen is any other GAS antigen; at least one nucleic acid molecule encoding the two antigens, or at least two antibodies which specifically bind to the two antigens. Some compositions comprise one or more additional GAS antigens, a nucleic acid molecule encoding the additional antigen(s), or an antibody which specifically binds to the additional antigen(s); of these antigens, GAS117 is preferred.
As described above, some compositions of the invention comprise a nucleic acid molecule which encodes the at least two GAS antigens and, optionally, other antigens which can be included in the composition (see below). See, e.g., Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Ann. Rev Immunol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9:471-480; Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther 2:441-447; Ilan (1999) Curr Opin Mol Ther 1:116-120; Dubensky et al. (2000) Mol Med 6:723-732; Robinson & Pertmer (2000) Adv Virus Res 55:1-74; Donnelly et al. (2000) Am J Respir Crit. Care Med 162(4 Pt 2):S190-193; Davis (1999) Mt. Sinai J. Med. 66:84-90. Typically the nucleic acid molecule is a DNA molecule, e.g., in the form of a plasmid.
Compositions for treating S. pyogenes infections comprise at least one antibody which specifically binds to a GAS antigen and, optionally, an antibody which specifically binds to a non-GAS antigen. Some compositions of the invention are immunogenic and comprise one or more polypeptide antigens, while other immunogenic compositions comprise nucleic acid molecules which encode a surface-exposed and/or surface-associated GAS antigen and, optionally, a secondary GAS antigen or a non-GAS antigen. See, e.g., Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Ann. Rev Immunol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9:471-480; Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther 2:441-447; Ilan (1999) Curr Opin Mol Ther 1:116-120; Dubensky et al. (2000) Mol Med 6:723-732; Robinson & Pertmer (2000) Adv Virus Res 55:1-74; Donnelly et al. (2000) Am J Respir Crit. Care Med 162(4 Pt 2):S190-193Davis (1999) Mt. Sinai J. Med. 66:84-90. Typically the nucleic acid molecule is a DNA molecule, e.g., in the form of a plasmid.
Other compositions of the invention comprise at least one active agent. Compositions for preventing S. pyogenes infection can comprise as an active agent either a polypeptide comprising a GAS antigen of the invention or a nucleic acid molecule which encodes the polypeptide.
In some embodiments, compositions of the invention can include one or more additional active agents. Such agents include, but are not limited to, (a) another GAS antigen of the invention, preferably a surface-exposed antigen, (b) a polypeptide antigen which is useful in a pediatric vaccine, (c) a polypeptide antigen which is useful in a vaccine for elderly or immunocompromised individuals, (d) a nucleic acid molecule encoding (a)-(c), and an antibody which specifically binds to (a)-(c).
Additional Antigens
Compositions of the invention may be administered in conjunction with one or more antigens for use in therapeutic, prophylactic, or diagnostic methods of the present invention. Preferred antigens include those listed below. Additionally, the compositions of the present invention may be used to treat or prevent infections caused by any of the below-listed pathogens. In addition to combination with the antigens described below, the compositions of the invention may also be combined with an adjuvant as described herein.
Antigens for use with the invention include, but are not limited to, one or more of the following antigens set forth below, or antigens derived from one or more of the pathogens set forth below:
A. Bacterial Antigens
Bacterial antigens suitable for use in the invention include proteins, polysaccharides, lipopolysaccharides, and outer membrane vesicles which may be isolated, purified or derived from a bacteria. In addition, bacterial antigens may include bacterial lysates and inactivated bacteria formulations. Bacteria antigens may be produced by recombinant expression. Bacterial antigens preferably include epitopes which are exposed on the surface of the bacteria during at least one stage of its life cycle. Bacterial antigens are preferably conserved across multiple serotypes. Bacterial antigens include antigens derived from one or more of the bacteria set forth below as well as the specific antigens examples identified below.
Neisseria meningitides: Meningitides antigens may include proteins (such as those identified in References 1-7), saccharides (including a polysaccharide, oligosaccharide or lipopolysaccharide), or outer-membrane vesicles (References 8, 9, 10, 11) purified or derived from N. meningitides serogroup such as A, C, W135, Y, and/or B. Meningitides protein antigens may be selected from adhesions, autotransporters, toxins, Fe acquisition proteins, and membrane associated proteins (preferably integral outer membrane protein).
Streptococcus pneumoniae: Streptococcus pneumoniae antigens may include a saccharide (including a polysaccharide or an oligosaccharide) and/or protein from Streptococcus pneumoniae. Saccharide antigens may be selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Protein antigens may be selected from a protein identified in WO 98/18931, WO 98/18930, U.S. Pat. No. 6,699,703, U.S. Pat. No. 6,800,744, WO 97/43303, and WO 97/37026.
Streptococcus pneumoniae proteins may be selected from the Poly Histidine Triad family (PhtX), the Choline Binding Protein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins, pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 or Sp133.
Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcus antigens may include a protein identified in WO 02/34771 or WO 2005/032582 (including GAS 40), fusions of fragments of GAS M proteins (including those described in WO 02/094851, and Dale, Vaccine (1999) 17:193-200, and Dale, Vaccine 14(10): 944-948), fibronectin binding protein (Sfb1), Streptococcal heme-associated protein (Shp), and Streptolysin S (SagA).
Moraxella catarrhalis: Moraxella antigens include antigens identified in WO 02/18595 and WO 99/58562, outer membrane protein antigens (HMW-OMP), C-antigen, and/or LPS.
Bordetella pertussis: Pertussis antigens include petussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also combination with pertactin and/or agglutinogens 2 and 3 antigen.
Staphylococcus aureus: Staphylococcus aureus antigens include S. aureus type 5 and 8 capsular polysaccharides optionally conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, or antigens derived from surface proteins, invasins (leukocidin, kinases, hyaluronidase), surface factors that inhibit phagocytic engulfment (capsule, Protein A), carotenoids, catalase production, Protein A, coagulase, clotting factor, and/or membrane-damaging toxins (optionally detoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin).
Staphylococcus epidermis: S. epidermidis antigens include slime-associated antigen (SAA).
Clostridium tetani (Tetanus): Tetanus antigens include tetanus toxoid (TT), preferably used as a carrier protein in conjunction/conjugated with the compositions of the present invention.
Cornynebacterium diphtheriae (Diphtheria): Diphtheria antigens include diphtheria toxin, preferably detoxified, such as CRM197. Additionally antigens capable of modulating, inhibiting or associated with ADP ribosylation are contemplated for combination/co-administration/conjugation with the compositions of the present invention. The diphtheria toxoids may be used as carrier proteins.
Haemophilus influenzae B (Hib): Hib antigens include a Hib saccharide antigen.
Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzz protein, P. aeruginosa LPS, more particularly LPS isolated from PAO1 (O5 serotype), and/or Outer Membrane Proteins, including Outer Membrane Proteins F (OprF) (Infect Immun. 2001 May; 69(5): 3510-3515).
Legionella pneumophila. Bacterial antigens may be derived from Legionella pneumophila.
Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcus antigens include a protein or saccharide antigen identified in WO 02/34771, WO 03/093306, WO 04/041157, or WO 2005/002619 (including proteins GBS 80, GBS 104, GBS 276 and GBS 322, and including saccharide antigens derived from serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII).
Neiserria gonorrhoeae: Gonorrhoeae antigens include Por (or porin) protein, such as PorB (see Zhu et al., Vaccine (2004) 22:660-669), a transferring binding protein, such as TbpA and TbpB (See Price et al., Infection and Immunity (2004) 71(1):277-283), a opacity protein (such as Opa), a reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see Plante et al., J Infectious Disease (2000) 182:848-855), also see e.g. WO99/24578, WO99/36544, WO99/57280, WO02/079243).
Chlamydia trachomatis: Chlamydia trachomatis antigens include antigens derived from serotypes A, B, Ba and C (agents of trachoma, a cause of blindness), serotypes L1, L2 & L3 (associated with Lymphogranuloma venereum), and serotypes, D-K. Chlamydia trachomas antigens may also include an antigen identified in WO 00/37494, WO 03/049762, WO 03/068811, or WO 05/002619, including PepA (CT045), LcrE (CT089), ArtJ (CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG (CT761).
Treponema pallidum (Syphilis): Syphilis antigens include TmpA antigen.
Haemophilus ducreyi (causing chancroid): Ducreyi antigens include outer membrane protein (DsrA).
Enterococcus faecalis or Enterococcus faecium: Antigens include a trisaccharide repeat or other Enterococcus derived antigens provided in U.S. Pat. No. 6,756,361.
Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX, HopY and/or urease antigen.
Staphylococcus saprophyticus: Antigens include the 160 kDa hemagglutinin of S. saprophyticus antigen.
Yersinia enterocolitica antigens include LPS (Infect Immun. 2002 August; 70(8): 4414).
E. coli: E. coli antigens may be derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E. coli (EHEC).
Bacillus anthracis (anthrax): B. anthracis antigens are optionally detoxified and may be selected from A-components (lethal factor (LF) and edema factor (EF)), both of which can share a common B-component known as protective antigen (PA).
Yersinia pestis (plague): Plague antigens include F1 capsular antigen (Infect Immun. 2003 January; 71(1)): 374-383, LPS (Infect Immun. 1999 October; 67(10): 5395), Yersinia pestis V antigen (Infect Immun. 1997 November; 65(11): 4476-4482).
Mycobacterium tuberculosis: Tuberculosis antigens include lipoproteins, LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6 optionally formulated in cationic lipid vesicles (Infect Immun. 2004 October; 72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associated antigens (Proc Natl Acad Sci USA. 2004 Aug. 24; 101(34): 12652), and/or MPT51 antigens (Infect Immun. 2004 July; 72(7): 3829).
Rickettsia: Antigens include outer membrane proteins, including the outer membrane protein A and/or B (OmpB) (Biochim Biophys Acta. 2004 Nov. 1; 1702(2):145), LPS, and surface protein antigen (SPA) (J. Autoimmun. 1989 June; 2 Suppl:81).
Listeria monocytogenes. Bacterial antigens may be derived from Listeria monocytogenes.
Chlamydia pneumoniae: Antigens include those identified in WO 02/02606.
Vibrio cholerae: Antigens include proteinase antigens, LPS, particularly lipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specific polysaccharides, V. cholera 0139, antigens of IEM108 vaccine (Infect Immun. 2003 October; 71(10):5498-504), and/or Zonula occludens toxin (Zot).
Salmonella typhi (typhoid fever): Antigens include capsular polysaccharides preferably conjugates (Vi, i.e. vax-TyVi).
Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (such as OspA, OspB, Osp C and Osp D), other surface proteins such as OspE-related proteins (Erps), decorin-binding proteins (such as DbpA), and antigenically variable VI proteins., such as antigens associated with P39 and P13 (an integral membrane protein, Infect Immun. 2001 May; 69(5): 3323-3334), VlsE Antigenic Variation Protein (J Clin Microbiol. 1999 December; 37(12): 3997).
Porphyromonas gingivalis: Antigens include P. gingivalis outer membrane protein (OMP).
Klebsiella: Antigens include an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid.
Further bacterial antigens of the invention may be capsular antigens, polysaccharide antigens or protein antigens of any of the above. Further bacterial antigens may also include an outer membrane vesicle (OMV) preparation. Additionally, antigens include live, attenuated, and/or purified versions of any of the aforementioned bacteria. The antigens of the present invention may be derived from gram-negative or gram-positive bacteria. The antigens of the present invention may be derived from aerobic or anaerobic bacteria.
Additionally, any of the above bacterial-derived saccharides (polysaccharides, LPS, LOS or oligosaccharides) can be conjugated to another agent or antigen, such as a carrier protein (for example CRM197). Such conjugation may be direct conjugation effected by reductive amination of carbonyl moieties on the saccharide to amino groups on the protein, as provided in U.S. Pat. No. 5,360,897 and Can J Biochem Cell Biol. 1984 May; 62(5):270-5. Alternatively, the saccharides can be conjugated through a linker, such as, with succinamide or other linkages provided in Bioconjugate Techniques, 1996 and CRC, Chemistry of Protein Conjugation and Cross-Linking, 1993.
B. Viral Antigens
Viral antigens suitable for use in the invention include inactivated (or killed) virus, attenuated virus, split virus formulations, purified subunit formulations, viral proteins which may be isolated, purified or derived from a virus, and Virus Like Particles (VLPs). Viral antigens may be derived from viruses propagated on cell culture or other substrate. Alternatively, viral antigens may be expressed recombinantly. Viral antigens preferably include epitopes which are exposed on the surface of the virus during at least one stage of its life cycle. Viral antigens are preferably conserved across multiple serotypes or isolates. Viral antigens include antigens derived from one or more of the viruses set forth below as well as the specific antigens examples identified below.
Orthomyxovirus: Viral antigens may be derived from an Orthomyxovirus, such as Influenza A, B and C. Orthomyxovirus antigens may be selected from one or more of the viral proteins, including hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membrane protein (M2), one or more of the transcriptase components (PB1, PB2 and PA). Preferred antigens include HA and NA.
Influenza antigens may be derived from interpandemic (annual) flu strains. Alternatively influenza antigens may be derived from strains with the potential to cause pandemic a pandemic outbreak (i.e., influenza strains with new haemagglutinin compared to the haemagglutinin in currently circulating strains, or influenza strains which are pathogenic in avian subjects and have the potential to be transmitted horizontally in the human population, or influenza strains which are pathogenic to humans).
Paramyxoviridae viruses: Viral antigens may be derived from Paramyxoviridae viruses, such as Pneumoviruses (RSV), Paramyxoviruses (PIV) and Morbilliviruses (Measles).
Pneumovirus: Viral antigens may be derived from a Pneumovirus, such as Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkey rhinotracheitis virus. Preferably, the Pneumovirus is RSV. Pneumovirus antigens may be selected from one or more of the following proteins, including surface proteins Fusion (F), Glycoprotein (G) and Small Hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P and L and nonstructural proteins NS1 and NS2. Preferred Pneumovirus antigens include F, G and M. See e.g., J Gen Virol. 2004 November; 85(Pt 11):3229). Pneumovirus antigens may also be formulated in or derived from chimeric viruses. For example, chimeric RSV/PIV viruses may comprise components of both RSV and PIV.
Paramyxovirus: Viral antigens may be derived from a Paramyxovirus, such as Parainfluenza virus types 1-4 (PIV), Mumps, Sendai viruses, Simian virus 5, Bovine parainfluenza virus and Newcastle disease virus. Preferably, the Paramyxovirus is PIV or Mumps. Paramyxovirus antigens may be selected from one or more of the following proteins: Hemagglutinin-Neuraminidase (HN), Fusion proteins F1 and F2, Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrix protein (M). Preferred Paramyxovirus proteins include HN, F1 and F2. Paramyxovirus antigens may also be formulated in or derived from chimeric viruses. For example, chimeric RSV/PIV viruses may comprise components of both RSV and PIV. Commercially available mumps vaccines include live attenuated mumps virus, in either a monovalent form or in combination with measles and rubella vaccines (MMR).
Morbillivirus: Viral antigens may be derived from a Morbillivirus, such as Measles. Morbillivirus antigens may be selected from one or more of the following proteins: hemagglutinin (H), Glycoprotein (G), Fusion factor (F), Large protein (L), Nucleoprotein (NP), Polymerase phosphoprotein (P), and Matrix (M). Commercially available measles vaccines include live attenuated measles virus, typically in combination with mumps and rubella (MMR).
Picornavirus: Viral antigens may be derived from Picornaviruses, such as Enteroviruses, Rhinoviruses, Hepamavirus, Cardioviruses and Aphthoviruses. Antigens derived from Enteroviruses, such as Poliovirus are preferred.
Enterovirus: Viral antigens may be derived from an Enterovirus, such as Poliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11 to 27 and 29 to 34 and Enterovirus 68 to 71. Preferably, the Enterovirus is poliovirus. Enterovirus antigens are preferably selected from one or more of the following Capsid proteins VP1, VP2, VP3 and VP4. Commercially available polio vaccines include Inactivated Polio Vaccine (IPV) and Oral poliovirus vaccine (OPV).
Heparnavirus: Viral antigens may be derived from an Heparnavirus, such as Hepatitis A virus (HAV). Commercially available HAV vaccines include inactivated HAV vaccine.
Togavirus: Viral antigens may be derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus. Antigens derived from Rubivirus, such as Rubella virus, are preferred. Togavirus antigens may be selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. Togavirus antigens are preferably selected from E1, E2 or E3. Commercially available Rubella vaccines include a live cold-adapted virus, typically in combination with mumps and measles vaccines (MMR).
Flavivirus: Viral antigens may be derived from a Flavivirus, such as Tick-borne encephalitis (TBE), Dengue (types 1, 2, 3 or 4), Yellow Fever, Japanese encephalitis, West Nile encephalitis, St. Louis encephalitis, Russian spring-summer encephalitis, Powassan encephalitis. Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens are preferably selected from PrM, M and E. Commercially available TBE vaccine include inactivated virus vaccines.
Pestivirus: Viral antigens may be derived from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV).
Hepadnavirus: Viral antigens may be derived from a Hepadnavirus, such as Hepatitis B virus. Hepadnavirus antigens may be selected from surface antigens (L, M and S), core antigens (HBc, HBe). Commercially available HBV vaccines include subunit vaccines comprising the surface antigen S protein.
Hepatitis C virus: Viral antigens may be derived from a Hepatitis C virus (HCV). HCV antigens may be selected from one or more of E1, E2, E1/E2, NS345 polyprotein, NS 345-core polyprotein, core, and/or peptides from the nonstructural regions (Houghton et al., Hepatology (1991) 14:381).
Rhabdovirus: Viral antigens may be derived from a Rhabdovirus, such as a Lyssavirus (Rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigens may be selected from glycoprotein (G), nucleoprotein (N), large protein (L), nonstructural proteins (NS). Commercially available Rabies virus vaccine comprise killed virus grown on human diploid cells or fetal rhesus lung cells.
Caliciviridae; Viral antigens may be derived from Calciviridae, such as Norwalk virus, and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus.
Coronavirus: Viral antigens may be derived from a Coronavirus, SARS, Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV). Coronavirus antigens may be selected from spike (S), envelope (E), matrix (M), nucleocapsid (N), and Hemagglutinin-esterase glycoprotein (HE). Preferably, the Coronavirus antigen is derived from a SARS virus. SARS viral antigens are described in WO 04/92360;
Retrovirus: Viral antigens may be derived from a Retrovirus, such as an Oncovirus, a Lentivirus or a Spumavirus. Oncovirus antigens may be derived from HTLV-1, HTLV-2 or HTLV-5. Lentivirus antigens may be derived from HIV-1 or HIV-2. Retrovirus antigens may be selected from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigens may be selected from gag (p24gag and p55gag), env (gp160 and gp41), pol, tat, nef, rev vpu, miniproteins, (preferably p55 gag and gp140v delete). HIV antigens may be derived from one or more of the following strains: HIVIIIb, HIVSF2, HIVLAV, HIVLAI, HIVMN, HIV-1CM235, HIV-1US4.
Reovirus: Viral antigens may be derived from a Reovirus, such as an Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Reovirus antigens may be selected from structural proteins λ1, λ2, λ3, μ1, μ2, σ1, σ2, or σ3, or nonstructural proteins σNS, μNS, or σls. Preferred Reovirus antigens may be derived from a Rotavirus. Rotavirus antigens may be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 and VP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Preferred Rotavirus antigens include VP4 (or the cleaved product VP5 and VP8), and VP7.
Parvovirus: Viral antigens may be derived from a Parvovirus, such as Parvovirus B19. Parvovirus antigens may be selected from VP-1, VP-2, VP-3, NS-1 and NS-2. Preferably, the Parvovirus antigen is capsid protein VP-2.
Delta hepatitis virus (HDV): Viral antigens may be derived HDV, particularly 6-antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814).
Hepatitis E virus (HEV): Viral antigens may be derived from HEV.
Hepatitis G virus (HGV): Viral antigens may be derived from HGV.
Human Herpesvirus Viral antigens may be derived from a Human Herpesvirus, such as Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). Human Herpesvirus antigens may be selected from immediate early proteins (α), early proteins (β), and late proteins (γ). HSV antigens may be derived from HSV-1 or HSV-2 strains. HSV antigens may be selected from glycoproteins gB, gC, gD and gH, fusion protein (gB), or immune escape proteins (gC, gE, or gI). VZV antigens may be selected from core, nucleocapsid, tegument, or envelope proteins. A live attenuated VZV vaccine is commercially available. EBV antigens may be selected from early antigen (EA) proteins, viral capsid antigen (VCA), and glycoproteins of the membrane antigen (MA). CMV antigens may be selected from capsid proteins, envelope glycoproteins (such as gB and gH), and tegument proteins
Papovaviruses: Antigens may be derived from Papovaviruses, such as Papillomaviruses and Polyomaviruses. Papillomaviruses include HPV serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 and 65. Preferably, HPV antigens are derived from serotypes 6, 11, 16 or 18. HPV antigens may be selected from capsid proteins (L1) and (L2), or E1-E7, or fusions thereof. HPV antigens are preferably formulated into virus-like particles (VLPs). Polyomyavirus viruses include BK virus and JK virus. Polyomavirus antigens may be selected from VP1, VP2 or VP3.
Further provided are antigens, compositions, methods, and microbes included in Vaccines, 4th Edition (Plotkin and Orenstein ed. 2004); Medical Microbiology 4th Edition (Murray et al. ed. 2002); Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), which are contemplated in conjunction with the compositions of the present invention.
C. Fungal Antigens
Fungal antigens for use in the invention may be derived from one or more of the fungi set forth below.
Fungal antigens may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme.
Fungal pathogens may be derived from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiunm insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.
Processes for producing a fungal antigens are well known in the art (see U.S. Pat. No. 6,333,164). In a preferred method a solubilized fraction extracted and separated from an insoluble fraction obtainable from fungal cells of which cell wall has been substantially removed or at least partially removed, characterized in that the process comprises the steps of: obtaining living fungal cells; obtaining fungal cells of which cell wall has been substantially removed or at least partially removed; bursting the fungal cells of which cell wall has been substantially removed or at least partially removed; obtaining an insoluble fraction; and extracting and separating a solubilized fraction from the insoluble fraction.
D. STD Antigens
The compositions of the invention may include one or more antigens derived from a sexually transmitted disease (STD). Such antigens may provide for prophylactis or therapy for STD's such as chlamydia, genital herpes, hepatits (such as HCV), genital warts, gonorrhoea, syphilis and/or chancroid (See, WO00/15255). Antigens may be derived from one or more viral or bacterial STD's. Viral STD antigens for use in the invention may be derived from, for example, HIV, herpes simplex virus (HSV-1 and HSV-2), human papillomavirus (HPV), and hepatitis (HCV). Bacterial STD antigens for use in the invention may be derived from, for example, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, E. coli, and Streptococcus agalactiae. Examples of specific antigens derived from these pathogens are described above.
E. Respiratory Antigens
The compositions of the invention may include one or more antigens derived from a pathogen which causes respiratory disease. For example, respiratory antigens may be derived from a respiratory virus such as Orthomyxoviruses (influenza), Pneumovirus (RSV), Paramyxovirus (PIV), Morbillivirus (measles), Togavirus (Rubella), VZV, and Coronavirus (SARS). Respiratory antigens may be derived from a bacteria which causes respiratory disease, such as Streptococcus pneumoniae, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxella catarrhalis. Examples of specific antigens derived from these pathogens are described above.
F. Pediatric Vaccine Antigens
The compositions of the invention may include one or more antigens suitable for use in pediatric subjects. Pediatric subjects are typically less than about 3-years old, or less than about 2 years old, or less than about 1 years old. Pediatric antigens may be administered multiple times over the course of 6 months, 1, 2 or 3 years. Pediatric antigens may be derived from a virus which may target pediatric populations and/or a virus from which pediatric populations are susceptible to infection. Pediatric viral antigens include antigens derived from one or more of Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella), Enterovirus (polio), HBV, Coronavirus (SARS), and Varicella-zoster virus (VZV), Epstein Barr virus (EBV). Pediatric bacterial antigens include antigens derived from one or more of Streptococcus pneumoniae, Neisseria meningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani (Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae (Group B Streptococcus), and E. coli. Examples of specific antigens derived from these pathogens are described above.
G. Antigens Suitable for Use in Elderly or Immunocompromised Individuals
The compositions of the invention may include one or more antigens suitable for use in elderly or immunocompromised individuals. Such individuals may need to be vaccinated more frequently, with higher doses or with adjuvanted formulations to improve their immune response to the targeted antigens. Antigens which may be targeted for use in Elderly or Immunocompromised individuals include antigens derived from one or more of the following pathogens: Neisseria meningitides, Streptococcus pneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani (Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Legionella pneumophila, Streptococcus agalactiae (Group B Streptococcus), Enterococcus faecalis, Helicobacter pylori, Clamydia pneumoniae, Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella), Enterovirus (polio), HBV, Coronavirus (SARS), Varicella-zoster virus (VZV), Epstein Barr virus (EBV), Cytomegalovirus (CMV). Examples of specific antigens derived from these pathogens are described above.
H. Antigens Suitable for Use in Adolescent Vaccines
The compositions of the invention may include one or more antigens suitable for use in adolescent subjects. Adolescents may be in need of a boost of a previously administered pediatric antigen. Pediatric antigens which may be suitable for use in adolescents are described above. In addition, adolescents may be targeted to receive antigens derived from an STD pathogen in order to ensure protective or therapeutic immunity before the beginning of sexual activity. STD antigens which may be suitable for use in adolescents are described above.
I. Antigen Formulations
In other aspects of the invention, methods of producing microparticles having adsorbed antigens are provided. The methods comprise: (a) providing an emulsion by dispersing a mixture comprising (i) water, (ii) a detergent, (iii) an organic solvent, and (iv) a biodegradable polymer selected from the group consisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate. The polymer is typically present in the mixture at a concentration of about 1% to about 30% relative to the organic solvent, while the detergent is typically present in the mixture at a weight-to-weight detergent-to-polymer ratio of from about 0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about 0.1:1, about 0.001:1 to about 0.1:1, or about 0.005:1 to about 0.1:1); (b) removing the organic solvent from the emulsion; and (c) adsorbing an antigen on the surface of the microparticles. In certain embodiments, the biodegradable polymer is present at a concentration of about 3% to about 10% relative to the organic solvent.
Microparticles for use herein will be formed from materials that are sterilizable, non-toxic and biodegradable. Such materials include, without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride, PACA, and polycyanoacrylate. Preferably, microparticles for use with the present invention are derived from a poly(α-hydroxy acid), in particular, from a poly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide or glycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or “PLGA”), or a copolymer of D,L-lactide and caprolactone. The microparticles may be derived from any of various polymeric starting materials which have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide:glycolide ratios, the selection of which will be largely a matter of choice, depending in part on the coadministered macromolecule. These parameters are discussed more fully below.
Further antigens may also include an outer membrane vesicle (OMV) preparation. Additional formulation methods and antigens (especially tumor antigens) are provided in U.S. patent Ser. No. 09/581,772.
J. Antigen References
The following references include antigens useful in conjunction with the compositions of the present invention:
The contents of all of the above cited patents, patent applications and journal articles are incorporated by reference as if set forth fully herein.
Where a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier protein in order to enhance immunogenicity. See Ramsay et al (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168; Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii; Goldblatt (1998) J. Med. Microbiol. 47:563-567; European patent 0 477 508; U.S. Pat. No. 5,306,492; WO98/42721; Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114; Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM197 diphtheria toxoid is particularly preferred.
Other carrier polypeptides include the N. meningitidis outer membrane protein (EP-A-0372501), synthetic peptides (EP-A-0378881 and EP-A 0427347), heat shock proteins (WO 93/17712 and WO 94/03208), pertussis proteins (WO 98/58668 and EP A 0471177), protein D from H. influenzae (WO 00/56360), cytokines (WO 91/01146), lymphokines, hormones, growth factors, toxin A or B from C. difficile (WO 00/61761), iron-uptake proteins (WO 01/72337), etc. Where a mixture comprises capsular saccharide from both serigraphs A and C, it may be preferred that the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Different saccharides can be conjugated to the same or different type of carrier protein. Any suitable conjugation reaction can be used, with any suitable linker where necessary.
Toxic protein antigens may be detoxified where necessary e.g., detoxification of pertussis toxin by chemical and/or genetic means.
Pharmaceutically Acceptable Carriers
Compositions of the invention will typically, in addition to the components mentioned above, comprise one or more “pharmaceutically acceptable carriers.” These include any carrier which does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers typically are large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. A composition may also contain a diluent, such as water, saline, glycerol, etc. Additionally, an auxiliary substance, such as a wetting or emulsifying agent, pH buffering substance, and the like, may be present. A thorough discussion of pharmaceutically acceptable components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th ed., ISBN: 0683306472.
Immunoregulatory Agents
Adjuvants
Vaccines of the invention may be administered in conjunction with other immunoregulatory agents. In particular, compositions will usually include an adjuvant. Adjuvants for use with the invention include, but are not limited to, one or more of the following set forth below:
A. Mineral Containing Compositions
Mineral containing compositions 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 (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulfates, etc. (e.g. see chapters 8 & 9 of Vaccine Design . . . (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.), or mixtures of different mineral compounds (e.g. a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption to the salt(s) being preferred. The mineral containing compositions may also be formulated as a particle of metal salt (WO00/23105).
Aluminum salts may be included in vaccines of the invention such that the dose of Al3+ is between 0.2 and 1.0 mg per dose.
In one embodiment the aluminum based adjuvant for use in the present invention is alum (aluminum potassium sulfate (AlK(SO4)2)), or an alum derivative, such as that formed in-situ by mixing an antigen in phosphate buffer with alum, followed by titration and precipitation with a base such as ammonium hydroxide or sodium hydroxide.
Another aluminum-based adjuvant for use in vaccine formulations of the present invention is aluminum hydroxide adjuvant (Al(OH)3) or crystalline aluminum oxyhydroxide (AlOOH), which is an excellent adsorbant, having a surface area of approximately 500 m2/g. Alternatively, aluminum phosphate adjuvant (AlPO4) or aluminum hydroxyphosphate, which contains phosphate groups in place of some or all of the hydroxyl groups of aluminum hydroxide adjuvant is provided. Preferred aluminum phosphate adjuvants provided herein are amorphous and soluble in acidic, basic and neutral media.
In another embodiment the adjuvant of the invention comprises both aluminum phosphate and aluminum hydroxide. In a more particular embodiment thereof, the adjuvant has a greater amount of aluminum phosphate than aluminum hydroxide, such as a ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or greater than 9:1, by weight aluminum phosphate to aluminum hydroxide. More particular still, aluminum salts in the vaccine are present at 0.4 to 1.0 mg per vaccine dose, or 0.4 to 0.8 mg per vaccine dose, or 0.5 to 0.7 mg per vaccine dose, or about 0.6 mg per vaccine dose.
Generally, the preferred aluminum-based adjuvant(s), or ratio of multiple aluminum-based adjuvants, such as aluminum phosphate to aluminum hydroxide is selected by optimization of electrostatic attraction between molecules such that the antigen carries an opposite charge as the adjuvant at the desired pH. For example, aluminum phosphate adjuvant (isoelectric point=4) adsorbs lysozyme, but not albumin at pH 7.4. Should albumin be the target, aluminum hydroxide adjuvant would be selected (iep 11.4). Alternatively, pretreatment of aluminum hydroxide with phosphate lowers its isoelectric point, making it a preferred adjuvant for more basic antigens.
B. Oil-Emulsions
Oil-emulsion compositions 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 into submicron particles using a microfluidizer). See WO90/14837. See also, Podda, Vaccine (2001) 19: 2673-2680; Frey et al., Vaccine (2003) 21:4234-4237. MF59 is used as the adjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.
Particularly preferred adjuvants for use in the compositions are submicron oil-in-water emulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN™ 80□ (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% SPAN 85™ (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphosphoryloxy)-ethylamine (MTP-PE), for example, the submicron oil-in-water emulsion known as “MF59” (International Publication No. WO90/14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, and Ott et al., in Vaccine Design The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene (e.g 4.3%), 0.25-0.5% w/v TWEEN™ 80, and 0.5% w/v SPAN 85™ and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE may be present in an amount of about 0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100 μg/dose. As used herein, the term “MF59-0” refers to the above submicron oil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes a formulation that contains MTP-PE. For instance, “MF59-100” contains 100 μg MTP-PE per dose, and so on. MF69, another submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v TWEEN™ 80, and 0.75% w/v SPAN 85™ and optionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 0.4% TWEEN™ 80, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion. MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 μg MTP-PE per dose.
Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325.
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention.
C. Saponin Formulations
Saponin formulations, may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins isolated from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponins can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-TLC) and Reversed 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-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO96/33739).
Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulating 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 EP0109942, WO96/11711 and WO96/33739. Optionally, the ISCOMS may be devoid of (an) additional detergent(s). See WO00/07621.
A review of the development of saponin based adjuvants can be found in Barr, et al., Advanced Drug Delivery Reviews (1998) 32:247-271. See also Sjolander, et al., Advanced Drug Delivery Reviews (1998) 32:321-338.
D. Virosomes and Virus Like Particles (VLPs)
Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1). VLPs are discussed further in WO03/024480, WO03/024481, and Niikura et al, Virology (2002) 293:273-280; Lenz et al., Journal of Immunology (2001) 5246-5355; Pinto, et al., Journal of Infectious Diseases (2003) 188:327-338; and Gerber et al., Journal of Virology (2001) 75(10):4752-4760. Virosomes are discussed further in, for example, Gluck et al., Vaccine (2002) 20:B10-B16. Immunopotentiating reconstituted influenza virosomes (IRIV) are used as the subunit antigen delivery system in the intranasal trivalent INFLEXAL™ product {Mischler & Metcalfe (2002) Vaccine 20 Suppl 5:B 17-23} and the INFLUVAC PLUS™ product.
E. Bacterial or Microbial Derivatives
Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as:
(1) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS)
Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is 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 mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g RC 529. See Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.
(2) Lipid A Derivatives
Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Meraldi et al., Vaccine (2003) 21:2485-2491; and Pajak, et al., Vaccine (2003) 21:836-842.
(3) Immunostimulatory Oligonucleotides
Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine may be replaced with an analog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al., Nucleic Acids Research (2003) 31(9): 2393-2400; WO02/26757 and WO99/62923 for examples of possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, Nature Medicine (2003) 9(7): 831-835; McCluskie, et al., FEMS Immunology and Medical Microbiology (2002) 32:179-185; WO98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199.
The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al., J. Immunol. (2003) 170(8):4061-4068; Krieg, TRENDS in Immunology (2002) 23(2): 64-65 and WO01/95935. Preferably, the 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 may be attached at their 3′ ends to form “immunomers”. See, for example, Kandimalla, et al., BBRC (2003) 306:948-953; Kandimalla, et al., Biochemical Society Transactions (2003) 31(part 3):664-658; Bhagat et al., BBRC (2003) 300:853-861 and WO03/035836.
(4) ADP-ribosylating toxins and detoxified derivatives thereof.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin “LT), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211 and as parenteral adjuvants in WO98/42375. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references: Beignon, et al., Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al, Vaccine (2001) 19:2534-2541; Pizza, et al., Int. J. Med. Microbiol. (2000) 290(4-5):455-461; Scharton-Kersten et al., Infection and Immunity (2000) 68(9):5306-5313; Ryan et al., Infection and Immunity (1999) 67(12):6270-6280; Partidos et al., Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al., Vaccines (2003) 2(2):285-293; and Pine et al., (2002) J. Control Release (2002) 85(1-3):263-270. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol. (1995) 15(6):1165-1167.
F. Bioadhesives and Mucoadhesives
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) or mucoadhesives such as cross-linked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention. See WO99/27960.
G. Microparticles
Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide co glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).
H. Liposomes
Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.
I. Polyoxyethylene ether and Polyoxyethylene Ester Formulations
Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters. WO99/52549. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152).
Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
J. Polyphosphazene (PCPP)
PCPP formulations are described, for example, in Andrianov et al., “Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payne et al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug. Delivery Review (1998) 31(3):185-196.
K. Muramyl Peptides
Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), and N acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
L. Imidazoquinoline Compounds.
Examples of imidazoquinoline compounds suitable for use adjuvants in the invention include Imiquimod and its analogues, described further in Stanley, Clin Exp Dermatol (2002) 27(7):571-577; Jones, Curr Opin Investig Drugs (2003) 4(2):214-218; and U.S. Pat. Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612.
M. Thiosemicarbazone Compounds.
Examples of thiosemicarbazone compounds, as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants in the invention include those described in WO04/60308. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
N. Tryptanthrin Compounds.
Examples of tryptanthrin compounds, as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants in the invention include those described in WO04/64759. The tryptanthrin compounds are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention:
(1) a saponin and an oil-in-water emulsion (WO99/11241);
(2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g 3dMPL) (see WO94/00153);
(3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol;
(4) a saponin (e.g., QS21)+3dMPL+IL 12 (optionally+a sterol) (WO98/57659);
(5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231);
(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.
(7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoX™); and
(8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dPML).
(9) one or more mineral salts (such as an aluminum salt)+an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif).
O. Human Immunomodulators
Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.
Aluminum salts and MF59 are preferred adjuvants for use with injectable influenza vaccines. Bacterial toxins and bioadhesives are preferred adjuvants for use with mucosally-delivered vaccines, such as nasal vaccines.
The contents of all of the above cited patents, patent applications and journal articles are incorporated by reference as if set forth fully herein.
The invention provides methods for inducing or increasing an immune response to a GAS antigen using the compositions described above. The immune response is preferably protective and can include antibodies and/or cell-mediated immunity (including systemic and mucosal immunity). Immune responses include booster responses. Compositions comprising antibodies can be used to treat S. pyogenes infections.
Teenagers and children, including toddles and infants, can receive a vaccine for prophylactic use; therapeutic vaccines typically are administered to teenagers or adults. A vaccine intended for children may also be administered to adults e.g., to assess safety, dosage, immunogenicity, etc.
Diseases caused by Streptococcus pyogenes which can be prevented or treated according to the invention include, but are not limited to, pharyngitis (such as streptococcal sore throat), scarlet fever, impetigo, erysipelas, cellulitis, septicemia, toxic shock syndrome, necrotizing fasciitis, and sequelae such as rheumatic fever and acute glomerulonephritis. The compositions may also be effective against other streptococcal bacteria, e.g., GBS.
Tests to Determine the Efficacy of the Immune Response
One way of assessing efficacy of therapeutic treatment involves monitoring GAS infection after administration of the composition of the invention. One way of assessing efficacy of prophylactic treatment involves monitoring immune responses against the GAS antigens in the compositions of the invention after administration of the composition.
Another way of assessing the immunogenicity of the component proteins of the immunogenic compositions of the present invention is to express the proteins recombinantly and to screen patient sera or mucosal secretions by immunoblot. A positive reaction between the protein and the patient serum indicates that the patient has previously mounted an immune response to the protein in question; i.e., the protein is an immunogen. This method may also be used to identify immunodominant proteins and/or epitopes.
Another way of checking efficacy of therapeutic treatment involves monitoring GAS infection after administration of the compositions of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses both systemically (such as monitoring the level of IgG1 and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the GAS antigens in the compositions of the invention after administration of the composition. Typically, GAS serum specific antibody responses are determined post-immunization but pre-challenge whereas mucosal GAS-specific antibody body responses are determined post-immunization and post-challenge.
The vaccine compositions of the present invention can be evaluated in in vitro and in vivo animal models prior to host, e.g., human, administration. Particularly useful mouse models include those in which intraperitoneal immunization is followed by either intraperitoneal challenge or intranasal challenge. A model in which intraperitoneal immunization is followed by intraperitoneal challenge is illustrated in
The efficacy of immunogenic compositions of the invention can also be determined in vivo by challenging animal models of GAS infection, e.g., guinea pigs or mice, with the immunogenic compositions. The immunogenic compositions may or may not be derived from the same serotypes as the challenge serotypes. Preferably the immunogenic compositions are derivable from the same serotypes as the challenge serotypes. More preferably, the immunogenic composition and/or the challenge serotype are derivable from the group of GAS serotypes consisting of M1, M3, M23 and/or combinations thereof.
In vivo efficacy models include but are not limited to: (i) a murine infection model using human GAS serotypes; (ii) a murine disease model which is a murine model using a mouse-adapted GAS strain, such as the M23 strain which is particularly virulent in mice, and (iii) a primate model using human GAS isolates.
The immune response may be one or both of a TH1 immune response and a TH2 response. The immune response may be an improved or an enhanced or an altered immune response. The immune response may be one or both of a systemic and a mucosal immune response. Preferably the immune response is an enhanced system and/or mucosal response.
An enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response. Preferably, the enhanced immune response includes an increase in the production of IgG1 and/or IgG2a and/or IgA.
Preferably the mucosal immune response is a TH2 immune response. Preferably, the mucosal immune response includes an increase in the production of IgA.
Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections. Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immune response may result in the production of IgG1, IgE, IgA and memory B cells for future protection.
A TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1, IgE, IgA and memory B cells. Preferably, the enhanced TH2 immune response will include an increase in IgG1 production.
A TH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. Preferably, the enhanced TH1 immune response will include an increase in IgG2a production.
Immunogenic compositions of the invention, in particular, immunogenic composition comprising one or more GAS antigens of the present invention may be used either alone or in combination with other GAS antigens optionally with an immunoregulatory agent capable of eliciting a Th1 and/or Th2 response.
The invention also comprises an immunogenic composition comprising one or more immunoregulatory agent, such as a mineral salt, such as an aluminium salt and an oligonucleotide containing a CpG motif. Most preferably, the immunogenic composition includes both an aluminium salt and an oligonucleotide containing a CpG motif. Alternatively, the immunogenic composition includes an ADP ribosylating toxin, such as a detoxified ADP ribosylating toxin and an oligonucleotide containing a CpG motif. Preferably, one or more of the immunoregulatory agents include an adjuvant. The adjuvant may be selected from one or more of the group consisting of a TH1 adjuvant and TH2 adjuvant, further discussed below.
The compositions of the invention will preferably elicit both a cell mediated immune response as well as a humoral immune response in order to effectively address a GAS infection. This immune response will preferably induce long lasting (e.g., neutralizing) antibodies and a cell mediated immunity that can quickly respond upon exposure to one or more GAS antigens.
In one particularly preferred embodiment, the immunogenic composition comprises one or more GAS antigen(s) which elicits a neutralizing antibody response and one or more GAS antigen(s) which elicit a cell mediated immune response. In this way, the neutralizing antibody response prevents or inhibits an initial GAS infection while the cell-mediated immune response capable of eliciting an enhanced Th1 cellular response prevents further spreading of the GAS infection. Preferably, the immunogenic composition comprises one or more GAS surface antigens and one or more GAS cytoplasmic antigens. Preferably the immunogenic composition comprises one or more GAS40 surface antigens or the like and one or other antigens, such as a cytoplasmic antigen capable of eliciting a Th1 cellular response.
Compositions of the invention will generally be administered directly to a patient. The compositions of the present invention may be administered, either alone or as part of a composition, via a variety of different routes. Certain routes may be favored for certain compositions, as resulting in the generation of a more effective immune response, preferably a CMI response, or as being less likely to induce side effects, or as being easier for administration.
Delivery methods include parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial injection) and rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal (e.g., see WO 99/27961), transcutaneous (e.g., see WO02/074244 and WO02/064162), intranasal (e.g., see WO03/028760), ocular, aural, and pulmonary or other mucosal administration.
By way of example, the compositions of the present invention may be administered via a systemic route or a mucosal route or a transdermal route or it may be administered directly into a specific tissue. As used herein, the term “systemic administration” includes but is not limited to any parenteral routes of administration. In particular, parenteral administration includes but is not limited to subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous, intraarterial, or kidney dialytic infusion techniques. Preferably, the systemic, parenteral administration is intramuscular injection. As used herein, the term “mucosal administration” includes but is not limited to oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal and ophthalmic administration.
Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
The compositions of the invention may be prepared in various forms. For example, a composition can be prepared as an injectable, either as a liquid solution or a suspension. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition). A composition can be prepared for oral administration, such as a tablet or capsule, as a spray, or as a syrup (optionally flavored). A composition can be prepared for pulmonary administration, e.g., as an inhaler, using a fine powder or a spray. A composition can be prepared as a suppository or pessary. A composition can be prepared for nasal, aural or ocular administration e.g., as drops. A composition can be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more GAS or other antigens in liquid form and one or more lyophilized antigens.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of GAS or other antigens (or nucleic acid molecules encoding the antigens) or antibodies, as well as any other components, as needed, such as antibiotics. An “immunologically effective amount” is an amount which, when administered to an individual, either in a single dose or as part of a series, increases a measurable immune response or prevents or reduces a clinical symptom.
The immunogenic compositions of the present invention may be administered in combination with an antibiotic treatment regime. In one embodiment, the antibiotic is administered prior to administration of the antigen of the invention or the composition comprising the one or more GAS antigens of the invention.
In another embodiment, the antibiotic is administered subsequent to the administration of the one or more surface-exposed and/or surface-associated GAS antigens of the invention or the composition comprising the one or more surface-exposed and/or surface-associated GAS antigens of the invention. Examples of antibiotics suitable for use in the treatment of a GAS infection include but are not limited to penicillin or a derivative thereof or clindamycin, cephalosporins, glycopeptides (e.g., vancomycin), and cycloserine.
The amount of active agent in a composition varies, however, depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. The amount will fall in a relatively broad range which can be determined through routine trials.
The invention also provides kits comprising one or more containers of compositions of the invention. Compositions can be in liquid form or can be lyophilized, as can individual antigens. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other buffers, diluents, filters, needles, and syringes. The kit can also comprise a second or third container with another active agent, for example an antibiotic.
The kit can also comprise a package insert containing written instructions for methods of inducing immunity against S. pyogenes or for treating S. pyogenes infections. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
A set of 73 proteins were identified in silico as surface-expressed proteins of the GASSF370 strain (M1) genome using BLAST, FASTa, MOTIFS, FINDPATTERNS, PSORT, and searches of the Propom, Pfam, and Blocks databases. These programs were used to predict features typical of surface-associated proteins, such as transmembrane domains, leader peptides, homologies to known surface proteins, lipoprotein signatures, outer membrane anchoring motifs, and host-cell binding domains such as RGD. The results are shown in Table 3.
Commercially available E. coli expression vectors pET and pGex were used to express GAS antigens either as HIS-tagged proteins or as HIS-GST fusions, i.e., an amino-terminal histidine tag and a carboxy terminal GST (wherever “GST” is not specified, only the HIS-tagged antigen was expressed. See
Two of the identified proteins (GAS87, 171) were not expressed. Mice were immunized with 70 of the other proteins. Sera of these mice were used for FACS analysis on native bacterial cells, and surface exposure was tested on 20 GAS strains of different M types (see Table 2). The presence or absence of each protein on the bacterial cell surface was assessed by calculating the difference (Delta Mean) between the FACS value obtained with the immune serum and that obtained with the preimmune serum. An arbitrary cut-off of Delta Mean ≧80 was used to classify a protein as “surface exposed.” Three of the tested antigens did not meet this threshold (GAS88, 208, and 210). The results for the other GAS antigens tested are shown in Tables 4A-4R and
Genomic DNAs were prepared from GAS strains of different M types, and the complete protein sequence of the full-length GAS40 antigen was obtained. The results are shown in
FACS analysis was carried out as described in Example 1 to demonstrate that GAS40 proteins are surface-exposed. The results are shown in
Mice were immunized with various GAS40 antigens. Sera of these mice were used for FACS analysis on native bacterial cells of various S. pyogenes strains. The ability of these antisera to detect GAS40 protein on the bacterial cell surface was assessed by calculating the difference between the FACS value obtained with the immune sera and that obtained with the preimmune sera. The results are shown in
“40 native” (
“GST40” (
“40a” (
“40aCH” (
“40/117” (
“117/40” (
“40aRR” (
“40aNH” (
“40aRRNH” (
Groups of 10 CD1 female mice aged between 6 and 7 weeks are immunized with two or more GAS antigens of the invention, (20 μg of each recombinant GAS antigen), suspended in 100 μl of suitable solution. Each group received 3 doses at days 0, 21, and 45. Immunization was performed through intra-peritoneal injection of the protein with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses. In each immunization scheme negative and positive control groups were used. See
For the negative control group, mice were immunized with E. coli proteins eluted from the purification columns following processing of total bacterial extract from an E. coli strain containing either the pET21b or the pGEX-NNH vector (thus expressing GST only) without any cloned GAS ORF (groups can be indicated as HisStop or GSTStop respectively). For the positive control groups, mice were immunized with purified GAS M cloned from either GAS SF370 or GAS DSM 2071 strains (groups indicated as 192SF and 192DSM respectively).
Pooled sera from each group was collected before the first immunization and two weeks after the last one. Mice were infected with GAS about a week after.
Immunized mice were infected using GAS strain 2071, a different strain from that used for the cloning of the selected proteins. (German Collection of Microorganisms and Cell Cultures, DSMZ).
For infection experiments, DSM 2071 was grown at 37° C. in THY broth until the OD600 was 0.4. Bacteria were pelletted by centrifugation, washed once with PBS, suspended, and diluted with PBS to obtain the appropriate concentration of bacteria/ml and administered to mice by intraperitoneal injection. Between 50 and 100 bacteria were given to each mouse, as determined by plating aliquots of the bacterial suspension on 5 THY plates. Animals were observed daily and checked for survival. The results obtained after intraperitoneal challenge are shown in
Using the model described above, selected GAS antigens were tested and some of them showed statistically significant protection rates (
Bacteria were grown in THY to OD600=0.4, washed twice with PBS, suspended in NCS (Newborn Calf Serum, Sigma), incubated for 20 min at RT, and dispensed in a 96-well plate (20 μl/well). Eighty μl of preimmune or immune mouse sera, diluted in PBS-0.1% BSA, were added to the bacterial suspension to a final dilution of 1:200 and incubation was performed on ice for 30 min. After washing twice with PBS-0.1% BSA, bacteria were incubated on ice for 30 min in 10 μl of Goat Anti-Mouse IgG, F(ab′)2 fragment-specific-R-Phycoerythrin-conjugated (Jackson Immunoresearch Laboratories Inc.) in PBS-0.1% BSA-20% NCS to a final dilution of 1:100.
Following incubation, bacteria were washed with PBS-0.1% BSA, suspended in 200 μl PBS and analyzed using a FACS Calibur cytometer (Becton Dikinson, Mountain View, Calif. USA) and Cell Quest Software (Becton Dikinson, Mountain View, Calif. USA). The results are shown in
Immunogold labeling and electron microscopy GAS were grown in THYE medium to mid-log phase, washed and resuspended in PBS. Formvar-carbon-coated nickel grids were floated on drops of GAS suspensions for 5 min, fixed in 2% PFA for 5 min, and placed in blocking solution (PBS containing 1% normal rabbit serum and 1% BSA) for 30 min. The grids were then floated on drops of primary antiserum diluted 1:20 in blocking solution for 30 min at RT, washed, and floated on secondary antibody conjugated to 10 nm gold particles diluted 1:10 in 1% BSA for 30 min.
The grids were washed with PBS then distilled water and air dried and examined using a TEM GEOL 1200EX II transmission electron microscope. Preimmune serum from the same animals were used as a negative control. The results are shown in
Preparation of Bacterial Inoculum
Bacterial cells were grown in THY medium until they reached the middle exponential phase (OD600 0.4) at 37° C. Bacteria were washed twice in chilled saline solution and suspended in MEM medium with the volume being adjusted for each strain depending on the amount of bacteria used. Bacterial cells were kept in ice until used in the assay.
Preparation of Peripheral Mononuclear Cells (PMN)
PMN were prepared from buffy coats of heparinized blood from healthy volunteers. The buffy coat was incubated for 30 minutes in a solution containing dextran, NaCl and Heparin (rate 1:1). After incubation the supernatant, enriched in leukocytes, was removed, transferred to a clean tube, and centrifuged at 700×g for 20 minutes. A short wash in water was performed to break red blood cells, and then a solution of NaCl was added to restore the appropriate salt concentration. After this step cells were centrifuged, washed, and suspended in MEM at a suitable concentration.
Opsonophagocytosis Assay
GAS strains were incubated with heat inactivated immune mice serum (or preimmune for the control), human PMN, and baby rabbit complement for 1 hour of incubation at 37° C. Samples taken immediately before and after the incubation were plated on THY blood agar plates. Phagocytosis was evaluated comparing the difference in the number of colonies at the two times for the preimmune and the immune serum. Data were reported as logarithm number of grown colonies at t=0−logarithm number of grown colonies at t=60. See
Bacterial Growth Inhibition
Complete heparinized blood from mice immunized with GAS40 was incubated with bacterial cells grown as described above. Blood of mice immunized only with protein buffer was used as a control. The samples were rotated end over end for 3 hours at 37° C. Reactions were plated on THY blood agar plates, and CFU were counted. Growth inhibition was evaluated by comparing the number of colonies in the samples and in the control. See
To test whether the GAS40 gene, which appears to be well conserved, was actually expressed in different strains, total cell extracts from a panel of distinct GAS strains were loaded on SDS-PAGE and probed with immune sera raised against the recombinant GAS40. As shown in
Cell extracts were obtained by growing the cells at 37° C. to OD600 0.32 in 10 ml of THY. The cell pellet was washed in PBS, resuspended in lysis buffer (40% sucrose, 0.1 M KPO4 pH 6.2, MgCl2 10 mM, and Roche's COMPLETE™ EDTA-free), and digested for 3 hours with 400U of mutanolysin and 2 mg/ml lysozyme. The insoluble fraction was separated by centrifugation and the supernatant was analyzed.
Computational structural studies based on the amino acid sequence of GAS40 identified two potential coiled coil regions, one at the C-terminus and one at the N-terminus (see also WO 05/032582). This prediction was used to clone and express two isolated protein domains, one of predicted 305 (40N) amino acids and one of 568 amino acids (40C). Two sets of primers (see below) were used to amplify the two distinct coding regions by PCR, each containing one of the predicted coiled-coil domains:
The DNA fragments were then cloned into pET21b+ and pGEX vectors and expressed in E. coli. Only the N-terminal domain gave a product of the expected size (40N). See
Note the blank vertical gel under the heading “GAS40N” and the reactivity with the GAS40 protein (see “GAS40” column). These results indicate that these GAS40 monoclonal antibodies were not raised against a GAS40N-specific epitope.
The FACS graphs shown in
Bacterial strains and culture. Streptococcus pyogenes bacteria from the hypocapsulated M1 wild-type strain SF370 (M1 serotype) (Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98, 4658-63, 2001), CDC SS-90 (M3 serotype), and 2071 (M23 serotype) were grown in Todd-Hewiff broth (THB) at 37° C. and 5% CO2, until an OD600=0.4 was reached (exponential growth phase). After culture, bacteria were harvested by centrifugation at 3,500×g for 10 minutes at 4° C., washed three times with phosphate-buffered saline (PBS), and used in the experiments described below. Genomic data for M1 are available at the TIGR website (URL address: http file type, www host server, domain name tigr.org), together with bioinformatics prediction results on function.
Bacterial surface digestion. Protease digestion of the bacterial surface was carried out separately with two different enzymes: Sequencing Grade Modified Trypsin (Promega, Madison, Wis.) and proteinase K (Promega). Cells from a 100 ml initial culture were resuspended in 0.8 ml phosphate-based buffer (2.7 mM KCl, 1.5 mM KH2PO4, 13.7 mM NaCl, 8.1 mM Na2HPO4) containing 40% sucrose.
Digestions were carried out in the presence of 5 mM DTT for 30 minutes at 37° C. with either 20 μg trypsin (ph 7.4) or 10 μg proteinase K (pH 6.0). The digestion mixtures were centrifuged at 3,500×g for 10 minutes at 4° C. The supernatants containing the digested peptides were filtered through a Millipore filter with a pore size of 0.22 μm. Protease reactions were stopped by adding 10% v/v of 1% formic acid. Prior to analysis, salts in samples were removed by off-line HPLC, with a 7 min gradient of 2-80% acetonitrile (ACN) in 0.1% formic acid. Fractions collected were pooled and concentrated with a Speed-vac. Supernatants were kept at −20° C. until further analysis.
Multidimensional protein identification technology (MudPIT). Two different platforms were used for the chromatographic separation of peptides and further identification by tandem mass spectrometry (MS/MS).
In the first platform, peptides were separated by two-dimensional nano-liquid chromatography (2-D LC), spotted directly onto a MALDI target, and analyzed by MALDI TOF-TOF (off-line coupled 2-D LC/MALDI MS/MS). The chromatographic system (Dionex, Amsterdam, The Netherlands) consisted of a FAMOS autosampler, an UltiMate micropump with UV detector and a Switchos column-switching device, as described in (Mitulovic et al., Proteomics. 2004 September; 4(9):2545-57). The UltiMate pump was set to operate at a flow rate 300 mL/min. The flow of the Switchos loading pump, which was used to carry the sample from the sample loop to the first column, was set to operate at 30 μL/min.
Briefly, peptide separation was performed as follows. In the first dimension, peptides were loaded on a strong cation exchange (SCX) column (10 cm×320 μm i. d.) and eluted isocratically by applying 5 increasing NaCl concentrations (0.01, 0.05, 0.1, 0.5 and 1 M). In the second dimension, peptides were separated by a reversed phase C18 analytical column (15 cm×75 μm i. d., C18 PEPMAP100™, 3 μm, 100 Å) through a C18 trap column (PEPMAP™ C18 μ-precolumn, 300 μm i.d.×1 mm, Dionex). Peptides were eluted with a 45-min gradient from 5 to 50% of 80% ACN in 0.1% formic acid at a flow rate of 300 nl/min.
Eluates were continuously spotted onto an ANCHOR-CHIP® MALDI target (Bruker Daltoniks, Bremen, Germany) every 60 s using a Proteineer FC robot (Bruker Daltoniks) prepared with a thin layer of a saturated solution of α-cyano-4-hydroxycynnamic acid in acetone, every 60 s using a Proteineer FC robot (Bruker Daltoniks). Prior to spotting, the target was prepared with a thin layer of a saturated solution of α-cyano-4-hydroxycynnamic acid in acetone. After fraction collection, every spot was manually recrystallized with 0.6 μl of ethanol/acetone/0.1% trifluoroacetic acid (6:3:1).
Mass spectrometry analysis was performed automatically with an Ultraflex MALDI TOF-TOF instrument, under the control of the WARP LC software (Bruker Daltoniks). First, MS spectra of all the spotted fractions were acquired for peak selection and further MS/MS spectra acquisition of selected peaks. Searching and identification of peptides were performed in batch mode with a licensed version of MASCOT in a local database. The MASCOT search parameters were: (1) species: S. pyogenes strain SF370; (2) allowed number of missed cleavages (only for trypsin digestion): 6; (3) variable post-translational modifications: methionine oxidation; (4) peptide tolerance: ±300 ppm; (5) MS/MS tolerance: ±1.5 Da and (6): peptide charge: +1.
In the second platform, peptides were separated by nanoLC-MS/MS on a CapLC HPLC system (Waters, Milford, Mass., USA) connected to a Q-ToF Micro ESI mass spectrometer equipped with a nanospray source (Waters, Milford, Mass., USA). Samples were resuspended in 5% (v/v) ACN, 0.1% (v/v) formic acid (Solvent A) and loaded on a C18 trap column (300 μm i.d.×5 mm, LC Packings, Amsterdam, The Netherlands). After 3 min, the flow was switched to an Atlantis C18 NanoEase column (100 μm i.d.×100 mm, Waters, Milford, Mass., USA) and a solvent gradient was started. The applied flow rate was of 4 μL/min and a flow splitter was set up to direct a nanoflow of 400 mL/min through the analytical column.
Peptides were eluted applying a linear gradient in 50 min from 2% to 60% Solvent B (95% (v/v) ACN, 0.1% (v/v) formic acid). The eluted peptides were subjected to an automated data-dependent acquisition program, using the MassLynx software (Waters, Milford, Mass., USA), where a MS survey scan was used to automatically select multi-charged peptides to be subjected to MS/MS fragmentation. Up to three different components where subjected to MS/MS fragmentation at the same time. For all the samples a second nanoLC-MS/MS analysis was carried out for the selective fragmentation of mono-charged peptide species.
All the acquired MS/MS spectra were converted in PKL file format and protein identification was achieved by database searching using licensed version of MASCOT running on local database. The applied searching criteria were the following: peptide tolerance ±500 ppm, MS/MS tolerance ±0.3Da, missed cleavage 6, peptide charge states from 1+ to 4+.
Cloning, expression and purification of recombinant proteins, and preparation of preimmune and immune sera, was done as described in Maione et al., Science 309, 148-50, 2005.
FACS analysis. FACS analysis was performed as follows. About 105 bacteria were washed with 200 μl of PBS, centrifuged for 10 minutes at 3,500×g, at 4° C., and then resuspended in 20 μl of PBS-0.1% BSA. Eighty μl of either pre-immune or immune mouse serum diluted in PBS-0.1% BSA were added to the bacterial suspension to a final dilution of either 1:100, 1:250, or 1:500, and incubated on ice for 30 min.
Bacteria were washed once by adding 100 μl of PBS-0.1% BSA, centrifuged for 10 minutes at 3,500×g, at 4° C., resuspended in 200 μl of PBS-0.1% BSA, and centrifuged again. The bacteria were resuspended in 10 μl of phycoerythrin-conjugated goat anti-mouse IgG F(ab′)2 fragment (Jackson Immunoresearch Laboratories Inc.) in PBS-0.1% BSA to a final dilution of 1:100, and incubated on ice for 30 minutes in the dark.
Bacteria were then washed by adding 180 μl of PBS-0.1% BSA, and centrifuged for 10 minutes at 3,500×g, at 4° C., and resuspended in 200 μl of PBS.
The bacterial resuspension was then analyzed using a FACS Calibur instrument (Becton Dickinson, Mountain View, Calif. USA), and 10,000 events were acquired. Data were analyzed using Cell Quest Software (Becton Dickinson) by drawing a morphological dot plot (using forward and side scatter parameters) on bacterial signals. A histogram plot was then created on FL2 intensity of fluorescence log scale.
The approach described above unambiguously identified 72 proteins from a total of 177 tryptic peptides and 107 peptides generated by proteinase K digestion. Ten proteins were identified from proteinase K-derived peptides, and 19 proteins were identified from both trypsin and proteinase K peptides. Table 9 shows the list of the proteins identified by applying both LC/MS/MS platforms, based on ESI-q-TOF and MALDI-TOF/TOF technologies. The protein list is the result of joining data from three independent surface digestion experiments for each protease. At least two chromatographic runs, followed by MS/MS analysis, were performed per sample and platform. Approximately 5 pmol of peptides were loaded into both 2-D LC/MALDI MS/MS and LC/ESI MS/MS systems. Assignment of subcellular location and specific protein features was made by means of PSORT software.
By scanning the sequence of each identified protein for the presence of signatures indicative of specific cellular localization, the 72 proteins could be grouped into 4 major families: the cell wall-anchored protein family containing LPXTG (SEQ ID NO:931)-like motifs (12 proteins), the lipoprotein family (11 proteins), the transmembrane protein family carrying one or more transmembrane spanning regions (37 proteins) and the family of secreted proteins (8 proteins). Based on genome computer analysis, the total number of GAS SF370 proteins which could be attributed to each of these protein families are 17, 28, 489 and 67, respectively. Therefore, while a large proportion of all predicted cell-wall anchored proteins and lipoproteins were identified, less than 7% of secreted proteins and membrane spanning proteins were found exposed on the cell surface.
This discrepancy was expected for the secreted proteins, being consistent with the notion that most of them are released out of the cell and only a fraction remain partially associated to the cell wall. On the contrary, the small number of identified transmembrane proteins was somehow surprising and suggested that a large fraction of these proteins are either deeply embedded in the membrane, or poorly expressed or both. Interestingly, only 4 PSORT-predicted cytoplasmic proteins were found associated to the external side of the cell (Table 9), and all of them belong to the category of cytoplasmic proteins reported to be membrane-associated in most, if not all, bacteria so far analyzed. They included the elongation factor Tu, reported to be membrane-associated in other bacteria (Marques et al., Infect. Immun. 66, 2625-31, 1998; Dallo et al., Mol. Microbiol. 46, 1041-51, 2002), two ribosomal proteins (Spence & Clark, Infect. Immun. 68, 5002-10, 2000; Kurar & Splitter, Vaccine 15, 1851-57, 1997), and a hypothetical protein possibly involved in cell wall localization and side chain formation.
See also Tables 7 and 8 and
Most of the proteins (60) were identified from peptides generated by trypsin digestion. Proteinase K released peptides corresponding to 30 proteins; 19 proteins were identified by both enzymes. Proteinase K was especially useful for the recovery of peptides corresponding to membrane proteins with a high number of transmembrane domains (TMD): e.g., proteins NT01SP0454, NT01SP0906 and NT01SP1664, with 7, 10 and 4 TMD, respectively, which were not identified from trypsin digestion. The number of identified peptides per protein ranged from one to several tens.
The 11 tryptic-generated peptides and their alignment along the GAS190 protein sequence are shown in
Not all the proteins identified were as extensively covered by their respective generated peptides. Proteins containing cell wall-anchoring motifs showed the highest degree of coverage. Proteins with at least one predicted TMD (according to PSORT prediction) were identified, in general, from a low number of peptides (Table 9).
Bacterial culture and antibiotic treatment. Streptococcus pyogenes SF370 cells were grown in THB at 37° C. and 5% CO2, until an OD600=0.4 was reached (exponential growth phase). Growth medium was harvested after centrifugation at 3,500×g for 10 minutes at 4° C. Bacterial suspension was diluted twofold by adding THB containing antibiotics (0.7 μg/ml penicillin; 10 μg/ml vancomycin, final concentration) and the culture was left for 80 min.
Recovery of membrane-delimited structures. Supernatant was filtered (0.22 μm) and membrane-delimited structures were recovered by ultracentrifuge at 200,000×g for 90 minutes at 4° C. The pellet was then washed once in PBS and then resuspended in the same buffer.
Proteomic analysis of membrane-delimited structures. Ultracentrifugation pellets were subjected to SDS-PAGE. The bands thus separated were picked, destained, trypsin-digested, desalted by using Zip-Tips (Millipore), and analyzed by MALDI-TOF using an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker Daltoniks). MASCOT software was used for spectra analysis and identification.
Proteins identified by this method are shown in Table 8. The majority of the identified proteins are surface-exposed proteins (secreted, membrane-bound, or lipoproteins). No cell wall proteins were observed either in this fraction or in the membrane-delimited structures. Thus, most of the protein content of the membrane-delimited structures is of potential interest for vaccine development.
Bacterial pellet preparation. Bacteria (GAS SF370) were grown in 250 ml THB at 37° C. and 5% CO2 until an OD600=0.4 was reached (exponential growth phase). After culture, bacteria were harvested by centrifugation at 3,500×g for 10 minutes at 4° C. and washed with phosphate-buffered saline (PBS). Bacteria were re-suspended in 20 ml of 6M guanidinium (urea or SDS also could be substituted for guanidinium), 200 mM Tris HCl and disrupted with 3 cycles at 1.7 kBar in a Basic Z 0.75V Model Cell Disrupter equipped with an “one shot head” (Constant System Ltd, Northants, England). The lysate was then centrifuged at 20,000×g for 20 minutes at 4° C. The resulting supernatant was filtered through a 0.22 μm membrane and spun in an ultracentrifuge at 200,000×g for 2 hours at 4° C. The pellet was washed with PBS (200,000×g for 30 minutes at 4° C.).
Total digestion. Three hundred microliters containing 10 μg of trypsin in 50 mM ammonium bicarbonate, 5 mM DTT was added to the pellet. Digestion was allowed to proceed overnight at 37° C.
Separation by SDS-PAGE. Three hundred microliters containing 9 μg of mutanolysin in 50 mM Tris-HCl pH7.5 was added to the pellet. Digestion was allowed to proceed 3 hours at 37° C., which permits the proteins to enter the gel. Proteins were separated by electrophoresis in a 12% polyacrylamide gel.
In-gel protein digestion and sample preparation for mass spectrometry analysis. Protein spots bands were excised from the gels, washed with 100 mM ammonium bicarbonate/acetonitrile 50/50 (V/V), and dried using a SpeedVac centrifuge (Savant, Holbrook, N.Y.). Dried spots were digested 2 hours at 37° C. in 12 μl of 0.012 μg/μl sequencing grade modified trypsin (Promega, Madison, Wis.) in 50 mM ammonium bicarbonate. After digestion, 5 μl of 0.1% trifluoacetic acid was added, and the peptides were desalted and concentrated with ZIP-TIPs (C18, Millipore).
Peptides were eluted with 2 μl of 5 g/l 2,5-dihydroxybenzoic acid in 50% acetonitrile/0.1% trifluoroacetic acid onto the mass spectrometer Anchorchip 384 (400 μm, Bruker Daltonics, Bremen, Germany), allowed to air dry at room temperature, and analysed by matrix-assisted matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Mass spectra were collected on a Bruker UltraFlex mass spectrometer, calibrated using a peptide calibration standard (1000-4000 Da) from Bruker (part no206195). Peptide masses were determined using FlexAnalysis (Version 2.2, Bruker). Protein spot identifications were carried out by both automatic and manual comparison of experimentally generated monoisotopic values of peptides in the mass range of 1000-3500 Da with computer-generated fingerprints using Mascot software.
Results. Fragmentation of peptide of m/z 1372.7, 1202.6 identified the C5A peptidase precursor (15675796). Fragmentation of peptides of m/z 1706.8, 1880.1 identified the M protein type 1 (15675799).
GAS is surrounded by a hyaluronic acid-based capsule whose thickness can vary from strain to strain. Capsule plays an important role in bacterial virulence, and in general the highly capsulated strains are the most virulent strains. It is expected that the number of proteins with accessible external domains will depend on the thickness of the surrounding capsule. To verify that, we characterized the surfome of the highly capsulated strain M3.
The strain was grown as described above and the capsule content was determined by measuring the amount of hyaluronic acid recovered as described below. Under these conditions, M3 produced approximately 51 fg of hyaluronic acid per cfu, three times as much as SF370 strain. See Table 10 and
As shown in Table 11, only 10 proteins could be detected upon proteolytic digestion and mass spectrometry analysis; all but one (Elongation Factor Tu) were predicted to be surface-associated. They include 5 LPXTG (SEQ ID NO:931)-carrying proteins, two membrane proteins and two secreted proteins. Interestingly, 5 of these proteins belong to the hypothetical/unknown protein family. Furthermore, with the exception of the F2-like protein, whose coding gene is absent in SF370, and of the putative penicillin binding protein and the hypothetical protein SPs1270, all the other proteins also belong to the SF370 surfome.
In conclusion, the presence of capsule does interfere with surface accessibility of proteins, as judged by the reduced number of peptides which could be generated upon proteolytic digestion of whole cells.
The almost complete absence of cytoplasmic proteins in both surfomes suggested that our procedure was selective for surface-exposed protein identification. To further confirm the robustness of the procedure we carried out two types of analysis.
First, we subjected the 37 trans-membrane proteins of SF370 surfome to topological prediction using PSORT (see URL address http file type, www host server, domain name nibb.ac.ip, form directory) and asked whether the peptides generated by the proteolytic cleavage and identified by MS/MS were located, as it would be expected in the case of a correct topological prediction, within the external domains. As shown in
This contradiction prompted us to manually inspect the topological and functional annotations of each of these 11 proteins. We concluded that it was desirable to revisit the predicted trans-membrane organization of at least 6 out of 11 proteins. In particular, the two peptides derived from the putative cell division protein NT01SP0014, homologous to the FtsH protein family, are located within a well conserved protein domain known to be extracellular in FtsH proteins (Tomoyasu et al., J. Bacteriol. 175, 1352-57, 1993; Akiyama et al., J. Biol. chem. 271, 22326-33, 1998; Amara et al., Microbiol. 144, 1197-1203, 1998) was found to carry a well conserved protein domain known to be extracellular in these proteins.
The four peptides of NT01SP1255, a second putative cell division protein homologous to FtsZ, are located within the C-terminal part of the molecule, a region that in Bartonella bacilliformis was found immunogenic and surface-exposed (Padmalayam et al., J. Bacteriol. 179, 4545-52, 1997).
The two hypothetical proteins NT01SP0289 and NT01SP1789 carry transmembrane regions (TMR) with a very poor PSORT score and, having a leader peptide cleavage sites next to a Cys residue, are likely to be lipoproteins rather than integral membrane proteins NT01SP0947 has two TMRs the second of which with a very poor PSORT score (−0.32 as opposed to −8.12 of the first TMR). If the second TMR were in fact not real, the C-terminal part of the molecule, which carries a typical sortase domain, would be exposed on the surface, which would be consistent with the sortases mechanism of action (Paterson & Mitchell, Trends Microbiol. 12, 89-95, 2004).
Finally, NT01SP0154, a putative glycine-betaine binding permease protein, is predicted to have 6 TMRs, one of which with a poor score. Again, if the weak TMR is neglected, the topological organization would change and the C-terminal region, where the two MS/MS identified peptides fall, would become surface-exposed. Indeed, polyclonal antibodies against the C-terminal domain of the protein efficiently bound GAS SF370 whole cells when tested by FACS.
The second type of study we used to validate our surfome analysis was based on FACS analysis using protein-specific antibodies. Polyclonal antibodies were produced against 51 recombinant proteins selected among the SF370 surfome, and the antibodies were tested for their capacity to bind to whole bacteria. All but 7 sera were positive in the assay, indicating that each corresponding protein was sufficiently exposed on the bacterial surface to be accessible to antibody binding (see also Table 9). Similarly, polyclonal antibodies against 4 of the 10 proteins belonging to the M3 surfome were tested by FACS and three of them were capable of binding to M3 cells (See
From these data we concluded that our approach for surfome analysis is accurate in determining which proteins are entirely or partially exposed on bacterial cell surface.
From previous experience with Meningococcus B and Group B Streptococcus we know that for an antigen to be a vaccine candidate it is desirable that it be well expressed and exposed on the surface of the bacterial cell (Pizza et al., Science 287, 1816-20, 2000; Maione et al., Science. 2005 Jul. 1; 309(5731):148-50.). Because surfomes, by definition, include this category of proteins, surfome analysis is an ideal approach to identification of new vaccine candidates. In support of this is the observation that 6 of the 10 reported GAS protective antigens whose genes are present in SF370 are part of the SF370 surfome. See Table 12 and references cited therein.
Because several of the SF370 surfome proteins have never been tested for protection, we investigated whether some of them could elicit protective responses in the mouse model. Unfortunately, SF370 is not virulent in the mouse, the LD50 dose being over 108 CFUs. Because we expect the GAS surfome to vary somewhat from strain to strain depending upon protein expression level, capsule thickness, and gene variability, before testing the SF370 surfome proteins in protection studies against a different strain, we investigated which of these proteins were also exposed on the surface of the challenge strain.
To this end, we defined the surfome of M23 DSM2071, one of the GAS strains we routinely used for mouse challenge. Because the genome sequence of M23 DSM2071 is not available, only the exposed proteins that are in common to SF370 or to the other six GAS strains whose sequences are available in the public databases (URL address: http file type, www host server, domain names tigr.org and ncbi.nlm.nih.gov) are identified, whereas the M23 DSM2071-specific proteins remain uncharacterized.
As shown in Table 7, a total of 17 proteins were unambiguously identified: 5 cell wall anchored proteins, 4 lipoproteins, 5 membrane proteins, 2 secreted proteins and 1 cytoplasmic protein. All these proteins have an analogue in SF370 and all but two (the putative zinc-containing alcohol dehydrogenase and the putative, RofA-related, regulatory protein) were also included in the SF370 surfome (Table 9). Interestingly, most (13 out of 17) of the identified proteins belong to the family of putative/hypothetical proteins.
Of the 17 proteins belonging to the M23 DSM2071 surfome, 14 proteins were successfully expressed in E. coli as either soluble His-fusions or GST-fusions (Table 7). Proteins NT01SP0908 and NT01SP0485 were not considered for expression because they have significant homology with human proteins. Five-week-old female CD 1 mice (10 mice/group) were immunized with 20 μg recombinant protein administered intraperitoneally (i.p.) at 0, 21 and 35 days with complete Freund's adjuvant (CFA) the first time and with incomplete Freund's adjuvant (IFA) the two following times. Blood samples were collected before the first and after the third immunizations. Immunized mice were challenged intranasally (i.n.) with 106 colony forming units (CFUs) of DSM 2071 strain grown in THY broth at OD600=0.4. CFU titer of the infecting dose was verified by plating on 5 THY/blood plates. Mice were monitored daily, and the final survival rate was calculated after 10 days.
As shown in Table 14, two proteins were protective in this model: the M protein (90% survival rate) and NT01SP0336 (70% survival rate). NT01SP0336, which corresponds to GAS57 in the SF370 serotype, is a putative cell envelope proteinase carrying a typical cell anchoring LPXTG (SEQ ID NO:931) motif.
Interestingly, GAS57 provided little or no protection in a mouse model in which intraperitoneal immunization was followed by intraperitoneal challenge. GAS40 was protective in both models, even though the survival rate in the intranasal challenge model was higher.
Cells from a 10-ml exponential-phase culture (OD600=0.4) are washed twice with water and then resuspended in 500 μl of water. Capsule is released by shaking with 1 ml of clorophorm. After clarifying the sample by centrifugation, the hyaluronic acid content of 50 μl of the aqueous phase is determined by measuring absorbance at 640 nm after adding to the sample 1 ml of a solution containing 20 mg of Stains-All product (Sigma Chemical Co.) and 60 μl of glacial acetic acid in 100 ml of 50% formamide. Absorbance values are compared with a standard curve generated using known concentrations of hyaluronic acid.
In silico prediction algorithms initially identified 684 genes encoding for products likely to be secreted or associated with the bacterial surface. See Pizza et al., Science 287, 1816-20, 2000; Tettelin et al., Proc. Natl. Acad. Sci. U.S.A. 99, 12391-96, 2002. Of these, 207 were predicted to contain more than two transmembrane spanning regions. The protein sequences were searched for isolated domains of at least 50 amino acids which were predicted to lay on the surface of the cell (e.g., extracellular loops, amino-terminal, or carboxy-terminal domains). Surface exposure was assessed with the aid of the on-line web server TMPRED (see URL address: http file type, www host server, domain name.ch.embnet.org, software/TMPRED_form.directory), which is able to predict membrane-spanning regions and their orientation using an algorithm based on the statistical analysis of TMbase, a database of naturally occurring transmembrane proteins.
Each of the identified domains was cloned in parallel in two vectors containing either sequences coding for 6 histidine residues. Recombinant products were successfully expressed and purified from E. coli.
Groups of 10 or more CD1 female mice aged between 6 and 7 weeks were immunized with 20 μg of a recombinantly produced surface-exposed GAS antigen suspended in 100 μl of suitable solution. Mice of each group received 3 doses, at days 0, 21 and 45. Immunization was performed through intraperitoneal injection of the protein with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and with Incomplete Freund's Adjuvant (IFA) for the following two doses. Negative and positive control groups were used in each immunization scheme.
Mice in the negative control group were immunized with E. coli proteins eluted from the purification columns following processing of total bacterial extract from an E. coli strain containing either the pET21b or the pGEX-NNH vector (thus expressing GST only) without any cloned GAS ORF (indicated as HisStop or GSTStop, respectively).
Mice in the positive control groups were immunized with purified GAS M cloned from either GAS SF370 or GAS DSM 2071 strains (groups indicated as 192SF and 192DSM respectively).
Serum from each mouse was collected before the first immunization and two weeks after the last immunization. The sera of mice in each group were pooled. Mice were infected with one of GAS strains 2071 (M23), 3348 (M1), or 2728 (M12) about a week after the last immunization. For infection, GAS strains were grown at 37° C. in THY broth until OD600 0.4. Bacteria were collected by centrifugation, washed once with PBS, suspended, and diluted with PBS to obtain the appropriate concentration of bacteria/ml and administered to mice by intraperitoneal injection. Between 50 and 100 bacteria were given to each mouse, as determined by plating aliquots of the bacterial suspension on 5 THY plates. Animals were observed daily and checked for survival.
The results are shown in
The results demonstrate that each of the tested domains—GAS35, GAS414, GAS426, GAS433, GAS434, GAS437, GAS438, GAS439, GAS461, GAS465-2, GAS469, GAS472, GAS473, GAS475, GAS477, GAS478, GAS495, GAS538, GAS543, GAS553, GAS561, GAS576, GAS577-2, GAS587, GAS591, GAS593, GAS636, GAS643, GAS649, and GAS663—is exposed on the surface of at least one of the three GAS strains tested. Some of the tested domains show a variable delta mean across the strains used (M1, M23 and M12), possibly because of the different “visibility” of these domains due to capsule masking (for instance M23 is a highly encapsulated strain). Domains GAS35, GAS414, GAS437, GAS438, GAS461, GAS465-2, GAS469, GAS472, GAS473, GAS475, GAS478, GAS495, GAS538, GAS553, GAS561, GAS577-2, GAS591, GAS593, GAS636, GAS643, GAS649, and GAS663 are surface-exposed on the surface of at least two of the three GAS strains tested. Domains GAS472, GAS473, and GAS553 are surface-exposed on all three of the tested GAS strains.
Protein chips allow for the identification of clinical immunogenic prevalence of pathogenic proteins in human sera. Using protein chips we tested serum samples from 6 healthy donors, two of which had had a recent documented pharyngitis associated to GAS infection (SC and TM, see
Experimental Procedure
112 GAS proteins (19 GST fusions, 91 His-tagged and 2 native proteins) were purified, diluted in PBS at a concentration of 0.5 mg/ml and dispensed (6 μl each) in 384 well polypropylene micro plates. Four replicates of the protein solutions were spotted on nitrocellulose coated Fast Slides chips (Schleier & Schuell) using the VERSARRAY CHIPWRITER Pro System (BIO-RAD) equipped with TeleChem quill pins (TeleChem International Sunnyvale, Calif., USA). Following the first printing of each protein, the pins were washed 7 times (6 seconds each), subjected to sonication (1 second) and dried under vacuum (2 seconds). Each chip contains at least one immunoglobulin and two BSA (Cy3 and Cy5-labelled, Amersham Bioscences) standard curves. After each printing process, each slide was scanned to check the signals of the Cy3 and Cy5-labeled BSA curves.
Slides were pre-incubated overnight at 4° C. in the dark with agitation in 3% Top Block (Fluka-BioChemiKa, Cat. no 37766) and 0.1% TPBS (0.1% Tween 20 in PBS). Slides were then incubated with human sera (1:1000 final dilution) for 1 hr at room temperature in the dark and were then washed 3 times (5 minutes each time) in 0.1% TPBS. Cy3 or Cy5 anti-human IgG (1:800), IgA or IgM (1:1000) were added and incubation was prolonged for 1 hr at room temperature in the dark.
Slides were washed two times with TPBS (5 minutes each time), once with PBS (10 minutes), once with milliQ sterile water (30 seconds) and were then dried either at 37° C. for 10-20 minutes in the dark or using a nitrogen stream.
Fluorescence signals were detected with a ScanArray 5000 Unit (Packard, Billerica, Mass., USA) at high resolution (10 μm pixel size) and quantified with the ImaGene 6.0 software (Biodiscovery Inc, Ca, Usa)
Elaboration of the collected data was performed using software which normalizes the data by interpolating the least-mean squares of the Ig controls to a sigmoid curve. The titer value corresponding to each experimental intensity signal is then referred to such sigmoid and normalized to a theoretic sigmoid curve extending over the whole dynamic range of the scanner.
In silico-predicted surface-exposed proteins
This application claims the benefit of and incorporates by reference co-pending provisional application Ser. No. 60/616,854 filed Oct. 8, 2004; Ser. No. 60/652,736 filed Feb. 15, 2005; Ser. No. 60/701,121 filed Jul. 21, 2005; and Ser. No. 60/705,209 filed Aug. 4, 2005.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2005/036009 | 10/11/2005 | WO | 00 | 11/25/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60616854 | Oct 2004 | US | |
| 60652736 | Feb 2005 | US | |
| 60701121 | Jul 2005 | US | |
| 60705209 | Aug 2005 | US |
