Patent Application
20150165019
All documents cited herein are incorporated by reference in their entirety.
This invention is in the field of vaccines and relates to new compositions comprising two or more saccharide antigens conjugated to a polyepitope carrier protein comprising T cell epitopes from multiple pathogenic proteins. The invention also relates to methods for making said compositions and to uses for said compositions.
Polyvalent vaccines are known in the art. One such example is a tetravalent vaccine of capsular polysaccharides from serogroups A, C, Y and W135 of N. meningitidis which has been known for many years [1, 2] and has been licensed for human use. However, although effective in adolescents and adults, it induces a poor immune response and short duration of protection and cannot be used in infants [e.g. 3]. This is because polysaccharides are T cell-independent antigens that generally induce a weak immune response that cannot be boosted. Concerns have often arisen regarding the widespread use of polyvalent vaccines because they are subject to a significant decrease in immune function known as immunosuppression. Immunosuppression may result when the amount of antigen introduced into the subject exceeds the ability of the immune system to respond. Such a condition is termed antigen-overload. Immunosuppression may also occur as a result of one antigen component preventing the immune system from responding to another antigen component of a polyvalent vaccine. This latter form of immunosuppression is termed vaccine interference.
In the last 20 years, conjugate vaccines, comprising bacterial capsular polysaccharides conjugated to protein carriers have developed. Examples include the Haemophilus influenzae type b (Hib) conjugate vaccine [4] as well as conjugate vaccines against Streptococcus pneumoniae [5] and serogroup C Neisseria meningitidis (MenC) [6].
The carrier proteins used in licensed vaccines include tetanus toxoid (TT), diphtheria toxoid (DT), the nontoxic CRM197 mutant of diptheria toxin, and the outer membrane protein complex from group B N. meningitidis. Since more conjugated vaccines are being introduced into the medical practice, infants could receive multiple injections of the carrier protein, either as a vaccine itself (e.g. TT or DT) or as a carrier protein present in a conjugate vaccine. As these proteins are highly immunogenic at both the B- and T-cell level, carrier overload may induce immune suppression in primed individuals [7]. This phenomenon, termed carrier-induced epitopic suppression, is thought to be due to carrier specific antibodies and intramolecular antigenic competition [8]. Ideally, a carrier protein should induce strong helper effect to a conjugated B-cell epitope (e.g. polysaccharide) without inducing an antibody response against itself. The use of universal epitopes, which are immunogenic in the context of most major histocompatability complex class II molecules, is one approach towards this goal [9]. Such epitopes have been identified within TT and other proteins. However, there remains the need for further improvements.
It is therefore the object of the invention to provide improved saccharide conjugates.
It has been discovered that polyepitope carrier proteins are particularly useful as carriers for combinations of saccharides. Furthermore, it has been discovered that only a low immunogenic response is seen against these carrier proteins even though they comprise a number of known pathogenic epitopes, whereas it would have been expected that the immunogenic response would increase proportionally to the number of pathogenic epitopes.
In some embodiments, the invention therefore provides a composition comprising a combination of two or more monovalent conjugates (e.g. 2, 3, 4, 5, 6 or more. See
Although each carrier protein molecule in each monovalent conjugate may be conjugated to more than one saccharide antigen molecule (e.g. 1, 5, 10, 20 or more) due to the multiple attachment sites on each carrier protein molecule (
As an alternative, in some embodiments, the invention provides a multivalent conjugate comprising two or more (e.g. 2, 3, 4, 5, 6 or more) antigenically distinct saccharide antigens conjugated to the same carrier protein molecule (
As a further alternative, the invention provides a composition comprising one or more (e.g. 1, 2, 3, 4, 5, 6 or more) monovalent conjugate(s) and one or more (e.g. 1, 2, 3, 4, 5, 6 or more) multivalent conjugate(s) as described above.
The carrier protein may comprise 2 or more T cell epitopes (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more). Preferably the carrier protein comprises 6 or more, or 10 or more epitopes. More preferably the carrier protein comprises 19 or more epitopes. Each carrier protein may only have one copy of a particular epitope or may have more than one copy of a particular epitope. Preferably the epitopes are CD4+ T cell epitopes. Preferably the carrier protein comprises at least one bacterial epitope and at least one viral epitope. Preferably the epitopes are derived from antigens to which the human population is frequently exposed either by natural infection or vaccination, for example, epitopes may be derived from Hepatitis A virus, Hepatitis B virus, Measles virus, Influenza Virus, Varicella-zoster virus, heat shock proteins from Mycobacterium bovis and M. leprae and/or Streptococcus strains etc. Preferably the epitopes are selected from Tetanus toxin (TT), Plasmodium falciparum CSP (PfCs), Hepatitis B virus nuclear capsid (HBVnc), Influenza haemagglutinin (HA), HBV surface antigen (HBsAg) and Influenza matrix (MT). The epitopes used in the carrier protein are preferably selected from P23TT (SEQ ID NO: 1), P32TT (SEQ ID NO: 2), P21TT (SEQ ID NO: 3), PfCs (SEQ ID NO: 4), P30TT (SEQ ID NO: 5), P2TT (SEQ ID NO: 6), HBVnc (SEQ ID NO: 7), HA (SEQ ID NO: 8), HBsAg (SEQ ID NO: 9) and MT (SEQ ID NO: 10).
Preferably the epitopes are joined by spacers. Preferably the spacer is a short (e.g. 1, 2, 3, 4 or 5) amino acid sequence which is not an epitope. A preferred spacer comprises one or more glycine residues, e.g -KG-. Preferably the carrier protein comprises a N- or C-terminal region comprising a six-His tail, an immunoaffinity tag useful for screening for the carrier protein (for example the sequence “MDYKDDDD” [SEQ ID NO: 12] may be used), and/or a protease cleavage sequence. Preferably the proteolytic sequence is the factor Xa recognition site.
Preferably the carrier comprises no suppressor T cell epitopes.
Preferably the carrier protein is N19 (SEQ ID NO: 11). It has been shown that a genetically engineered protein, termed N19 [10], expressed in Escherichia coli and having several human CD4+ T-cell universal epitopes, behaves as a strong carrier when conjugated to Hib polysaccharide [11]. The N-terminal region of the N19 consists of (i) a six His tail that may be exploited during purification, (ii) a flag peptide Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp sequence (SEQ ID NO: 12) recognized by a rabbit polyclonal antibody that can be used for the screening of positive colonies during the cloning procedure, (iii) the Ile-Glu-Gly-Arg (SEQ ID NO: 13) Factor Xa recognition site for ready elimination of the tag. N19 is a duplication of the first nine epitopes listed in Table 1 plus the influenza matrix CD4+ epitope MT. The epitopes are separated by a Lys-Gly spacer to provide flexibility to the molecule and to allow the subsequent conjugation of the polysaccharide to the primary ε-amino groups of Lys residues.
In addition to CD4+ epitopes, carrier proteins may comprise other peptides or protein fragments, such as epitopes from immunomodulating cytokines such as interleukin-2 (IL-2) or granulocyte-macrophage colony stimulating factor (GM-CSF).
P. falciparum
Preferably, the saccharide antigen conjugated to the carrier protein in a composition of the invention is a bacterial saccharide and in particular a bacterial capsular saccharide.
Examples of bacterial capsular saccharides which may be included in the compositions of the invention include capsular saccharides from Neisseria meningitidis (serogroups A, B, C, W135 and/or Y), Streptococcus pneumoniae (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, particularly 4, 6B, 9V, 14, 18C, 19F and/or 23F), Streptococcus agalactiae (types Ia, Ib, II, III, IV, V, VI, VII, and/or VIII, such as the saccharide antigens disclosed in references 20-23), Haemophilus influenzae (typeable strains: a, b, c, d, e and/or f), Pseudomonas aeruginosa (for example LPS isolated from PA01, O5 serotype), Staphylococcus aureus (from, for example, serotypes 5 and 8), Enterococcus faecalis or E. faecium (trisaccharide repeats), Yersinia enterocolitica, Vibrio cholerae, Salmonella typhi, Klebsiella spp., etc. Other saccharides which may be included in the compositions of the invention include glucans (e.g. fungal glucans, such as those in Candida albicans), and fungal capsular saccharides e.g. from the capsule of Cryptococcus neoformans.
The N. meningitidis serogroup A (MenA) capsule is a homopolymer of (α1→6)-linked N-acetyl-D-mannosamine-1-phosphate, with partial O-acetylation in the C3 and C4 positions. The N. meningitidis serogroup B (MenB) capsule is a homopolymer of (α2→8)-linked sialic acid. The N. meningitidis serogroup C (MenC) capsular saccharide is a homopolymer of (α2→9) linked sialic acid, with variable O-acetylation at positions 7 and/or 8. The N. meningitidis serogroup W135 saccharide is a polymer having sialic acid-galactose disaccharide units [→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α-(1→]. It has variable O-acetylation at the 7 and 9 positions of the sialic acid [24]. The N. meningitidis serogroup Y saccharide is similar to the serogroup W135 saccharide, except that the disaccharide repeating unit includes glucose instead of galactose [→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Glc-α-(1→]. It also has variable O-acetylation at positions 7 and 9 of the sialic acid.
The compositions of the invention comprise mixtures of saccharide antigens. Preferably the compositions comprise 2, 3, 4 or more different saccharide antigens. The antigens may be from the same or from antigenically distinct pathogens. Preferably, compositions of the invention comprise saccharide antigens from more than one serogroup of N. meningitidis, e.g. compositions may comprise saccharides conjugates from serogroups A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, C+W135+Y, A+C+W135+Y, etc. Preferred compositions comprise saccharides from serogroups C and Y. Other preferred compositions comprise saccharides from serogroups C, W135 and Y. Particularly preferred compositions comprise saccharides from serogroups A, C, W135 and Y.
Where a mixture comprises meningococcal saccharides from serogroup A and at least one other serogroup saccharide, the ratio (w/w) of MenA saccharide to any other serogroup saccharide may be greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Ratios between 1:2 and 5:1 are preferred, as are ratios between 1:1.25 and 1:2.5. Preferred ratios (w/w) for saccharides from serogroups A:C:W135:Y are: 1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2; 8:4:1:2; 4:2:2:1; 2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; and 2:2:2:1.
Further preferred compositions of the invention comprise a Hib saccharide conjugate and a saccharide conjugate from at least one serogroup of N. meningitidis, preferably from more than one serogroup of N. meningitidis. For example, a composition of the invention may comprise a Hib saccharide and saccharides from one or more (i.e. 1, 2, 3 or 4) of N. meningitidis serogroups A, C, W135 and Y. Other combinations of saccharide conjugates from the pathogens mentioned above are also provided.
The invention also provides, in some embodiments, combinations of conjugates where the carrier protein is not the same for each conjugate.
Further preferred compositions of the invention comprise a first conjugate and a second conjugate. The first conjugate is a polyepitope conjugate as described above and the second conjugate comprises a saccharide antigen conjugated to a carrier protein different from that used in the first conjugate. For example the second conjugate may be a saccharide antigen conjugated to the carrier CRM197. The saccharide antigen(s) in the second conjugate may be the same as or different from the saccharide antigen(s) in the first conjugate.
Methods for the preparation of capsular saccharide antigens are well known. For example, ref. 25 describes the preparation of saccharide antigens from N. meningitidis. The preparation of saccharide antigens from H. influenzae is described in chapter 14 of ref. 26. The preparation of saccharide antigens and conjugates from S. pneumoniae is described in the art. For example, Prevenar™ is a 7-valent pneumococcal conjugate vaccine. Processes for the preparation of saccharide antigens from S. agalactiae are described in detail in refs. 27 and 28. Capsular saccharides can be purified by known techniques, as described in several references herein.
The saccharide antigens may be chemically modified. For instance, they may be modified to replace one or more hydroxyl groups with blocking groups. This is particularly useful for meningococcal serogroup A where the acetyl groups may be replaced with blocking groups to prevent hydrolysis [29]. Such modified saccharides are still serogroup A saccharides within the meaning of the present invention.
The saccharide may be chemically modified relative to the capsular saccharide as found in nature. For example, the saccharide may be de-O-acetylated (partially or fully), de-N-acetylated (partially or fully), N-propionated (partially or fully), etc. De-acetylation may occur before, during or after conjugation, but preferably occurs before conjugation. Depending on the particular saccharide, de-acetylation may or may not affect immunogenicity e.g. the NeisVac-C™ vaccine uses a de-O-acetylated saccharide, whereas Menjugate™ is acetylated, but both vaccines are effective. The effect of de-acetylation etc. can be assessed by routine assays.
Capsular saccharides may be used in the form of oligosaccharides. These are conveniently formed by fragmentation of purified capsular polysaccharide (e.g. by hydrolysis), which will usually be followed by purification of the fragments of the desired size. Fragmentation of polysaccharides is preferably performed to give a final average degree of polymerisation (DP) in the oligosaccharide of less than 30. DP can conveniently be measured by ion exchange chromatography or by colorimetric assays [30].
If hydrolysis is performed, the hydrolysate will generally be sized in order to remove short-length oligosaccharides [31]. This can be achieved in various ways, such as ultrafiltration followed by ion-exchange chromatography. Oligosaccharides with a degree of polymerisation of less than or equal to about 6 are preferably removed for serogroup A meningococcus, and those less than around 4 are preferably removed for serogroups W135 and Y.
Conjugates of the invention may include small amounts of free (i.e. unconjugated) carrier. When a given carrier protein is present in both free and conjugated form in a composition of the invention, the unconjugated form is preferably no more than 5% of the total amount of the carrier protein in the composition as a whole, and more preferably present at less than 2% (by weight).
After conjugation, free and conjugated saccharides can be separated. There are many suitable methods, including hydrophobic chromatography, tangential ultrafiltration, diafiltration etc. [see also refs. 32 & 33, etc.].
Any suitable conjugation reaction can be used, with any suitable linker where necessary. Attachment of the saccharide antigen to the carrier is preferably via a —NH2 group e.g. in the side chain of a lysine residue in a carrier protein, or of an arginine residue. Where a saccharide has a free aldehyde group then this can react with an amine in the carrier to form a conjugate by reductive amination. Attachment may also be via a —SH group e.g. in the side chain of a cysteine residue. Alternatively the saccharide antigen may be attached to the carrier via a linker molecule.
The saccharide will typically be activated or functionalised prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate [34, 35, etc.]). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, MC, TSTU (see also the introduction to reference 36).
Linkages via a linker group may be made using any known procedure, for example, the procedures described in references 37 and 38. One type of linkage involves reductive amination of the saccharide, coupling the resulting amino group with one end of an adipic acid linker group, and then coupling the carrier protein to the other end of the adipic acid linker group [39, 40]. Other linkers include B-propionamido [41], nitrophenyl-ethylamine [42], haloacyl halides [43], glycosidic linkages [44], 6-aminocaproic acid [45], ADH [46], C4 to C12 moieties [47] etc. As an alternative to using a linker, direct linkage can be used. Direct linkages to the protein may comprise oxidation of the polysaccharide followed by reductive amination with the protein, as described in, for example, references 48 and 49.
A process involving the introduction of amino groups into the saccharide (e.g. by replacing terminal=O groups with —NH2) followed by derivatisation with an adipic diester (e.g. adipic acid N-hydroxysuccinimido diester) and reaction with carrier protein is preferred.
A bifunctional linker may be used to provide a first group for coupling to an amine group in the saccharide and a second group for coupling to the carrier (typically for coupling to an amine in the carrier).
The first group in the bifunctional linker is thus able to react with an amine group (—NH2) on the saccharide. This reaction will typically involve an electrophilic substitution of the amine's hydrogen. The second group in the bifunctional linker is able to react with an amine group on the carrier. This reaction will again typically involve an electrophilic substitution of the amine.
Where the reactions with both the saccharide and the carrier involve amines then it is preferred to use a bifunctional linker of the formula X-L-X, where: the two X groups are the same as each other and can react with the amines; and where L is a linking moiety in the linker. A preferred X group is N-oxysuccinimide. L preferably has formula L′-L2-L′, where L′ is carbonyl. Preferred L2 groups are straight chain alkyls with 1 to 10 carbon atoms (e.g. C1, C2, C3, C4, C5, C6, C7, C8, C9, C10) e.g. —(CH2)4—.
Other X groups are those which form esters when combined with HO-L-OH, such as norborane, p-nitrobenzoic acid, and sulfo-N-hydroxysuccinimide.
Further bifunctional linkers for use with the invention include acryloyl halides (e.g. chloride) and haloacylhalides.
The linker will generally be added in molar excess to modified saccharide.
After conjugation, free and conjugated saccharides can be separated. There are many suitable methods, including hydrophobic chromatography, tangential ultrafiltration, diafiltration etc. [see also refs. 50 & 51, etc.].
Where the composition of the invention includes a depolymerised saccharide, it is preferred that depolymerisation precedes conjugation.
Compositions of the invention may comprise one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) further antigens, such as:
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 meningitidis: meningococcal antigens may include proteins (such as those identified in references 52-58), saccharides (including a polysaccharide, oligosaccharide or lipopolysaccharide), or outer-membrane vesicles [59-62] purified or derived from a N. meningitidis serogroup such as A, C, W135, Y, and/or B. Meningococcal protein antigens may be selected from adhesins, autotransporters, toxins, iron acquisition proteins, and membrane associated proteins (preferably integral outer membrane proteins). See also refs. 63-71.
Streptococcus pneumoniae: S. pneumoniae antigens may include a saccharide (including a polysaccharide or an oligosaccharide) and/or protein from S. pneumoniae. Protein antigens may be selected, for example, from a protein identified in any of refs. 72-77. S. 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. See also refs. 78-84.
Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcus antigens may include a protein identified in reference 85 or 86 (including GAS40), fusions of fragments of GAS M proteins (including those described in refs. 87-89), fibronectin binding protein (Sfb1), Streptococcal heme-associated protein (Shp), and Streptolysin S (SagA). See also refs. 85, 90 and 91.
Moraxella catarrhalis: Moraxella antigens include antigens identified in refs. 92 & 93, outer membrane protein antigens (HMW-OMP), C-antigen, and/or LPS. See also ref. 94.
Bordetella pertussis: Pertussis antigens include pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 antigen. See also refs. 95 & 96.
Staphylococcus aureus: S. 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 engulfinent (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). See also ref. 97.
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.
Corynebacterium diphtheriae (Diphtheria): Diphtheria antigens include diphtheria toxin or detoxified mutants thereof, 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. These diphtheria antigens may be used as carrier proteins.
Haemophilus influenzae: H influenzae antigens include a saccharide antigen from type B, or protein D [98].
Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzz protein and/or Outer Membrane Proteins, including Outer Membrane Proteins F (OprF) [99].
Legionella pneumophila. Bacterial antigens may be derived from Legionella pneumophila.
Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcus antigens include protein antigens identified in refs. 85 and 100-103. For example, the antigens include proteins GBS80, GBS104, GBS276 and GBS322.
Neisseria gonorrhoeae: Gonococcal antigens include Por (or porin) protein, such as PorB [104], a transferring binding protein, such as TbpA and TbpB [105], an opacity protein (such as Opa), a reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations [106]. See also refs. 52-54 & 107.
Chlamydia trachomatis: C. 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. C. trachomatis antigens may also include an antigen identified in refs. 103 & 108-110, 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). See also ref. 111.
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 ref. 112.
Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX, HopY and/or urease antigen. [113-123].
Staphylococcus saprophyticus: Antigens include the 160 kDa hemagglutinin of S. saprophyticus antigen.
Yersinia enterocolitica Antigens include LPS [124].
Escherichia 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) strains.
Bacillus anthracia (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). See refs. 125-127.
Yersinia pestis (plague): Plague antigens include F1 capsular antigen [128], LPS [129], V antigen [130].
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 [131], Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associated antigens [132], and/or MPT51 antigens [133].
Rickettsia: Antigens include outer membrane proteins, including the outer membrane protein A and/or B (OmpB) [134], LPS, and surface protein antigen (SPA) [135].
Listeria monocytogenes: Bacterial antigens may be derived from Listeria monocytogenes.
Chlamydia pneumoniae: Antigens include those identified in refs. 108 & 136 to 141.
Vibrio cholerae: Antigens include proteinase antigens, particularly lipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specific polysaccharides, V. cholera 0139, antigens of IEM108 vaccine [142], and/or Zonula occludens toxin (Zot).
Salmonella typhi (typhoid fever): Antigens include capsular polysaccharides preferably conjugates (Vi, e.g. 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, [143]) and VlsE Antigenic Variation Protein [144].
Porphyromonas gingivalis: Antigens include the outer membrane protein (OMP). See also ref. 145.
Klebsiella: Antigens include an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid.
Further bacterial antigens may be capsular antigens, saccharide 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 used in the present invention may be derived from gram-negative and/or gram-positive bacteria. The antigens used in the present invention may be derived from aerobic and/or anaerobic bacteria.
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 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). [146-148].
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, for example, ref. 149. 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, Heparnavirus, Cardioviruses and Aphthoviruses. Antigens derived from Enteroviruses, such as Poliovirus are preferred. See refs. 150 & 151.
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. [152,153].
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 [153,154].
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 [155,156].
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 comprises killed virus grown on human diploid cells or fetal rhesus lung cells [157,158].
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/or Hemagglutinin-esterase glycoprotein (HE). Preferably, the Coronavirus antigen is derived from a SARS virus. SARS viral antigens are described in ref. 159.
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, gp120 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 σ1s. 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, and/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/or NS-2. Preferably, the Parvovirus antigen is capsid protein VP-2.
Delta hepatitis virus (HDV): Viral antigens may be derived HDV, particularly 8-antigen from HDV (see, e.g., ref. 160).
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.
Fungal antigens 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 include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowii, 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, Pythiumn 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 [161]. 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.
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 [162]. 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, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Escherichia coli, and Streptococcus agalactiae. Examples of specific antigens derived from these pathogens are described above.
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 anthracia, and Moraxella catarrhalis. Examples of specific antigens derived from these pathogens are described above.
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 meningitidis, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani (Tetanus), Corynebacterium diphtheriae (Diphtheria), Haemophilus influenzae type B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae (Group B Streptococcus), and Escherichia coli. Examples of specific antigens derived from these pathogens are described above.
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 meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani (Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilus influenzae type B (Hib), Pseudomonas aeruginosa, Legionella pneumophila, Streptococcus agalactiae (Group B Streptococcus), Enterococcus faecalis, Helicobacter pylori, Chlamydia 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.
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.
One embodiment of the invention involves a tumor antigen or cancer antigen. Tumor antigens can be, for example, peptide-containing tumor antigens, such as a polypeptide tumor antigen or glycoprotein tumor antigens. A tumor antigen can also be, for example, a saccharide-containing tumor antigen, such as a glycolipid tumor antigen or a ganglioside tumor antigen. The tumor antigen can further be, for example, a polynucleotide-containing tumor antigen that expresses a polypeptide-containing tumor antigen, for instance, an RNA vector construct or a DNA vector construct, such as plasmid DNA.
Tumor antigens appropriate for the practice of the present invention encompass a wide variety of molecules, such as (a) polypeptide-containing tumor antigens, including polypeptides (which can range, for example, from 8-20 amino acids in length, although lengths outside this range are also common), lipopolypeptides and glycoproteins, (b) saccharide-containing tumor antigens, including poly-saccharides, mucins, gangliosides, glycolipids and glycoproteins, and (c) polynucleotides that express antigenic polypeptides.
The tumor antigens can be, for example, (a) full length molecules associated with cancer cells, (b) homologs and modified forms of the same, including molecules with deleted, added and/or substituted portions, and (c) fragments of the same. Tumor antigens can be provided in recombinant form. Tumor antigens include, for example, class I-restricted antigens recognized by CD8+ lymphocytes or class II-restricted antigens recognized by CD4+ lymphocytes.
Numerous tumor antigens are known in the art, including: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-ab1 (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer), (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase related protein-2/TRP2 (associated with, e.g., melanoma), (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer, (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example), and (g) other tumor antigens, such as polypeptide- and saccharide-containing antigens including (i) glycoproteins such as sialyl Tn and sialyl Lex (associated with, e.g., breast and colorectal cancer) as well as various mucins; glycoproteins may be coupled to a carrier protein (e.g., MUC-1 may be coupled to KLH); (ii) lipopolypeptides (e.g., MUC-1 linked to a lipid moiety); (iii) polysaccharides (e.g., Globo H synthetic hexasaccharide), which may be coupled to a carrier proteins (e.g., to KLH), (iv) gangliosides such as GM2, GM12, GD2, GD3 (associated with, e.g., brain, lung cancer, melanoma), which also may be coupled to carrier proteins (e.g., KLH).
Additional tumor antigens which are known in the art include p15, Hom/Me1-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like. These as well as other cellular components are described for example in reference 163 and references cited therein.
Polynucleotide-containing antigens in accordance with the present invention typically comprise polynucleotides that encode polypeptide cancer antigens such as those listed above. Preferred polynucleotide-containing antigens include DNA or RNA vector constructs, such as plasmid vectors (e.g., pCMV), which are capable of expressing polypeptide cancer antigens in vivo.
Tumor antigens may be derived, for example, from mutated or altered cellular components. After alteration, the cellular components no longer perform their regulatory functions, and hence the cell may experience uncontrolled growth. Representative examples of altered cellular components include ras, p53, Rb, altered protein encoded by the Wilms' tumor gene, ubiquitin, mucin, protein encoded by the DCC, APC, and MCC genes, as well as receptors or receptor-like structures such as neu, thyroid hormone receptor, platelet derived growth factor (PDGF) receptor, insulin receptor, epidermal growth factor (EGF) receptor, and the colony stimulating factor (CSF) receptor. These as well as other cellular components are described for example in ref. 164 and references cited therein.
Additionally, bacterial and viral antigens, may be used in conjunction with the compositions of the present invention for the treatment of cancer. In particular, carrier proteins, such as CRM197, tetanus toxoid, or Salmonella typhimurium antigen can be used in conjunction/conjugation with compounds of the present invention for treatment of cancer. The cancer antigen combination therapies will show increased efficacy and bioavailability as compared with existing therapies.
Additional information on cancer or tumor antigens can be found, for example, in reference 165 (e.g. Tables 3 & 4), in reference 166 (e.g. Table 1) and in references 167 to 189.
Immunisation can also be used against Alzheimer's disease e.g. using Abeta as an antigen[190].
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 PLO, 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.
Additional formulation methods and antigens (especially tumor antigens) are provided in ref. 191.
Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated. The compositions may be formulated as vaccines that are particularly useful for vaccinating children and teenagers. They may be delivered by systemic and/or mucosal routes.
Typically, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Direct delivery of the compositions will generally be parenteral (e.g. by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue). The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e.g. see ref. 192), needles, and hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).
Vaccines of the invention are preferably sterile. They are preferably pyrogen-free. They are preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Where a vaccine comprises an aluminium hydroxide salt, it is preferred to use a histidine buffer [193].
Vaccines of the invention may comprise detergent (e.g. a Tween, such as Tween 80) at low levels (e.g. <0.01%). Vaccines of the invention may comprise a sugar alcohol (e.g. mannitol) or trehalose e.g. at around 15 mg/ml, particularly if they are to be lyophilised.
Optimum doses of individual antigens can be assessed empirically. In general, however, saccharide antigens of the invention will be administered at a dose of between 0.1 and 100 μg of each saccharide per dose, with a typical dosage volume of 0.5 ml. The dose is typically between 5 and 20 μg per saccharide per dose. These values are measured as saccharide.
Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection), but will typically be prophylactic.
The invention provides a conjugate of the invention for use in medicine.
The invention also provides a method of raising an immune response in a patient, comprising administering to a patient a conjugate according to the invention. The immune response is preferably protective against meningococcal disease, pneumococcal disease or H. influenzae and may comprise a humoral immune response and/or a cellular immune response. The patient is preferably a child. The method may raise a booster response, in a patient that has already been primed against meningococcus, pneumococcus or H. influenzae.
The invention also provides the use of a conjugate of the invention in the manufacture of a medicament for raising an immune response in a patient, wherein said patient has been pre-treated with a different saccharide antigen to that comprised within the composition conjugated to a carrier.
The invention also provides the use of a conjugate in the manufacture of a medicament for raising an immune response in a patient, wherein said patient has been pre-treated with the same saccharide antigen as that comprised within the composition conjugated to a different carrier.
The medicament is preferably an immunogenic composition (e.g. a vaccine). The medicament is preferably for the prevention and/or treatment of a disease caused by a Neisseria (e.g. meningitis, septicaemia, gonorrhoea etc.), by H. influenzae (e.g. otitis media, bronchitis, pneumonia, cellulitis, pericarditis, meningitis etc.) or by pneumococcus (e.g. meningitis, sepsis, pneumonia, etc). The prevention and/or treatment of bacterial meningitis is thus preferred.
Vaccines can be tested in standard animal models (e.g. see ref. 194).
The invention further provides a kit comprising: a) a first conjugate of the invention and b) a second conjugate of the invention.
Adjuvants Conjugates of the invention may be administered in conjunction with other immunoregulatory agents. In particular, compositions will usually include an adjuvant. Adjuvants which may be used in compositions of the invention include, but are not limited to:
Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. Such mineral compositions may include mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 195], 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 [196].
Aluminum salts may be included in compositions of the invention such that the dose of Al3+ is between 0.2 and 1.0 mg per dose.
A typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with PO4/Al molar ratio between 0.84 and 0.92, included at 0.6 mg Al3+/ml. Adsorption with a low dose of aluminium phosphate may be used e.g. between 50 and 100 μg Al3+ per conjugate per dose. Where an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution (e.g. by the use of a phosphate buffer).
Oil emulsion compositions suitable for use as adjuvants with conjugates of 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) [Chapter 10 of ref. 195; see also refs. 197-199]. MF59 is used as the adjuvant in the FLUAD™ influenza virus trivalent subunit vaccine. The MF59 emulsion advantageously includes citrate ions e.g. 10 mM sodium citrate buffer.
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-hydroxyphosphophoryloxy)-ethylamine (MTP-PE). 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 references 197 & 200-201.
An emulsion of squalene, a tocopherol, and Tween 80 can be used. The emulsion may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably ≦1 as this provides a more stable emulsion. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm.
An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100) can be used.
An emulsion of squalane, polysorbate 80 and poloxamer 401 (“Pluronic™ L121”) can be used. The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the “SAF-1” adjuvant [202] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the “AF” adjuvant [203] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants.
Saponin formulations may also be used as adjuvants of conjugates of 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. Saponin can also be commercially obtained from Smilax ornata (sarsaparilla), 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. QS21 is marketed as Stimulon™.
Saponin compositions have been purified using HPLC and 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 ref. 204. Saponin formulations may also comprise a sterol, such as cholesterol [205].
Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs) [chapter 23 of ref. 195]. 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 QuilA, QHA and QHC. ISCOMs are further described in refs. 205-207. Optionally, the ISCOMS may be devoid of additional detergent(s) [208].
A review of the development of saponin based adjuvants can be found in refs. 209 & 210.
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 refs. 211-216. Virosomes are discussed further in, for example, ref. 217
Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
Non-toxic derivatives of LPS 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 ref. 218. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 μm membrane [218]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [219,220].
Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in refs. 221 & 222.
Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and 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. References 223, 224 and 225 disclose possible analog substitutions e.g. replacement of guanosine with 2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in refs. 226-231.
The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [232]. 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 refs. 233-235. 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, refs. 232 & 236-238.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (E. coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 239 and as parenteral adjuvants in ref. 240. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivates thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 241-248. 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 ref. 249, specifically incorporated herein by reference in its entirety.
Compounds of formula I, II or III, or salts thereof, can also be used as adjuvants:
as defined in reference 250, such as ‘ER 803058’, ‘ER 803732’, ‘ER 804053’, ‘ER 804058’, ‘ER 804059’, ‘ER 804442’, ‘ER 804680’, ‘ER 804764’, ER 803022 or ‘ER 804057’ e.g.:
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 [251], IL-23, IL-27 [252] etc.) [253], interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor and macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta [254].
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres [255] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [256].
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).
Examples of liposome formulations suitable for use as adjuvants are described in refs. 257-259.
Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [260]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [261] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [262]. 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.
PCPP (poly[di(carboxylatophenoxy)phosphazene]) formulations are described, for example, in refs. 263 and 264.
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-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention include Imiquamod and its homologues (e.g. “Resiquimod 3M”), described further in refs. 265 and 266.
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 ref. 267. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
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 ref. 268. The tryptanthrin compounds are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
Various nucleoside analogs can be used as adjuvants, such as (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):
and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds disclosed in references 269 to 271; (f) a compound having the formula:
wherein:
Adjuvants containing lipids linked to a phosphate-containing acyclic backbone include the TLR4 antagonist E5564 [272,273]:
SMIPs include:
One adjuvant is an outer membrane protein proteosome preparation prepared from a first Gram-negative bacterium in combination with a liposaccharide preparation derived from a second Grain-negative bacterium, wherein the outer membrane protein proteosome and liposaccharide preparations form a stable non-covalent adjuvant complex. Such complexes include “IVX-908”, a complex comprised of Neisseria meningitidis outer membrane and lipopolysaccharides. They have been used as adjuvants for influenza vaccines [274].
Other substances that act as immunostimulating agents are disclosed in references 195 and 275. Further useful adjuvant substances include:
A formulation of a cationic lipid and a (usually neutral) co-lipid, such as aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine (“Vaxfectin™”) or aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine (“GAP-DLRIE:DOPE”). Formulations containing (+)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium salts are preferred [286].
The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following combinations may be used as adjuvant compositions in the invention: (1) a saponin and an oil-in-water emulsion [287]; (2) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [288]; (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) [289]; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [290]; (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™); (8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dMPL); and (9) one or more mineral salts (such as an aluminum salt)+an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif).
The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The term “about” in relation to a numerical value x means, for example, x±10%. All numerical values herein can be considered to be qualified by “about”, unless the context indicates otherwise.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
E. coli strains carrying the recombinant plasmids pQE-N19 were grown O/N on LB-agar plates, 100 μg/ml ampicillin at 37° C. The grown bacteria were then inoculated in 500 ml LB medium, 100 μg/ml ampicillin and grown 0/N at 37° C. The 500 ml were then diluted in 5 l medium in a fermentator. The growth has been conducted in optimised conditions. When an OD600nm value of 4.2 was obtained, the expression of the polyepitope protein was induced for 3.5 hours by adding 1 mM IPTG (iso-propyl-thio-galactoside) until an OD600nm 7.2. Two samples of the bacterial culture supernatant were collected, at time zero before adding IPTG (t0 OD 4.2) and the end time point of expression (tend OD 7.2). The pellet obtained was resuspended in sample buffer and loaded onto a 12.5% SDS-PAGE in serial dilution corresponding to different bacterial culture ODs. The whole bacterial culture was centrifuged at 5000 g in a JA10 rotor (Beckman, Fullerton, Calif.) for 20 min at 4° C. The cellular pellet obtained of 60 g was suspended in 500 ml lysis buffer (6 M guanidine-HCl, 100 mM NaH2PO4, 2 mM TCEP (Pierce) pH 8, stirred for 1 h at RT and then incubated for 1 h at 37° C. The supernatant containing the dissolved protein was collected by centrifugation at 12000 rpm in a J20 rotor (Beckman) for 20 min at RT and subjected to Immobilized Metal Affinity Chromatography (IMAC). Before adsorbing the sample on the IMAC column, 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, Pierce) had been added, which showed previously to be essential during the purification, to avoid co-purification of contaminating substances bound covalently to N19 by disulphide bonds. The dissolved material was loaded onto a XK50 column containing 360 ml of Nickel activated IDA (iminodiacetic acid) Chelating Sepharose Fast Flow (Pharmacia, Uppsala, Sweden), the column was then washed with 5 volumes of lysis buffer. Then a 300 ml gradient was applied from guanidine-HCl 6 M pH 8 to urea 8 M pH 8 containing 1 mM TCEP. The column was washed with 3 volumes of buffer B (8 M urea, 100 mM NaH2PO4, pH 7) and the proteins were eluted with 1800 ml 0-200 mM imidazole gradient in buffer B. Fractions collected from the column were qualitatively analyzed on 12.5% SDS-PAGE (BioRad) and quantitatively by Bradford protein determination method (BioRad protein assay).
The selected gradient fractions containing the purified recombinant proteins were subjected to Cation Exchange Chromatography (CEC). The 600 ml pooled fractions were loaded on a XK50 column containing 120 ml SP-Sepharose Fast Flow resin (Pharmacia, Uppsala, Sweden). The column was washed with 5 volumes of buffer C (7 M urea, 20 mM NaH2PO4 pH 7, 10 mM β-Mercaptoethanol) and the proteins were eluted with 1300 ml 0-500 mM NaCl gradient in buffer C. The gradient fractions containing the purified recombinant proteins, selected by 12.5% SDS-PAGE analysis, (BioRad) were pooled and dialyzed against 10 mM NaH2PO4, 150 mM NaCl, 10% glycerol. The final protein concentration was determined by the micro BCA method according to the manufacturer's instructions (Pierce). Protein was analyzed on 12.5% SDS-PAGE (BioRad). Optical density of the bands has been measured for integrity evaluation (Image Master 1D Elite v4.00 LabScan Computer Program). The level of endotoxins in the final protein preparation was determined by the kinetic turbidimetric method of the limulus amebocyte lysate (LAL) by Quality Control Department (Chiron Vaccines Siena).
The group A, C, W, Y meningococcal polysaccharides were purified from Neisseria meningitidis strains by the standard procedure described for meningococcal vaccine production (291). Purified capsular polysaccharides were then depolymerised and activated in order to be coupled to the carrier protein as previously described (292, 293). Briefly we describe here the procedure for meningococcal serogroup C oligosaccharide preparation. The purified MenC capsular polysaccharide was submitted to hydrolysis in 10 mM sodium acetate buffer pH 5.0 to reduce the average degree of polymerization (DP). The reaction is conducted at 80° C. for ˜12 h until a DP of 10 was reached. The DP can be followed on-line during the hydrolysis by analysing total sialic acid content in the starting polysaccharide solution (constant during hydrolysis) and formaldehyde released from the terminal group of each chain after oxidation. This real-time DP measurement permitted the extrapolation of the end time of the hydrolysis. Oligosaccharides were sized by Q-Sepharose FF ion-exchange chromatography that retained the higher molecular weight polysaccharides on the column while the low molecular weight oligosaccharides (DP<6) were eluted from the column with 5 mM sodium acetate buffer, 100 mM in NaCl, pH 6.5. The desired oligosaccharide fraction was then eluted with 0.7 M tetrabutylammonium bromide (TAB), a positive counterion, which displaced the negatively charged oligosaccharides from the column. The products were then submitted to concentration/diafiltration against water on a 3K cut-off membrane to remove the excess of TAB and to concentrate the MenC oligosaccharide in preparation. After the diafiltration to retentate was dried by a rotary evaporation step. Thereafter the MenC oligosaccharide was subjected to reductive amination to yield an oligosaccharide with a terminal primary amino group. The reaction mixture was made up to 10% DMSO, 90% methanol, 50 mM ammonium acetate and 10 mM sodium cyanoborohydride and incubated for 24 h in a covered water bath at 50° C. The reaction mixture was then submitted to a rotary evaporation step to reduce the methanol content of the amination reaction mixture to avoid possible interaction with silicon tubing and diafiltration membranes in the following diafiltration step. The aminated oligosaccharides were then purified from reagents (cyanoborohydride, DMSO, methanol) by concentration/diafiltration against 8 volumes of 0.5 M NaCl, followed by 4 volumes of 20 mM NaCl. The purified aminated oligosaccharides were dried under vacuum in preparation for the activation step. The MenC oligosaccharide was solubilized in water followed by the addition to the mixture of DMSO. Triethylamine (TEA) was added to ensure sufficient deprotonation of the oligosaccharide primary amino group and of the di-N-hydroxysuccinimide (bis-NHS) ester of adipic acid. The bis-NHS was added in molar excess to favor the formation of the covalent linkage of a single oligosaccharide polymer to each molecule of bis-NHS ester. The activated oligosaccharide was precipitated by addition of acetone to the reaction mixture, which was also used to separate the oligosaccharides from DMSO, bis-NHS ester and the TEA. The precipitate was dried under vacuum, weight and stored at −20° C. until the use for conjugation.
The procedure for purification of the other PSs was basically the same with minor modifications in the reaction time and temperature [294].
After purification, sizing and activation oligosaccharides were used for the subsequent conjugation to N19 protein [295]. Before starting the conjugation experiment we evaluated preliminarily the potential a specific adsorption of the polysaccharides to the Ni-activated resin. In a typical conjugation experiment, 343.2 nmol of N19 carrier protein was dissolved in Guanidium-HCl pH 8, 100 mM Na2HPO4, and adsorbed to a previously packed 5 ml Ni-activated Sepharose Fast Flow resin (Pharmacia, Uppsala, Sweden) equilibrated in the same buffer. Guanidinium-HCl was removed by washing the resin with 50 ml of 100 mM phosphate buffer pH 7.5 and then 1 ml of 100 mM phosphate buffer pH 7.5 containing 6864 nmol of activated meningococcal oligosaccharide (MenA, MenC, MenW or MenY) was added to the column, recirculating at room temperature for 2 h. The column was washed with 50 ml of 100 mM Na2HPO4 pH 7.5 to remove the excess of unconjugated oligosaccharide. Finally, the conjugate product was eluted with 300 mM imidazole, pH 7, 100 mM NaH2PO4 and analyzed on 7.5% SDS-PAGE. The selected fractions containing the conjugate were pooled and dialyzed against PBS. The glyco-conjugates were analyzed for sugar and protein content. The saccharide content of MenC, MenW and MenY conjugates was quantified by sialic acid determination (143), while that of MenA conjugate by mannosamine-1-phosphate chromatographic determination (121). The protein content was measured by micro BCA assay (Pierce, Rockford, Ill.). The glycosylation degree was calculated from the sugar-to-protein ratio in weight. The CRM-based conjugate vaccines (CRM-MenA, CRM-MenC, CRM-MenW, CRM-MenY) taken as reference in this study were prepared by the Manufacturing Department (Chiron Vaccines Siena).
Unless otherwise specified groups of six female 7-week old mice BALB/c were used. In another experiment, four congenic strains of 7-week old female mice with the following H-2 haplotype were used: BALB/B (H-2b) congenic with BALB/c (H-2d) and B10.BR (H-2k), B10.D2N (H-2q), B10.D1 (H-2d) congenic with C57BL/6 (H-2b). The mice were purchased from Charles River (Calco, Italy) or from Jackson Laboratories (Bar Harbor, Me.).
Mice were immunized subcutaneously on days 0, 21 and 35 with N19 or CRM conjugates with different 0.5 ml formulations of monovalent, bivalent, tetravalent or bi-carrier conjugate vaccine based on saccharide content diluted in NaCl 0.9% buffer as specified below. Individual serum samples were taken at days −1 (pre), 20 (post-1), 34 (post-2) and 45 (post-3) and frozen at −20° C. until use. Spleens were collected from mice immunized with N19-conjugates for assessing T-cell proliferation as described in cell-mediated immune response section.
Mice were immunized with decreasing amounts of N19-MenC or CRM-MenC (from 2.5 to 0.039 μg of MenC/dose) in the presence of 0.5 mg aluminium hydroxide as adjuvant. Antibody titres were measured as detailed below.
The conjugate containing N19 was more immunogenic than the one with CRM (
Two and three immunizations with CRM-based conjugates induced strong anti-carrier antibody responses against CRM even at the lowest doses tested (i.e. 0.3 μg and lower). On the contrary, the N19-specific antibody response was always negligible and was detectable (even though at very low titers) only at the highest dose (i.e. 6 μg) (
Since protective immunity against MenC relies mainly on bactericidal antibodies that kill the bacteria in the presence of complement, the functional activity of the antibodies induced was measured. In agreement with the results obtained in ELISA,
Mice were immunized N19-MenA and N19-MenC separately and combined or CRM-MenA and CRM-MenC separately and combined (0.625, 0.156 or 0.039 μg of each MenPS/dose) in the presence of 0.06 mg aluminum phosphate as adjuvant. Antibody titres were measured by ELISA as described below.
As shown in upper panel in
When the anti-MenC antibody response was measured (lower panel in
Tetravalent formulations were prepared mixing together in equivalent saccharide amount N19-MenA, N19-MenC, N19-MenW and N19-MenY (N19-MenACWY). As reference we used clinical grade lots of CRM conjugate vaccine (Chiron Vaccines, Siena) formulated before use by mixing liquid CRM-MenCWY to lyophilised CRM-MenA. Mice received decreasing amounts of tetravalent formulations (from 2 μg to 0.074 μg of each MenPS/dose) in the presence of 0.06 mg aluminum phosphate as adjuvant.
N19-MenACWY was highly effective in inducing bactericidal antibodies against all four Men polysaccharides. In particular, bactericidal titers against group C were significantly higher at all given dosages after two doses of N19 conjugates than of CRM conjugates. Performing a dose escalation, the potency of N19 carrier was highlighted, since limiting the dose, N19 conjugates induced higher bactericidal antibody titers against all four polysaccharides than those induced by CRM conjugates. Bactericidal titers against MenC and MenW on single sera from mice immunized with the lowest dose (0.074 m) were analysed in particular.
A detailed analysis of functional activity of group A and C antibodies was conducted using a modified antigen-binding assay that measures only high affinity antibodies [296]. Results show in
To evaluate the influence of the carrier protein shared by four polysaccharides to induce antibodies against itself, we measured antibodies against both carrier proteins employed (
Tetravalent formulations were prepared mixing together MenA conjugated either to N19 or CRM with MenCWY conjugated either to CRM or N19 (N19-MenA+CRM-MenCWY and vice versa CRM-MenA+N19-MenCWY). Control groups received tetravalent formulations containing one carrier (N19-MenACWY or CRM-MenACWY). Mice received decreasing amounts of tetravalent formulations (from 0.67 μg to 0.074 μg of each Men polysaccharides/dose) in the presence of 0.06 mg aluminium phosphate as adjuvant. Antibody titres were determined using the methods described below.
N19-MenACWY produced, after the first dose, anti-MenA titers comparable to those obtained after two doses of CRM based vaccine (
3.5 Mouse Strains with Different Genetic Background.
In a preliminary experiment, two groups of mice BALB/c and C57BL/6 were immunized twice with 0.67 or 0.22 μg N19-MenACWY or CRM-MenACWY with of 0.06 mg aluminium phosphate. In another experiment, congenic strains of mice were immunized three times with tetravalent formulations N19-MenACWY or CRM-MenACWY (0.67 μg of each Men polysaccharide/dose) in the presence of 0.06 mg phosphate prepared as described above. BALB/c mice were used as control.
Based on the above results obtained in BALB/c mice, we decided to immunize mice only twice with two different dosages of tetravalent formulations containing N19 or CRM and the antibody responses against the four polysaccharides were measured (
As shown in
As shown in
Titration of MenA, MenC, MenW and MenY specific immunoglobulins G (IgG) was performed on individual sera from each mouse according to the assays already described [297]. Nunc Maxisorp 96-well flat-bottom plates were coated overnight at 4° C. separately with 5 μg/ml of purified N. meningitidis serogroup A, C, W or Y polysaccharides in the presence of 5 μg/ml methylated human serum albumin. The plates were washed three times with PBS containing 0.33% Brij-35 (PBS-Brij), then saturated with 200 μl/well of PBS containing 5% FCS and 0.33% Brij-35 (PBS-FCS-Brij) for 1 h at RT. Single sera were diluted in PBS-FCS-Brij and titrated against the four polysaccharides separately. Plates were incubated overnight at 4° C. On the following day, plates were washed with PBS-Brij, alkaline phosphatase conjugated goat anti-mouse IgG (Sigma Chemical Co., SA Louis, Mo.) diluted in PBS-FCS-Brij was added and plates were incubated 2 hours at 37° C. Bound antibodies were revealed using 1 mg/ml p-nitrophenyl-phosphate (Sigma Chemical Co., SA Louis, Mo.) in diethanolamine solution. After 20 min incubation, the absorbance was read out at 405 nm. Pre-immunization values gave consistently an OD value below 0.1. The results were expressed as titers relative to an in-house reference serum by parallel line analysis, to minimize plate-to plate variation. IgG titers were calculated by using Reference Line Assay [298] and expressed as the logarithm of EU/ml.
To measure anti-MenA and anti-MenC specific IgG1 and IgG2a antibodies, plates were coated overnight at 4° C. with 5 μg of methylated human serum albumin/ml and 5 μg of purified MenA or MenC per ml in PBS as described above for IgG ELISA. The plates were then washed and blocked with PBS-CS-Brij for 1 h at RT. Serum samples were diluted in PBS-FCS-Brij across two plates in parallel starting from 1:100 and incubated for 2 h at 37° C. Biotin-conjugated goat anti-mouse IgG1 or IgG2a antibodies (Southern Biotechnology Associates, Inc.) were added. After 2 h incubation at 37° C. horseradishperoxidase-conjugated streptavidin (DAKO) was added to the wells, and the plates were incubated for 1 h at 37° C. The plates were developed with the substrate 0-phenylenediamine dihydrochloride (Sigma). Titers were calculated as the reciprocal of the serum dilution at which the OD 0.5 (450 nm).
Titration of N19, CRM197 carrier proteins and its parent proteins, therein tetanus toxoid (TT), haemophilus influenzae (HA) and diphtheria toxoid (DT) was performed on pooled sera as described previously [299, 300]. Briefly, 96-well plates (Nunc Maxisorp) were coated overnight at 4° C. with 200 μl of a PBS solution containing separately 2 μg/ml of N19, TT, HA or CRM197 or 5 μg/ml of DT antigen. The plates were then washed and blocked with PBS-BSA 1% for 1 h at 37° C. Serum samples were diluted in PBS-BSA 1%-Tween20 0.05% across the plate starting from 1:100 and incubated for 2 h at 37° C. Alkaline phosphatase conjugated goat anti-mouse IgG and p-nitrophenyl-phosphate were used for detection. The presence of antigen-specific antibodies was revealed as described above. The results were expressed as titers relative to an in-house reference serum by parallel line analysis, to minimize plate-to plate variation.
The avidity of meningococcal group A and C specific IgG antibodies was assessed by ELISA elution assay of pooled sera using 75 mM of ammonium thiocyanate [NH4SCN] as chaotropic agent, according to the well-established method [301, 302]. Assay validation included the assessment of antigen stability following incubation with 4 M NH4SCN [303]. Nunc Maxisorp 96-well flat-bottom plates were coated overnight at 4° C. with 5 μg/ml of purified N. meningitidis serogroup A and C polysaccharides separately. The solution was aspirated and the wells were washed three times with PBS-Brij and blocked for 1 h at room temperature with blocking buffer (PBS-FCS-Brij). The plates were washed with wash buffer (PBS-Brij). Test and reference sera were diluted in dilution buffer PBS-FCS-Brij and duplicate twofold serial dilutions in one microplate were prepared. After 2 h incubation at 37° C., the plates were washed three times. Serum samples in one of the duplicate were incubated 15 minutes at room temperature with 75 mM NH4SCN in serum dilution buffer PBS-FCS-Brij, whereas the other duplicate was incubated with diluting buffer alone. After washing, the plates were incubated with alkaline phosphatase conjugated goat anti-mouse IgG antibodies (Sigma Chemical Co., SA Louis, Mo.) as in the above-mentioned ELISA assay. The amount of antibodies remaining bound to the plate after elution with 75 mM NH4SCN was calculated in ELISA units by reference to standard ELISA curves, corresponding to 100% bound antibodies. High-avidity IgG titers were represented in % of bound antibodies in function of the time.
The method used for measurement of bactericidal antibody titers has been previously described (94). N. meningitidis serogroup A (strain F8238), C (strain 11), W (strain 240070) or Y (strain 240539) target strains were grown overnight at 37° C. with 5% CO2 on chocolate agar plates (starting from a frozen stock). Colonies with an absorbance of 0.05-0.1 at 600 nm were suspended in 7 ml Mueller Hinton broth containing 0.25% glucose and incubated shaking for 1.5 hours at 37° C. with 5% CO2 to reach an absorbance of ˜0.24-0.4 at 600 nm. The bacterial cell suspensions were diluted in GBSS buffer (Gey's balanced salt solution) (SIGMA) and 1% BSA (assay buffer) to yield approximately 105 CPU/ml. Heat-inactivated (56° C. for 30 min) single or pooled serum samples (50 μl) were diluted serially diluted twofold (reciprocal starting dilution of 4) in buffer in 96-well flat-bottom tissue culture-treated plates (Costar, Inc., Cambridge, Mass.). Equal volumes of cell suspensions and pooled baby rabbit complement (25%) were gently mixed, and 25 μl was added to serially diluted sera. The final volume in each well was 50 μl. Controls included (i) bacteria-complement-buffer (complement-dependent control) and (ii) heat-inactivated test serum-bacteria-buffer (complement-independent control). Immediately after the addition of the baby rabbit complement, 10 μl of the controls were plated on Mueller-Hinton agar plates by the tilt method (time zero, t0). The microtiter plates were incubated for all serogroup target strains at 37° C. for 1 h with 5% CO2. After incubation, 10 μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10 μl of the controls were plated by the tilt method (time one, t1). Agar plates were incubated for 18 h at 37° C. with 5% CO2, and the colonies corresponding to t0 and t1 were counted. Colonies at t1 were a control of eventual toxicity of the complement or the serum and has to be 1.5 times colonies at t0. The bactericidal titers were expressed as the reciprocal serum dilution yielding ≧50% killing compared to the number of target cells present before incubation with serum and complement (t0). Titers were considered reliable if at least two following dilutions yield ≧90% bacterial killing.
Student's t test (2 tails) was used to compare antibody titers between groups and at different times. A P value of <0.05 was considered as statistically significant.
6.1 In Vitro Proliferation Assay with N19-Epitopes, N19 or N19-Conjugates of BALBc Mice Primed with N19-MenACWY.
To assess whether the immunization with N19-conjugates primed for carrier-epitope specific T cells, spleens from mice immunized two or three times with tetravalent N19-MenACWY (˜6 or 2 μg of protein/dose) as described above were removed 10 days after the last immunization and tested for their capacity to proliferate following in vitro stimulation with single peptides constituting N19 or N19 free or conjugated [304]. The purified N19 employed in this assay did not contain detectable LPS, which could have possibly interfered. Spleens of each mouse group were pooled and dispersed manually. Once washed and counted, cells were cultured at a density of 5×105 cells per well in RPMI (GIBCO BRL Life Technologies) supplemented with 25 mM HEPES buffer, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, 0.15 mM L-glutamine, sodium pyruvate, vitamins, sodium pyruvate and a cocktail of non-essential amino acids (GIBCO BRL Life Technologies 1% of a 100×stock) and 5% fetal calf serum (Hyclone) in flat-bottom 96-well cell culture plates (Corning N.Y.). The cells were cultured in triplicate in the presence of the individual peptides from 0.12 to 30 μM per well (two or three-fold dilutions) (˜0.15-50 μg/ml) or of free or conjugated N19 from 0.004 to 1 μM diluted in the same medium were added to triplicate wells to give a total of 200 μl per well. Controls were run with complete culture medium or 10 μg/ml Concanvalin A, to demonstrate the proliferative capacity of the cells. Plates were incubated at 37° C. in 5% CO2. After five days, cells were pulsed with 0.5 μCi of [3H] thymidine (Amersham Biosciences 1 mCi/ml stock) per well for additional 18 h and harvested with Filtermate Harvester and counted in a liquid scintillation counter (Packard Bioscience). Results of proliferative assays were expressed as stimulation index (SI), calculated by the ratio of counts per minute (cpm) in experimental cultures with the stimulus to counts per minute of control cultures (background) without stimulus. Triplicates of cultures were run in parallel. An SI>2 was considered positive.
To determine whether the strong T helper effect of N19 in the mouse system was mediated by any of the CD4+ epitopes originally included in N19, T-cell proliferation of splenocytes from BALB/c mice primed two or three times with N19-MenACWY (6 μg of N19/dose) was assessed. Spleen cells were stimulated in vitro with different concentrations of N19 peptides or with whole N19, either free or conjugated to the polysaccharides. As shown in
Furthermore we measured N19-specific T cell activation in congenic strains of mice to investigate if there was any MHC-restriction pattern. The activation was analyzed in vitro by measuring proliferative responses of spleen cells of mice with different genetic background in the presence of different concentrations of N19, either 1) free or 2) conjugated to the polysaccharides, or with 3) single N19 constituting peptides or with 4) free polysaccharide components. We observed that free N19 induced T cell activation in all strains, but N19 conjugates resulted in differential proliferative responses in the tested strains (
Synthetic peptides (P2TT, P21TT, P23TT, P30TT, P32TT, HA and HBsAg) with 95% purity were purchased from Primm s.r.l. (Italy). Groups of three BALB/c mice were immunized subcutaneously at the base of the tail with 50 μl volume per mouse containing 50 μg of a single peptide (P2TT, P30TT, P23TT, P32TT, HA, HBsAg) or N19 emulsified in complete Freund's adjuvant (CFA). Seven days later, mice were killed, inguinal and periaortic lymph nodes were removed and pooled form mice within each group, and a single-cell suspension was prepared. The cells were cultured at a density of 3×105 cells per well in complete medium (supplemented RPMI as described above for spleen cells) in flat-bottom 96-well cell culture plates (Costar Corp., Cambridge, Mass.). N19 or homologous peptide diluted in the same medium were added to triplicate wells of single mouse or pooled cultured cells at three different concentrations (15, 7.5 and 3.75 mM of all the peptides and 10, 1 and 0.1 μg/ml of N19). After five days incubation at 37° C. at 5% CO2, cells were pulsed with 0.5 μCi [3H] thymidine for 16 h and then harvested as described above. A non-related peptide CH60 (in silico predicted to bind HLA-A2) derived from surface protein Chlamydia pneumoniae as employed as negative control in these experiments.
It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0428394.1 | Dec 2004 | GB | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11793996 | Feb 2008 | US |
| Child | 14451907 | US |
