Effective vaccines, particularly vaccines against bacterial capsular polysaccharide (CPS) antigens, appear to require three components: (i) a CPS B-cell epitope to induce the production of antibody (Ab) that mediates opsonin-dependent phagocytosis and bacterial clearance; (ii) a T-cell epitope to induce an IgG isotype switch and immune memory; and (iii) an adjuvant to induce innate-cell receptor activation of a pro-inflammatory cytokine environment. Classical adjuvants contain bacterial or other ligands that stimulate Toll-like receptor (TLR) signaling of innate immune cells. Stimulation of TLR signaling enhances the production of pro-inflammatory cytokines and the up-regulation of MHC and co-stimulatory molecules. Thus, TLR stimulation plays a key role in the interface between innate and adaptive immunity. Indeed, TLR molecules are considered to be “adjuvant receptors”, and the ligands that activate them are “adjuvants”. Vaccines that incorporate ligands for TLR stimulation were shown to boost vaccine responses.
Heat Shock Protein 60 (HSP60)
HSP60 belongs to a family of chaperone molecules highly conserved throughout evolution; a similar HSP60 molecule is present in all cells, prokaryotes and eukaryotes. The human HSP60 molecule was formerly designated HSP65, but is now designated HSP60 in view of more accurate molecular weight information; by either designation, the protein is the same. Mammalian HSP60 is highly homologous to the bacterial cognates, showing about 50% amino acid identity. Thus, HSP60 is shared by the host and its parasites, and is immunogenic, cross-reactive, and universally expressed in inflammation. Furthermore, HSP60 is well recognized by the immune system and is a part of the set of self-molecules for which autoimmunity naturally exists; HSP60 is member of the immunologic homunculus. Heat shock, IFNγ, bacterial or viral infection, and inflammation, all result in the presentation of endogenous HSP60 epitopes on MHC class II molecules leading to the activation of HSP60-specific T cells, even in healthy individuals.
U.S. Pat. No. 7,157,089 discloses fusion proteins comprising influenza virus antigens and stress proteins useful for vaccination against influenza infection. U.S. Pat. No. 7,157,089 does not disclose conjugates comprising any specific peptide carrier derived from heat shock proteins such as HSP60.
WO 94/29459 to Young is directed to a fusion protein wherein a stress protein is fused to an antigen protein, particularly a fusion protein that contains a HSP70 and an HIV antigen.
WO 95/24923 to Srivastava et al. discloses complexes of stress proteins noncovalently associated with peptides. WO 95/24923 does not teach or suggest complexes comprising multimeric HSP60-derived peptide carriers.
WO 97/06821 to Rothman et al. relates to methods and compositions for inducing an immune response in a subject, wherein at least one heat shock protein in combination with one or more defined target antigens is administered to the subject. Particularly, WO 97/06821 discloses complexes of HSP70 and peptide antigens.
European Patent EP 262 710 and U.S. Pat. No. 5,154,923 to Van Eden et al. describe peptides having an amino acid sequence corresponding to positions 171-240 and 172-192, respectively, of a Mycobacterium boris BCG 64 kD polypeptide, that are useful as immunogens inducing resistance to autoimmune arthritis and similar autoimmune diseases.
PCT Pub. No. WO 90/10449 to some of the inventors of the present invention describes a peptide designated p277 having an amino acid sequence corresponding to positions 437-460 of the human HSP65 molecule that is useful as immunogen inducing resistance to insulin dependent diabetes mellitus (IDDM). A control peptide, designated p278, corresponding to positions 458-474 of human HSP65, did not induce resistance to IDDM.
Lussow et al. (1990) showed that the priming of mice with live Mycobacterium tuberculosis var. bovis (BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies. Later on, Lussow et al. (1991) showed that mycobacterial HSPs of 65 kDa (GroEL-type) and 70 kDa (DnaK-type) acted as carrier molecules in mice, previously primed with BCG, for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)40.
Barrios et al. (1992) have shown that mice immunized with peptides or oligosaccharides conjugated to the Mycobacterial 70 kDa HSP produced high titers of IgG antibodies in the absence of any previous priming with BCG. This adjuvant-free carrier effect of the 70 kDa HSP was T cell dependent, since no anti-peptide or anti-70 kDa IgG antibodies were induced in athymic nu/nu mice. In addition, preimmunization with the 65 kDa HSP could substitute for BCG in providing effective priming for the induction of anti-(NANP)40 antibodies. Finally, both the 65 kDa and 70 kDa Mycobacterial HSP acted as carrier molecules for the induction of IgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants.
Peptide p458 (corresponding to positions 458-474 and 437-453 of mammalian HSP60) was previously used to develop conjugate vaccines to Streptococcus pneumoniae, Salmonella typhi (then named CP1) and to a murine cytomegalovirus CTL-epitope (Amir-Kroll et al., 2003; Konen-Waisman et al., 1999; Konen-Waisman et al., 1995; Rouvio et al., 2005).
U.S. Pat. No. 5,736,146 to some of the inventors of the present invention discloses conjugates of poorly immunogenic antigens with a synthetic peptide carrier comprising a T cell epitope derived from the sequence of human heat shock protein HSP65, or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the antigen. The '146 patent discloses conjugates of a peptide corresponding to positions 458-474 and 437-453 of human or mouse HSP60 and homologs thereof with a wide variety of antigens including peptides, proteins and polysaccharides such as bacterial polysaccharide (e.g. capsular polysaccharide (CPS) Vi of Salmonella typhi), and antigens derived from HIV virus or from malaria antigen.
U.S. Pat. No. 5,869,058 to some of the inventors of the present invention discloses conjugates of poorly immunogenic antigens, e.g., peptides, proteins and polysaccharides, with a synthetic peptide carrier comprising a T cell epitope derived from the sequence of E. coli HSP65 (GroEL), or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the antigen. A suitable peptide according to the invention is Pep278e, which corresponds to positions 437-453 of the E. coli HSP65 molecule.
Neisseria meningitidis (N. meningitidis, meningococcus, Mn) and Streptococcus pneumoniae (S. pneumoniae, pneumococcus, Pn) are major causes of bacterial meningitis and pneumonia throughout the world, particularly in children. Thus, developing vaccines conferring immunity against these pathogens has been the focus of extensive research efforts.
Meningococcal Vaccines
The Meningococcal type B capsular polysaccharide (MnB), like other CPS, is a T-independent (TI) antigen. TI antigens are not recognized by helper T cells and so activate only B cells leading predominantly to a short-lived IgM response and to almost no immunological memory. Protein carriers have been conjugated to TI antigens to recruit T-cell help to induce B-cells to switch to IgG secretion and to generate immunological memory (Goldblatt et al., 2000). Indeed, an immunogenic carrier can induce high titers of IgG antibodies (Abs) to a conjugated capsular polysaccharide (CPS), for example, that of the Meningococcus type C (MnC) (Lindberg et al., 1999). New conjugated vaccines to MnC using Tetanus toxoid (TT) and Diphtheria toxoid as carriers are now available, and have been effective in inhibiting Meningococcus type C meningitis in populations at risk (Miller et al., 2001; Rappuoli, 2001; Trotter et al., 2004; Richmond et al., 2001).
Both MnB and MnC are homopolymers of sialic acid with a difference in their glycosidic linkage: α(2-8) sialic acid in MnB compared to α(2-9) sialic acid in MnC. This seemingly minor difference leads to a major difference in the immunogenicity of these antigens; MnB is a very poor immunogen while MnC is an immunogenic TI antigen. MnB has structural homology with a self-antigen, the polysialylated adhesion molecule NCAM (PSA-NCAM) that is expressed in mammalian fetal tissues (Devi et al., 1997). In addition, cross-reactivity between anti-MnB Abs and brain extracts has been reported (Finne et al., 1983). Apparently, the structural homology of MnB with mammalian tissues leads to its very poor immunogenicity (Lifely et al., 1987). Thus, there is currently no effective vaccine against Meningococcus type B based on the CPS. Vaccines to Meningococcus type B, based on the outer membrane vesicle protein have been used (Morley et al., 2001), and others are undergoing clinical trials (Robertson et al., 2001; Perkins et al., 1998; Haneberg et al., 1998). Nevertheless, a conjugated MnB vaccine would be useful for the induction of protection against all the bacterial serotypes that share this CPS.
Conjugated vaccines composed of MnB attached to TT or CRM197 have had limited success in inducing IgG Abs to MnB (Devi et al., 1997; Bartoloni et al., 1995). Abs were mostly raised after the third injection, but those Abs were short-lived (Devi et al., 1997). Another approach, in which a chemically modified N-propionylated polysialic acid was conjugated to Meningococcal B Porin, was able to elicit bactericidal Abs to the CPS moiety in mice and monkeys (Fusco et al., 1997). Native MnB was not effective in inhibiting the bactericidal effect.
Thirteen different serogroups of meningococci have been identified on the basis of the immunological specificity of their capsular polysaccharide. Of these thirteen serogroups, five cause the majority of meningococcal disease; these include serogroups A, B, C, W135, and Y.
Vaccines suitable for immunization against groups A, C, Y and W-135 are commercially available. For example, Menomune® A/C/Y/W-135, a freeze-dried preparation of the group-specific polysaccharide antigens from N. meningitidis group A, group C, group Y and group W-135, and Menactra®, a meningococcal polysaccharide (groups A, C, Y and W-135)-diphtheria toxoid conjugate vaccine, have been approved for sale in the United States.
Disclosures directed to specific vaccine formulations comprising epitopes derived from CPS of meningococcal strains other than MnB were described, for example, in U.S. Pat. No. 5,425,946, WO 2005/000345, WO 2005/004909, WO 02/058737, and WO 2004/103400.
While many disclosures are directed to group B N. meningitidis vaccines, no such vaccine is currently available commercially. For example, U.S. Pat. No. 5,597,572 discloses a vaccine for immunizing against infection caused by Group B N. meningitidis microorganism, which comprises a purified protein antigenic complex weighing from 65 to 95 kDa, vesicles, and capsular polysaccharide. This vaccine is extracted from the cell membranes of the live microorganisms using detergent and enzyme.
U.S. Pat. No. 6,080,589 discloses capsular polysaccharides containing multiple sialic acid residues, particularly the Group B polysaccharide of N. meningitidis, modified by chemical reaction to randomly introduce pendant reactive residues of heterobifunctional linker molecules to the polysaccharide backbone. The introduction of the linker molecules to the polysaccharide chain between the termini enables the polysaccharide to be linked to a carrier molecule, such as a protein, to enhance the immunogenicity of the polysaccharide.
U.S. Pat. No. 6,350,449 relates to chemically modified group B polysaccharides of N. meningitidis, vaccines in which the respective modified polysaccharides are conjugated to a protein carrier, as well as antibodies to these conjugate vaccines. More specifically, the '449 patent provides group B meningococcal unsaturated N-acyl derivative polysaccharides, conjugates thereof, pharmaceutical compositions comprising conjugate molecules of group B meningococcal unsaturated N-acyl derivative polysaccharide fragments covalently bound to proteins, and the use of these compositions as vaccines.
U.S. Pat. No. 6,642,354 is directed to bactericidal antibodies against N. menigitidis serogroup B, which either do not cross-react or minimally cross-react with host tissue polysialic acid, useful for identifying molecular mimetics of unique epitopes found on N. meningitidis serogroup B or E. coli K1. The '354 patent discloses examples of such peptide mimetics that may elicit serum antibody capable of activating complement-mediated bacteriolysis of the pathogen.
U.S. Pat. No. 6,656,472 discloses a method of forming a multivalent immunogenic molecule tag comprising: treating at least two different carbohydrate molecules to obtain carbohydrate fragments thereof, forming a lysine-branching peptide containing at least two different T-helper cell epitopes as a carrier molecule anchored to a polymeric anchor wherein at least two carrier peptide segments have different terminal protecting groups, selectively removing one of the protecting groups, coupling a first one of the oligosaccharide fragments to the unprotected carrier peptide segment, selectively removing another of the protecting groups, coupling a second one of the oligosaccharide fragments to the unprotected carrier peptide segment, and cleaving the resulting molecule from the polymeric anchor. The oligosaccharide fragments may be derived, inter alia, from N. meningitidis, and are sized from about 2 to about 5 kDa.
U.S. Pat. No. 6,936,261 is directed to methods and vaccines for the prevention of diseases caused by N. meningitidis bacteria, particularly serogroup B strains, comprising the steps of: administering to a mammal a first preparation of microvesicles (MVs) from a first N. meningitidis species that is a member of a first serotype and of a first serosubtype, in an amount sufficient to elicit an immune response to epitopes present in said first preparation; and administering to said mammal a second preparation of MVs from a second N. meningitidis species that is a member of a second serotype and of a second serosubtype, in an amount sufficient to elicit an immune response to epitopes present in said second preparation; wherein the serotype or serosubtype of each of the first, second, and third N. meningitidis species is different, and wherein administering of the first, second, and third preparations is sufficient to elicit an immune response in said mammal, wherein said immune response confers protective immunity against a disease caused by more than one strain of N. meningitidis species.
Polysaccharide Vaccines for Cancer
PSA-NCAM is expressed in different tumors (Qu et al., 1996; Fukuda, 1996; Komminoth et al., 1994), and was suggested to be involved as an adhesion molecule in tumor metastasis (Friedl et al., 2004). Several patent applications are directed to utilizing PSA for cancer therapy.
For example, WO 01/47552 relates to an immunogenic composition comprising an α-(2-8)-polysialic acid-carrier conjugate and a saponin, wherein the α-(2-8)-polysialic acid-carrier conjugate comprises one or more α-(2-8)-polysialic acid polymers covalently linked to an immunogenic carrier, and wherein the median number of sialic acid units in each of the polymers is at least about 10. Preferred immunogenic carriers include keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
WO 01/09298 is directed to modifying the sialic acid component of a sialic acid unit-containing cell surface marker characteristic of cancerous mammalian cells, such as alpha 2-8 polysialic acid, so that cells normally expressing such a marker express instead a modified sialic acid unit-containing cell surface marker, which is strongly immunogenic. The modification is suitably N-acylation of a precursor of the sialic acid, so that the N-acylated precursor becomes chemically incorporated in the polysialic acid during its intracellular biochemical synthesis. The '298 application is further directed to vaccines comprising said conjugates or antibodies raised against said conjugates, useful for managing cancer conditions which involve cancer cells characterized by expression of modified sialic acid unit containing marker.
Krug et al. (2004) discloses a clinical study aimed to elicit an immune response against polysialic acid to target clinically inapparent residual disease in patients with SCLC who had successfully completed initial therapy. Krug et al. reports that vaccination with N-propionylated-polysialic acid-KLH, but not unmodified polysialic acid-KLH, resulted in a consistent high titer antibody response.
Pneumococcal Vaccines
Vaccines against encapsulated bacteria such as S. pneumoniae have included the CPS coat, since the CPS acts as the major Pn virulence factor (AlonsoDeVelasco et al., 1995). However, the Pn CPS is, like other polysaccharides, poorly immunogenic, especially in young children, the elderly and immunosuppressed adults. Conjugate vaccines have been designed using various protein carriers like TT as a source for T-cell epitopes conjugated to the CPS with the aim of supplying the help needed for IgG switching and immune memory (Goldblatt et al., 2000; Lesinski et al., 2001). However, the conjugation of a CPS to a protein carrier is usually insufficient to evoke a strong immune response, and an added adjuvant is often needed.
U.S. Pat. No. 6,855,321 is directed to polyepitope carrier proteins that comprise at least five CD4+ T cell epitopes, for conjugation to capsular polysaccharides, including, inter alia, pneumococcal CPS.
U.S. Pat. No. 5,773,007 is directed to vaccine compositions comprising a long chain alkyl compound as an immunoadjuvant in combination with a bacterial polysaccharide protein conjugate.
U.S. Pat. No. 5,565,204 is directed to an immunogenic polysaccharide-protein conjugate obtained by reductive amination comprising (a) an oxidized polysaccharide derived from the capsular polysaccharide of S. pneumoniae, and (b) the pneumolysin protein of S. pneumoniae which is expressed recombinantly, where said pneumolysin is not toxoided or is not produced by site-specific mutagenesis prior to conjugation with said oxidized polysaccharide.
U.S. Pat. No. 7,018,637 discloses multivalent immunogenic molecules comprising a carrier molecule containing at least one functional T-cell epitope and multiple different carbohydrate fragments each linked to the carrier molecule and each containing at least one functional B-cell epitope. The carbohydrate fragments may be derived from pneumococcal or meningococcal CPS, among others.
Commercially available vaccines include e.g. Prevnar® and Pneumovax®. Prevnar® is a sterile solution of saccharides of the capsular antigens of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F individually conjugated to diphtheria CRM 197 protein. Pneumovax® 23 (Pneumococcal Vaccine Polyvalent) is a sterile, liquid vaccine for intramuscular or subcutaneous injection. It consists of a mixture of highly purified capsular polysaccharides from the 23 most prevalent or invasive pneumococcal types of S. pneumoniae, including the six serotypes that most frequently cause invasive drug-resistant pneumococcal infections among children and adults in the United States.
There remains an unmet medical need for providing additional effective vaccines conferring immunity against disease and infection. Improved vaccine compositions comprising conjugates of peptide carriers and antigen determinants, said conjugates having enhanced immunogenicity, would thus be highly beneficial.