Patent Application
20060276400
This invention relates to hybrid vaccine peptides comprising a helper T cell epitope portion and a B cell epitope-portion derived from the N-terminal region of cholesteryl ester transfer protein (CETP), which vaccine peptides are useful for eliciting an autoimmune response in an individual administered such a peptide against endogenous CETP activity. Control of CETP activity is beneficial in preventing or treating cardiovascular disease, such as atherosclerosis.
A promising new field of research has opened in the area of treatment and prevention of cardiovascular disease and in particular the prevention and treatment of atherosclerosis by directly controlling the activity of cholesteryl ester transfer protein (CETP). Human CETP is a hydrophobic glycoprotein having 476 amino acids and a molecular weight of approximately 66,000 to 74,000 daltons. (Hesler et al., J. Biol. Chem., 262: 2275-2282 (1987)). CETP mediates the transfer of cholesteryl esters from high density lipoproteins (HDL) to triglyceride (TG)-rich lipoproteins such as low density lipoproteins (LDL) and very low density lipoproteins (VLDL), and also the reciprocal exchange of TG from VLDL to HDL (Hesler et al., id.). The region of CETP defined by the carboxyl-terminal 26 amino acids, and in particular amino acids 470-475, has been shown to be especially important for neutral lipid binding involved in neutral lipid transfer. (Hesler et al, J. Biol. Chem., 263: 5020-5023 (1988)). CETP may play a role in modulating the levels of cholesteryl esters and TG associated with various classes of lipoproteins. A high CETP cholesteryl ester transfer activity has been correlated with increased levels of LDL-associated cholesterol and VLDL-associated cholesterol, which in turn are correlated with increased risk of cardiovascular disease (see, e.g., Tato et al., Arterioscler. Thromb. Vascular Biol., 15: 112-120(1995)).
Decreased susceptibility to cardiovascular disease, such as atherosclerosis, is generally correlated with increased absolute levels of circulating HDL-cholesterol (or HDLc, so-called “good cholesterol”) and also increased levels of HDLc relative to circulating levels of LDL-cholesterol (LDLc, so-called “bad cholesterol”). See, e.g., Castelli et al., J. Am. Med. Assoc., 256: 2835-2838 (1986).
Therefore, inhibition of endogenous CETP activity is an attractive therapeutic method for modulating the relative levels of lipoproteins, which, in turn, is effective for preventing the progression of or inducing regression of cardiovascular diseases, such as atherosclerosis, by increasing the ratio of circulating HDLc:LDLc in the bloodstream.
U.S. Pat. No. 6,410,022 and U.S. Pat. No. 6,284,533 describe antigenic vaccine peptides and plasmid-based vaccines, respectively, for use in the modulation or inhibition of CETP activity for the treatment or prevention of atherosclerosis. The disclosed vaccine peptides are comprised of a universal helper T cell epitope peptide linked to a B cell epitope-containing peptide from CETP. When administered to a mammal, the vaccine peptides cause an antibody response that provides native antibodies recognizing the mammal's own endogenous CETP, in turn causing a decrease in CETP activity. Data presented in these patents shows that use of a vaccine peptide including a B cell epitope from the C-terminal portion of CETP (i.e., amino acids 461-476 of human CETP) led to an autoimmune response producing anti-native CETP antibodies, a rise in HDL to LDL/VLDL ratio, a lowered level of circulating cholesterol, and a significant reduction in the development of atherosclerotic lesions in the arteries of test animals vaccinated with the vaccine peptide.
The foregoing developments are very promising for the development of an alternative approach to statin drugs for controlling cholesterol metabolism and therapeutically addressing cardiovascular disease. Other B cell epitopes upstream of the C-terminal sixteen amino acids of human CETP have been indicated in various studies (see, e.g., Swenson et al., J. Biol. Chem., 264(24): 14318-14326 (1989)), however no data have appeared showing another hybrid CETP B cell epitope/universal helper T cell epitope vaccine peptide that is effective for eliciting an immune response in a vaccinated individual that leads to the production of antibodies capable of neutralizing the lipid transfer activity of the individual's native, endogenous CETP. Therefore, the need still exists for the development of improved vaccine peptides or vaccine peptides that might be used as alternative or supplemental vaccines for the treatment of non-responders or low responders to previously reported vaccine peptides targeting CETP activity.
As disclosed in the present application, it has now been surprisingly discovered that a vaccine peptide comprising a B cell epitope from the N-terminal portion of the CETP molecule, when linked to a universal helper T cell epitope, provides an autoantigenic vaccine peptide that causes an anti-endogenous CETP response, leading to reduction in CETP activity in a mammalian subject receiving the vaccine peptide. The vaccine peptides of the present invention, when administered to a mammal, are effective to decrease the activity levels of CETP in the bloodstream and increase the levels of circulating HDL-cholesterol and are therefore also useful in the treatment of cardiovascular disease, in particular atherosclerosis. The vaccine peptides of this invention, utilizing B cell epitope portions from the N-terminal region of CETP, have comparable or even superior autoimmunization activity to previously reported vaccine peptides utilizing B cell epitope portions of the C-terminal region of CETP, which is somewhat surprising, since the importance of the C-terminal region in CETP-mediated neutral lipid binding and transfer has been documented. (See, Hesler et al., J. Biol. Chem., 263: 5020-5023 (1988).) The present invention provides compositions and methods useful for the inhibition of cholesteryl ester transfer protein (CETP) activity. In particular, vaccine peptides are described which, when administered to a mammal, raise an antibody response against the mammal's own endogenous CETP, resulting in a decrease in overall CETP activity, and/or an increase in serum HDLc levels, and/or a decrease in the level of circulating cholesterol, and/or a decrease in serum LDLc or VLDLc levels in the subject administered the vaccine. These vaccine peptides are useful for the treatment of atherosclerosis, as they are believed to inhibit the development of atherosclerotic lesions in the arteries of a subject vaccinated with the vaccine peptides.
Such vaccine peptides are comprised of a CETP B cell epitope portion and a universal (or “broad range”) helper T cell epitope portion. The B cell epitope portion is comprised of 6 to 21 consecutive amino acids of the N-terminal 21 amino acids of CETP, preferably human CETP; the universal helper T cell epitope portion is comprised of a universal immunogenic helper T cell epitope, which binds the antigen presenting site of multiple class II major histocompatability complex (MHC) molecules. The B cell epitope portion and the universal helper T cell epitope portion are linked together, preferably covalently linked, most preferably linked by a peptide or amide bond to form a fusion peptide. Preferred fusion peptides will have the B cell epitope portion linked upstream (N-terminal) to the universal helper T cell epitope portion, however the reverse arrangement is also contemplated. Multimers, especially dimers, of the vaccine peptides of this invention are also contemplated.
In a preferred embodiment, a universal helper T cell epitope portion of a vaccine peptide of this invention is an immunogenic segment of amino acids such as short segments of tetanus toxin or diptheria toxin, or immunogenic peptides known from pertussis vaccine, Bacile Calmette-Guerin (BCG), polio vaccine, measles vaccine, mumps vaccine, rubella vaccine, and purified protein derivative (PPD) of tuberculin. An immunogenic carrier protein such as keyhole limpet hemocyanin (KLH) also may be used. Furthermore, various universal helper T cell epitopes may be linked to one another to form multiple universal helper T cell epitope portions of the vaccine peptides of this invention. In addition to naturally occurring universal helper T cell epitopes, designed peptide epitopes composed either entirely of natural amino acids or peptides comprised of a combination of natural amino acids and non-natural or synthetically modified amino acid residues may be used. Such non-natural universal helper T cell epitopes include, e.g., pan-DR epitope peptides such as those known by the PADRE™ designation (see, Alexander et al., Immunity, 1:751-762 (1994)).
In a preferred embodiment of a vaccine peptide of this invention, a universal helper T cell epitope from tetanus toxin having the sequence QYIKANSKFIGITE (SEQ ID NO: 1) is covalently linked to the C-terminus of the B cell epitope portion, the B cell epitope portion comprising a peptide having the sequence of the 21 amino-terminal amino acids of human CETP, i.e., CSKGTSHEAGIVCRITKPALL (SEQ ID NO: 2). The tetanus toxin segment may include a terminal cysteine residue to allow for dimerization of the peptide. More preferably, the vaccine peptide according to the present invention comprises the amino acid sequence from tetanus toxin QYIKANSKFIGITE (SEQ ID NO: 1) linked, preferably covalently, to amino acids 2-21 from the N-terminus of the CETP protein, i.e., SKGTSHEAGIVCRITKPALL (SEQ ID NO:3), wherein the N-terminal cysteine residue of human CETP has been removed. The most preferred embodiments of the peptides of the present invention are fusion peptides comprising a B cell epitope portion and a universal helper T cell epitope portion comprising either of the following sequences: CSKGTSHEAGIVCRITKPALLQYIKANSKFIGITE (SEQ ID NO: 4) and SKGTSHEAGIVCRITKPALLQYIKANSKFIGITE (SEQ ID NO: 5). Alternative embodiments may employ shorter spans, such as 6-8 consecutive amino acids, of the CETP N-terminal twenty-one amino acids (SEQ ID NO:2). Alternative embodiments utilizing other universal helper T cell epitope portions will include, for example, vaccine peptides incorporating such PADRE™ peptides as X1KX2VAAWTLKAX1 (SEQ ID NO:42), X1KX2VAAWTLKAAX1 (SEQ ID NO:48), or AKX2VAAWTLKAAA (SEQ ID NO:49), wherein X1 is D-Ala and X2 is cyclohexylalanine.
Vaccine peptides of the present invention were demonstrated to reduce the level of CETP activity and increase the levels of serum HDLc in both rabbits and human CETP transgenic mice administered the peptides. Thus, the vaccine peptides according to the present invention are useful in the treatment of cardiovascular disease, such as atherosclerosis.
The vaccine peptides of this invention may be used alone or in combination with a pharmaceutically acceptable adjuvant for administration to a mammal. After an initial immunization, additional or “booster” administrations of the vaccine peptides according to the invention may advantageously be made, for example in order to achieve or maintain a beneficial anti-endogenous CETP antibody titer. The vaccine peptides disclosed herein may also be co-administered with vaccine peptides having a similar structure including a universal helper T cell epitope portion and a CETP B cell epitope portion, wherein the B cell epitope portion corresponds to a segment of CETP other than the N-terminal region of CETP (for example, the C-terminal region involved in neutral lipid binding; see, U.S. Pat. No. 6,410,022).
The present invention also contemplates a DNA plasmid-based vaccine comprising a plasmid DNA molecule including a DNA sequence encoding an autoantigenic fusion polypeptide that, when administered to a mammalian (preferably human) subject, will induce the production of autoantibodies specifically reactive with the subject's endogenous CETP. Such autoantibodies inhibit endogenous CETP activity or remove CETP from circulation, promote the formation and maintenance of an antiatherogenic serum lipoprotein profile (for example, increased HDLc levels, decreased LDLc levels, or decreased circulating cholesterol levels), and/or inhibit the development of atherosclerotic lesions in the vaccinated subject.
The DNA plasmid-based vaccine of the present invention is comprised of a synthetic gene encoding a vaccine peptide fusion protein wherein a DNA segment encoding at least one CETP B cell epitope portion is linked in-frame with a DNA segment encoding a universal helper T cell epitope portion. The synthetic gene is operably linked to suitable DNA expression control sequences for expression of the synthetic gene product in mammalian cells.
The B cell epitope portion-encoding segment of the synthetic gene of the DNA plasmid-based vaccine of the present invention is comprised of a DNA sequence encoding 6 to 21 consecutive amino acids of the amino-terminal 21 amino acids of CETP, preferably human CETP. In a preferred embodiment, the B cell epitope portion-encoding segment of the synthetic gene is comprised of a nucleotide sequence encoding the N-terminal 21 amino acids of mature human CETP or encoding amino acids 2-21 of the N-terminal 21 amino acids of mature human CETP.
The preferred DNA plasmid-based vaccine of the present invention includes the following nucleotide sequence encoding a universal helper T cell epitope portion: 5′-CAGTACATCAAGGCCAATAGCAAGTTCATCGGCATTACCGAG-3′ (SEQ ID NO: 6).
A preferred DNA plasmid-based vaccine includes the following nucleotide sequence encoding a CETP B cell epitope useful in the present invention: 5′-TGTAGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAAC CTGCCCTCCTG-3′ (SEQ ID NO: 45). Underlined bases represent silent nucleotide substitutions (i.e., not changing the encoded amino acid) as compared with the native mature human CETP nucleotide sequence (SEQ ID NO: 43). The substitutions are optimized for human codon usage, which should result in optimal expression levels in vivo in human cells.
Another preferred DNA plasmid-based vaccine of the present invention includes the nucleotide sequence encoding a CETP B cell epitope comprising amino acids 2-21 (SEQ ID NO: 3) of the mature human CETP: 5′-AGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAACCT GCCCTCCTG-3′ (SEQ ID NO: 46). Underlined bases represent silent nucleotide substitutions (i.e., not changing the encoded amino acid) as compared with the native mature human CETP nucleotide sequence (SEQ ID NO: 43). The substitutions are optimized for human codon usage.
A preferred nucleotide sequence encoding a universal helper T cell epitope and a CETP B cell epitope fusion peptide for use in the DNA plasmid-based vaccine of the present invention includes the sequence: 5′-TGTAGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAAC CTGCCCTCCTGCAGTACATCAAGGCCAATAGCAAGTTCATCGGCATTACCGAG-3′ (SEQ ID NO: 9). Underlined bases represent silent nucleotide substitutions (i.e., not changing the encoded amino acid) as compared with the native mature human CETP nucleotide sequence (SEQ ID NO: 43). The substitutions are optimized for human codon usage.
A further preferred nucleotide sequence encoding a universal helper T cell epitope and a CETP B cell epitope fusion peptide for use in a DNA plasmid-based vaccine of the present invention includes the nucleotide sequence encoding amino acids 2-21 from the N-terminal region of the full-length mature human CETP protein: 5′-AGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAACC TGCCCTCCTGCAGTACATCAAGGCCAATAGCAAGTTCATCGGCATTACCGAG-3′ (SEQ ID NO: 47). Underlined bases represent silent nucleotide substitutions (i.e., not changing the encoded amino acid) as compared with the native full-length mature human CETP nucleotide sequence (SEQ ID NO: 43). The substitutions are optimized for human codon usage.
Plasmid-based vaccines of the invention taken up by cells are transcribed and translated to produce autoantigenic fusion peptides in vivo. Expression at sufficient levels and for a sufficient period of time exposes the autoantigenic fusion peptide to the host immune system, which elicits production of autoantibodies that react specifically with endogenous CETP of the host and that serve to inhibit CETP-mediated hypercholesterolemia. This, in turn, promotes an an antiatherogenic serum lipoprotein profile and inhibits the deposit of atherosclerotic plaque in the lumen of blood vessels.
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CETP has been validated as a therapeutic target for raising levels of HDL-cholesterol, raising the ratio of HDL-cholesterol to LDL-cholesterol, and for treating atherosclerosis (Davidson et al., Atherosclerosis, 169(1): 113-117 (July 2003); U.S. Pat. No. 6,410,022). This invention is directed to the modulation of endogenous CETP activity by providing CETP vaccine peptides derived from the N-terminus of the protein, or DNA based plasmid vaccines encoding the N-terminal CETP peptides, which peptides are useful for inducing an immune response in individuals against their own endogenous CETP, i.e., autoantibodies, thereby promoting an improved serum lipoprotein profile, e.g., decreasing the level of CETP activity in the bloodstream, increasing the levels of circulating HDLc or decreasing the levels of circulating LDLc/VLDLc, all of which are correlated with a decreased risk of cardiovascular disease.
The present invention provides CETP vaccine peptides, for inducing the production of anti-endogenous CETP antibodies in a vaccinated mammal, which are synthetic (non-naturally-occurring) vaccine peptides comprising a helper T cell epitope portion, comprising an amino acid sequence of a universal helper T cell epitope (see, for example, SEQ ID NO: 1), and a B cell epitope portion, comprising an amino acid sequence from the amino-terminal region of CETP, specifically from the amino-terminal 21 amino acids of CETP (see, for example, SEQ ID NO: 2 showing the N-terminal 21 amino acids from the mature full-length human CETP protein). Such CETP vaccine peptides are “autoantigenic”, that is, when administered to a mammalian subject they elicit production of specific antibodies against that peptide (antigen) which also bind that mammal's endogenous CETP, i.e., the mammal's native protein. Thus, the vaccine peptides of this invention are hybrid (universal helper T cell epitope peptide+CETP B cell epitope peptide) immunogenic moieties that have the capacity to stimulate the formation of autoantibodies which specifically bind endogenous CETP and/or inhibit endogenous CETP activity in a mammal vaccinated with the hybrid peptide or a DNA-based plasmid vaccine capable of directing in vivo expression of such a hybrid peptide.
Universal Helper T Cell Epitope Portion
The universal helper T cell epitope portion of the vaccine peptides of the present invention comprises an amino acid sequence of a universally immunogenic or “broad range” helper T cell epitope, which is defined as a peptide which can be presented by multiple major histocompatibility complex (MHC) haplotypes and thereby activate helper T cells, which, in turn, stimulate B cell growth and differentiation. Many examples of what are termed “universal” or “broad range” helper T cell epitopes which have been used for human vaccination are known in the art and include, for example, peptide segments of tetanus toxin (tt) and diptheria toxin (dt) (see, e.g., Panina-Bordignon et al., Eur. J. Immunol., 19: 2237-2242 (1989); Etlinger, H. M., Immunol. Today, 13: 52-55 (1992); Valmori, et al., J. Immunol., 149: 717-721 (1992); Talwar et al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994)).
In addition to short segments of tt and dt, tetanus toxoid (i.e., formaldehyde-detoxified tetanus toxin) or diphtheria toxoid can be used as a broad range helpter T cell epitope portion. Other broad range helper T cell epitope sequences useful in this invention include immunogenic peptides known from pertussis vaccine, Bacile Calmette-Guerin (BCG), polio vaccine, measles vaccine, mumps vaccine, rubella vaccine, and purified protein derivative (PPD) of tuberculin (see, e.g., Etlinger, H. M., Immunol. Today, 13: 52-55 (1992)); incorporated herein by reference). Additional universal helper T cell epitopes include synthetic compounds known as pan-DR-binding epitope (PADRE™) peptides, such as the peptide having the formula: X1KX2VWANTLKAAX1 (SEQ ID NO:42), where X1=D-alanine and X2=cyclohexylalanine. (Alexander et al., Immunity, 1: 751-761 (1994)). Furthermore, two or more copies of the same or various different universal or broad range helper T cell epitopes may be linked to one another to form multiple or multivalent helper T cell epitope portions of the vaccine peptides of this invention. For example, a vaccine peptide of this invention can be synthesized containing a multiple or multivalent helper T cell epitope portion comprising an amino acid sequence of a tt helper T cell epitope segment and a dt helper T cell epitope segment.
An immunogenic carrier protein may also be used as the universal helper T cell epitope portion of the vaccine peptide. Such carrier proteins are selected because they have immunostimulatory properties presumably from the presence of several helper T cell epitope sites, and also include convenient binding site(s) for covalent attachment of one or more CETP B cell epitope portions. One such immunogenic carrier protein is keyhole limpet hemocyanin (KLH). KLH contains multiple lysine residues in its amino acid sequence, and each of these lysines is a potential site at which a B cell epitope peptide or a whole vaccine peptide as described herein could be linked (for example, using maleimide-activated KLH, Catalog No. 77106, Pierce Chemical Co., Rockford, Ill.). Other immunogenic carrier proteins useful in the present invention include heat shock proteins HSP70 and HSP65 from Mycobacterium tuberculosis.
In particular embodiments, sequences of complement protein C3d may be used in addition to the universal helper T cell epitope(s), to enhance the magnitutde of the B cell response, leading to higher antibody titers.
In embodiments of the present invention, the universal helper T cell epitope portion is not a segment of CETP. During maturation, an individual's immune system generally has eliminated from the T cell repertoire any T cells capable of recognizing the individual's own (self) proteins (including CETP), which otherwise would encounter the immune system and initiate an immune response. Auto-immune responses are typically regarded as inappropriate and detrimental, and can lead to the manifestation of disease states, with potentially harmful or even fatal consequences. Since the helper T cells that might be capable of recognizing endogenous CETP epitopes normally have been eliminated from an individual's immune system, CETP would not be expected to be a source of any universal helpter T cell epitopes, and the practice of the present invention would require selection of a universal helper T cell epitope portion from a source other than CETP. Thus, the universal helper T cell epitope portion is occasionally referred to herein as a “non-CETP-related” component of the vaccine peptides of the invention. This naturally means that CETP itself, or a fragment thereof, without further modification (i.e., by linking to a universal helper T cell epitope), is not an embodiment of the present invention. Also, because the components of the vaccine peptides of this invention include CETP-related and non-CETP related components, the vaccine peptides are aptly regarded as hybrid polypeptides, combining elements from at least two different sources.
Preferred universal helper T cell epitope portions for use in vaccine peptides of this invention comprise universal immunogenic peptide fragments of tetanus toxin or diphtheria toxin. In a more preferred embodiment, the peptides of this invention use tetanus toxin segments having the amino acid sequence: QYIKANSKFIGITE (SEQ ID NO: 1), or FNNFTVSFWLRVP KVSASHLE (SEQ ID NO:50), or repeats or combinations thereof. Most preferably, the vaccine peptides of this invention utilize the universal helper T cell epitope from tetanus toxin having the amino acid sequence QYIKANSKFIGITE (SEQ ID NO:1). In addition to the various examples of universal helper T cell epitopes discussed above, additional universal helper T cell epitopes can be determined using a standard proliferation assay for MHC class II (helper) T cell epitopes (see, for example, Current Protocols in Immunology, Vol. 1 (Coligan et al., eds.) (John Wiley & Sons, Inc., New York, N.Y., 1994), pages 3.12.9-3.12.14).
CETP B Cell Epitope Portion
The B cell epitope portion of a vaccine peptide according to the present invention comprises an amino acid sequence from the amino-terminal region of CETP. Specifically, the B cell epitope portion comprises a peptide of at least six consecutive amino acids of the twenty-one N-terminal amino acids of CETP. In a preferred embodiment the B cell epitope portion of a vaccine peptide of this invention comprises the amino terminal 21 amino acids of mature human CETP protein. The full-length amino acid sequence of mature human CETP is shown in SEQ ID NO:7, the amino-terminal 21 amino acids of mature human CETP are shown in SEQ ID NO:2, the full-length amino acid sequence of rabbit CETP is shown in SEQ ID NO: 8.
More preferably, the B cell epitope portion (or “CETP-related” portion) of the vaccine peptides of this invention may be any fragment of the amino-terminal region of CETP which is at least six, preferably at least eight, consecutive amino acids from the amino-terminal 21 amino acids of CETP.
In a preferred embodiment, the CETP B cell epitope portion of a vaccine peptide according to the present invention is comprised of amino acids 2-21 of the human CETP, i.e., the N-terminal amino acid sequence without the cysteine residue located at amino acid position one of the CETP sequence (see, SEQ ID NO: 3).
The B cell epitope portion may utilize a segment of at least six consecutive amino acids from any mammalian CETP N-terminal region, however it is preferred to utilize a segment from the CETP of the same species that the vaccine peptide is intended for. For example, where the subject to be autoimmunized with the vaccine peptide is a human, and endogenous human CETP is thus the target for immunomodulation, the vaccine peptide B cell epitope portion will preferably be a segment selected from the N-terminal 21 amino acids of mature human CETP (see, SEQ ID NO: 2). Although less preferred, however, a xenogeneic N-terminal CETP segment may also be used, so long as it is effective to elicit antibodies cross-reactive with the endogenous CETP of the vaccinated subject.
Preparation of Vaccine Peptides
The universal helper T cell epitope (non-CETP-related) and the B cell epitope (CETP-related) portions of the CETP vaccine peptides of this invention are linked together to form autoantigenic moieties. The universal helper T cell epitope and B cell epitope portions may be covalently linked, directly by peptide bonds or via a cross-linking molecule. Where cross-linking molecules are used, they must join the universal or broad range helper T cell epitope portion and B cell epitope portion of the vaccine peptide together, without causing the peptide to become toxic to the vaccinated subject or significantly interfering with or reducing the overall immunogenicity of the vaccine peptide. Suitable cross-linking agents and molecules include amino acids, for example, using one or more glycine residues to form a “glycine bridge” between the universal helper T cell epitope and B cell epitope portions of the vaccine peptides of this invention; disulfide bonds between cysteine residues that exist in the universal helper T cell epitope portion and B cell epitope portion; cross-linking molecules such as glutaraldehyde (Kom et al., J. Mol. Biol., 65: 525-529 (1972)), and other bifunctional cross-linker molecules to link a universal helper T cell epitope portion to a B cell epitope portion are suitable for use in the present invention. Bifunctional cross-linker molecules possess two distinct binding sites, one of the sites can form a covalent attachment to a reactive site on the universal helper T cell epitope portion, and the other cross-linker binding site can form a covalent attachment to a reactive site on a B cell epitope portion. General methods for the use of cross-linking molecules are reviewed in Means and Feeney, Bioconjugate Chem., 1: 2-12 (1990).
Preferably, the universal helper T cell epitope and CETP B cell epitope portions of the vaccine peptides of this invention are covalently linked end-to-end to form a continuous fusion peptide. See, e.g., the synthetic peptides designated CETi-2 and CETi-2.1 (SEQ ID NOs: 4 and 5, respectively). Most preferably, a selected universal helper T cell epitope portion forms the carboxyl terminal portion of the vaccine peptide with its amino terminal amino acid residue covalently linked in a peptide bond to the carboxyl terminal amino acid of a selected CETP-related amino acid sequence (B cell epitope portion) of the vaccine peptide. (See, for example, SEQ ID NOs: 4 and 5).
The peptides of this invention can be produced by any of the available methods known in the art for synthesizing peptides of defined amino acid sequence. Direct synthesis of the peptides of the invention may be accomplished using conventional techniques, including solid-phase peptide synthesis, solution-phase synthesis, etc. Solid-phase synthesis is preferred. See, Stewart et al., Solid-Phase Peptide Synthesis (1989), W. H. Freeman Co., San Francisco; Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963); Bodanszky and Bodanszky, The Practice of Peptide Synthesis (Springer-Verlag, New York 1984), incorporated herein by reference.
Polypeptides according to the invention may also be prepared commercially by companies providing peptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality Controlled Biochemicals, Inc., Hopkinton, Mass.).
Automated peptide synthesis machines, such as those manufactured by Perkin-Elmer Applied Biosystems, also are available.
Alternatively, the peptides of this invention may be produced using synthetic and recombinant nucleic acid technology. For example, one of ordinary skill in the art can design from the known genetic code a 5′ to 3′ nucleic acid sequence encoding a vaccine peptide of this invention. A DNA molecule including the coding sequences of the universal helper T cell epitope and CETP B cell epitope portions (and any linking peptide, such as polyglycine, or other additional residue(s), such as a C-terminal or N-terminal cysteine, if desired) can readily be synthesized either using an automated DNA synthesizer or by a commercial DNA synthesizing service. The synthesized DNA molecule can then be inserted into any of a variety of available gene expression systems (e.g., bacterial plasmids, bacteriophage expression vectors, retroviral expression vectors, baculoviral expression vectors) using standard methods available in the art (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3 (Cold Spring Harbor Laboratory, Cold Spring harbor, N.Y., (1989)). The expressed peptide is then isolated from the expression system using standard methods to purify peptides.
The polypeptide compound is preferably purified once it has been isolated or synthesized by either chemical or recombinant techniques. For purification purposes, there are many standard methods that may be employed including reversed-phase high-pressure liquid chromatography (RP-HPLC) using an alkylated silica column such as C4-, C8- or C18-silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can also be used to separate peptides based on their charge. Purification of the vaccine peptides of this invention may be expedited by employing affinity chromatography or immunoprecipitation, e.g., based on using antibodies or other ligands recognizing the particular universal helper T cell epitope portion or B cell epitope portion of the vaccine peptide to be purified. The degree of purity of the polypeptide may be determined by various methods, including identification of a major large peak on HPLC. A polypeptide that produces a single peak that is at least 95% of the input material on an HPLC column is preferred. Even more preferable is a polypeptide that produces a single peak that is at least 97%, at least 98%, at least 99% or even 99.5% or more of the input material on an HPLC column.
Uses of the Vaccine Peptides
The peptides of this invention are used as autoimmunogenic compositions that elicit production of endogenous autoantibodies which specifically bind to the immunized subject's endogenous CETP and/or modulate (i.e., decrease or inhibit) endogenous CETP activity in the immunized subject. A vaccine composition including one or more vaccine peptides of this invention may be used. For example, peptides having different universal helper T cell epitope portions (e.g., different universal helper T cell epitopes) and/or different CETP B cell epitope portions (e.g., different CETP-related portions comprised of the amino terminal 21 amino acids of CETP or fragments thereof spanning 6 or more amino acids) may be combined and administered as a single vaccine composition. In addition, vaccine peptides according to this invention may be combined with other CETP vaccine peptides, as disclosed in, for example, U.S. Pat. No. 6,410,022.
Pharmaceutically acceptable adjuvants, such as alum, may be mixed with vaccine peptides of this invention. Alum is the single adjuvant currently approved for use in administering vaccines to humans (see, Eldridge et al., In Immunobiology of Proteins and Peptides V: Vaccines: Mechanisms, Design, and Applications, Atassi, M. Z., ed. (Plenum Press, New York, 1989), page 192). Recently, alum was used in combination with a sodium phthalyl derivative of lipopolysaccharide to administer a vaccine shown to be effective against human chorionic gonadotropin to humans (see, Talwar et al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994)).
Other conventional adjuvants may be used as they are approved for a particular use. For example, biodegradable microspheres comprised of poly (DL-lactide-co-glycolide) have been studied as adjuvants for oral or parenteral administration of vaccine compositions (Eldridge et al., In Immunobiology of Proteins and Peptides V. Vaccines: Mechanisms, Design, and Applications, Atassi, M. Z., ed. (Plenum Press, New York, 1989), page 192).
Other adjuvants have been used for administering vaccines to non-human mammals. For example, Complete Freund's Adjuvant (Sigma Chemical Co., St. Louis, Mo.), Incomplete Freund's Adjuvant (Sigma Chemical Co., St. Louis, Mo.), and the MPL, RC-259 and Ribi Adjuvant System (RAS) available from Corixa Corp. (Seattle, Wash.) are well known adjuvants routinely used to administer antigens to mammalian subjects, which may also eventually be approved for use in humans. In addition, adjuvant structures may also be mixed with or, preferably, covalently incorporated into peptides of this invention, for example at the amino or carboxyl terminal amino acid residue of the peptides. Such incorporated adjuvants include lipophilic N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl)]-cysteine (“Pam3-Cys-OH”); glycopeptides such as N-acetyl-glucosaminyl-N-acetylmuramyl-alanyl-D-isoglutamine (“GMDP”), muramyl dipeptides, and alanyl-N-adamantyl-D-glutamine; and polyamide gel-based adjuvants which can easily be attached to peptides during their in vitro chemical synthesis (see, Synthetic Vaccines, Nicholson, B. H., ed. (Blackwell Scientific Publication, Cambridge, Mass., 1994), pp. 236-238).
In addition, the vaccine peptides of the present invention may be linked to other molecules that may enhance the immunogenicity of the peptides. For example, linking peptides of this invention to a surface of a larger molecule, such as serum albumin, may enhance immunogenicity because the epitopes of the vaccine peptides are presented to the immune system of an individual as adjacent multiple repeated copies (see, e.g., Tam, J. P., Proc. Natl. Acad. Sci. USA, 85: 5409-5413 (1988); Wang, C. Y., et al., Science, 254: 285-288 (1991); Marguerite, M., et al., Mol. Immunol., 29: 793-800 (1992)). Such “multiple” or “multivalent” arrangements of the vaccine peptides of this invention can be prepared using cross-linker molecules. For example, bifunctional cross-linker molecules possess two reactive sites, one of the sites can attach the linker to a vaccine peptide of this invention and the other site is available to react with a different molecule, e.g., a larger protein like serum albumin or a resin or polymeric bead. Thus, covalent cross-linker molecules may be used to link vaccine peptides to other proteins or substrates to form multicopy arrangements of the peptides (multicopy peptide assemblies). Linking vaccine peptides of this invention to another molecule or surface should be carried out in a manner that does not significantly disrupt or reduce the autoantigenic characteristics of the vaccine peptides. Preferably, the use of such linker molecules enhances the immunogenicity of the vaccine peptides of this invention as evidenced, for example, by a more rapid rise in anti-CETP antibody titer and/or production of higher affinity anti-CETP antibodies than when individuals are administered vaccine peptides that are not linked. Such cross-linker molecules may also be used to attach peptides of this invention to an “immunogenic enhancer” molecule such as granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been shown to serve as an effective immunogenic enhancer in generating the production of specific anti-tumor antibodies (e.g., Tao, M. H., et al., Nature, 362: 755-758 (1993)).
The vaccine peptides of this invention may be administered, either alone or in association with one or more pharmaceutically acceptable carriers or adjuvants, in the same manner as a conventional vaccine, such as, e.g., tetanus vaccine. Suitable means include, for example, subcutaneous, intramuscular or intravenous injection. However, in contrast to conventional vaccines, which elicit an immune reaction against a non-endogenous, “foreign” antigen, such as, e.g., tetanus toxoid, the vaccine peptides of the present invention elicit an autoantibody response against endogenous CETP of the vaccine recipient. In some embodiments of this invention, the vaccine peptides may also be combined and administered with vaccines for other diseases or disorders.
The immune response elicited against endogenous CETP should significantly inhibit endogenous CETP function, in particular the transfer of cholesteryl esters from HDL to LDL/VLDL, and thereby produce a change in the circulating levels of LDLc and/or VLDLc and/or HDLc, preferably increasing the levels of circulating HDLc, increasing the HDLc/LDLc ratio, and/or decreasing the levels of circulating LDLc/VLDLc.
Accordingly, the desired therapeutic effect of administering vaccine peptides according to this invention is evidenced by eliciting autoantibodies in an individual that bind endogenous CETP and/or inhibit CETP activity, or by a relative decrease in LDLc and/or VLDLc levels compared to HDLc levels, or by an elevation of absolute levels of circulating HDLc. Because the vaccine peptides of the present invention produce these therapeutically useful effects, the vaccine peptides are useful in the treatment of cardiovascular disease, particularly atherosclerosis. By “treatment” is meant inhibition of progression of a disease or amelioration or reduction of disease symptoms. It is expected that administration of the vaccine peptides of the present invention will prevent or retard the accumulation of atherosclerotic plaque in vaccinated subjects, will arrest the buildup of atherosclerotic plaque, or may even lead to regression of atherosclerotic lesions in treated subjects.
The CETP vaccine peptides of this invention may be administered by any route used for vaccination, including: parenterally such as intraperitoneally, interperitoneally, intradermally, subcutaneously, intramuscularly, or intravenously; or orally. If oral administration of a vaccine peptide is desired, any pharmaceutically acceptable oral excipient may be combined with the vaccine peptides of this invention, such as, for example, the solutions approved for use in the Sabin oral polio vaccine.
Repeat administrations of the vaccine peptides subsequent to the initial priming dose, also known as “booster” administrations, are also contemplated in order to raise or maintain desired circulating anti-CETP antibody titer levels. Biodegradable microspheres, such as those comprised of poly(DL-lactide-co-glycolide), have been shown to be useful for effective vaccine delivery and immunization via oral or parenteral routes (Eldridge et al., in Immunobiology of Proteins and Peptides V: Vaccines: Mechanisms, Design, and Applications, Atassi, M. Z., ed. (Plenum Press, New York, 1989), pp. 191-202)). Because endogenous CETP lacks helper T cell epitopes, endogenous CETP is not expected to boost the autoantibody response elicited by vaccine peptides according to the invention. This differs from traditional vaccines targeting exogenous antigens where re-exposure or challenge by the vaccine target can boost the immune response, for example, leading to elevated antibody titers. For the present invention, it is expected that booster immunizations will be necessary to maintain the autoantibody response capable of inhibiting endogenous CETP.
Appropriate dosages of the peptide vaccines of this invention are established by general vaccine methodologies used in the art based on measurable parameters for which the vaccine is proposed to affect, including monitoring for potential contraindications, such as hypersensitivity reaction, erythema, induration, tenderness (see, e.g., Physicians'Desk Reference, 49th ed., (Medical Economics Data Production Co., Mont Vale, N.J., 1995), pp. 1628, 2371 (referring to hepatitis B vaccine), pp. 1501, 1573, and 1575 (referring to measles, mumps, and/or rubella vaccines), pp. 904, 919, 1247, 1257, 1289, 1293, and 2363 (referring to diptheria, tetanus and/or pertussis vaccines). A common and traditional approach for vaccinating humans is to administer an initial dose of a particular vaccine to sensitize (“prime”) the immune system and then to follow up with one or more “booster” doses of the vaccine to elevate an anamnestic response by the immune system that was sensitized by the initial administration of the vaccine (vaccination). Such a “priming and booster” administration procedure has been well known and commonly used in the art, as for example, in developing and using measles, polio, tetanus, diptheria, and hepatitis B vaccines.
Initially, the amount of a vaccine peptide administered to an individual may be that required to neutralize the approximate level of endogenous CETP activity present in the individual prior to vaccination, as can be determined by measuring CETP activity in serum or plasma samples from the individual, for example as determined using a commercially available CETP assay. Plasma or serum samples from a vaccinated individual can also be monitored to determine whether a measurable increase in the levels of HDL-cholesterol is seen after administration of the vaccine peptide using commercially available assays. A rise in the concentration (titer) of circulating anti-CETP antibodies can be measured in plasma or serum samples, for example using an ELISA assay.
Thus, it is possible and recommended that initially it be established whether a rise in anti-CETP antibody can be correlated with an increase in the level of HDL-cholesterol (i.e., total cholesterol, including cholesteryl esters and unesterified cholesterol, associated with high density lipoprotein), or with a decrease in CETP activity. Thereafter, one needs only to monitor a rise in titer of anti-CETP antibody to determine whether a sufficient dosage of vaccine peptide has been administered or whether a “booster” dose is indicated to elicit an elevated level of anti-CETP antibody. This is the common procedure with various established vaccinations, such a vaccination against hepatitis B virus.
DNA-Based Vaccines
The present invention also contemplates DNA plasmid-based vaccines capable of expressing the autoantigenic peptides of the present invention in situ. Such DNA vaccines are prepared in the form of a plasmid for administration, e.g., by intramuscular injection, to a subject, after which the transcription and translation in vivo of the portion of the plasmid encoding a vaccine peptide according to the present invention leads to production of vaccine peptides that, in turn, elicit the desired autoimmune response described above. A plasmid-based vaccine according to the invention comprises: the structural coding sequence for an autoantigenic fusion polypeptide comprising a DNA sequence encoding at least one universal helper T cell epitope and a DNA sequence encoding at least one B cell epitope from the N-terminus of CETP as described above, which structural coding sequence is operatively linked to a promoter sequence or a promoter/enhancer sequence capable of directing transcription of the structural coding sequence in cells of a mammalian subject. It may also be desirable to include a bacterial origin of replication and a selectable marker(s), for example, to aid in the production of large quantities of the plasmid vaccine in bacterial culture.
A preferred nucleotide sequence encoding a universal helper T cell epitope for use according to the present invention, includes a nucleotide sequence encoding the 14-amino acid tetanus toxin fragment shown in SEQ ID NO:1. A preferred nucleotide sequence encoding this tetanus toxin segment is set forth below: 5′-CAGTACATCAAGGCCAATAGCAAGTTCATCGGCATTACCGAG-3′ (SEQ ID NO: 6).
A preferred nucleotide sequence encoding a CETP B cell epitope for use according to the present invention comprises a nucleotide sequence encoding the N-terminal 21 amino acids of mature human CETP shown in SEQ ID NO:2. A preferred coding sequence for such N-terminal peptide is shown below: 5′-TGTAGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAAC CTGCCCTCCTG-3′ (SEQ ID NO: 45). Underlined bases represent silent nucleotide substitutions (i.e., not changing the encoded amino acid) as compared with the native mature human CETP nucleotide sequence (SEQ ID NO:43). The substitutions are optimized for human codon usage.
Another preferred nucleotide sequence encoding a CETP B cell epitope for use in the present invention comprises the nucleotide sequence encoding the CETP N-terminal amino acids 2-21 (SEQ ID NO:3) of human CETP: 5′-AGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAACC TGCCCTCCTG-3′ (SEQ ID NO:46). Underlined nucleic acids represent silent nucleotide substitutions as compared with the native CETP nucleotide sequence (SEQ ID NO:43). The substitutions are optimized for human codon usage.
A preferred nucleotide sequence encoding a hybrid peptide for use in a DNA plasmid-based vaccine of the present invention comprises a plasmid insert including the nucleotide sequence encoding the N-terminal 21 amino acids of mature human CETP linked in-frame to the nucleotide sequence encoding a universal helper T cell epitope derived from tetanus toxin: 5′-TGTAGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAAC CTGCCCTCCTGCAGTACATCAAGGCCAATAGCAAGTTCATCGGCATTACCGAG-3′ (SEQ ID NO:9). Underlined nucleic acids within the nucleotide sequence encoding the B cell epitope portion represent silent nucleotide substitutions as compared with the native mature human CETP nucleotide sequence (SEQ ID NO:43). The substitutions are optimized for human codon usage.
Another preferred nucleotide sequence for use in a DNA plasmid-based vaccine of the present invention comprises a plasmid insert including a nucleotide sequence encoding amino acids 2-21 from the N-terminus of mature human CETP, linked in-frame to the nucleotide sequence encoding a universal helper T cell epitope derived from tetanus toxin: 5′-AGCAAGGGCACCTCTCACGAGGCCGGCATCGTGTGCCGGATCACCAAACCT GCCCTCCTGCAGTACATCAAGGCCAATAGCAAGTTCATCGGCATTACCGAG-3′ (SEQ ID NO:47). Underlined nucleic acids within the nucleotide sequence encoding the B cell epitope portion represent silent nucleotide substitutions as compared with the native mature human CETP nucleotide sequence (SEQ ID NO:43). The substitutions are optimized for human codon usage.
The DNA plasmid vaccines of the present invention include the nucleotide sequences necessary for in vivo expression of the encoded autoantigenic fusion polypeptide at levels sufficient to elicit production of autoantibodies to the vaccinated subject's endogenous CETP. Transcription of the gene coding for the autoantigenic fusion protein is under the control of a promoter/enhancer sequence. A variety of promoter and enhancer sequences are known in the art and will be suitable for use in the present invention. Preferred promoter/enhancer sequences that may be used in plasmids of this invention include, but are not limited to, the CMV promoter/enhancer sequence, adenovirus promoter/enhancer sequence, and β-actin promoter/enhancer sequence. Whether a particular promoter/enhancer is more or less useful than another promoter/enhancer sequence in the plasmids of this invention can be determined by evaluating the ability of a promoter/enhancer to cause expression of, or to increase the level of expression of a standard reporter gene, such as luciferase or B-galactosidase, and the level of production of antibodies reactive with the expressed reporter in an animal model for gene expression, such as in rabbits or mice.
Generally, the higher the level of expression of the reporter gene product and/or the higher the level of production of antibodies reactive with the expressed reporter gene product, the more useful that particular promoter/enhancer will be at directing transcription of the structural coding sequence for autoantigenic fusion proteins in plasmid-based vaccines according to the invention.
The plasmid-based vaccines according to the invention may be administered in any manner calculated to lead to in vivo expression of the vaccine peptide encoded in the plasmid by the vaccinated subject. Suitable methods of administration include, for instance, direct administration of plasmid DNA via intramuscular injection, intradermal injection or DNA-coated microprojectiles. The amount of vaccine administered will vary widely according to the method of administration, the tissue (for example, skeletal muscle vs. skin) into which the vaccine is administered, the desired titer of anti-CETP antibodies, the particular therapeutic needs of the subject to be immunized, etc. Very large amounts of DNA vaccine, on the order of 10 mg/kg of body weight of the subject, may be administered with injection into muscle tissue, whereas for coated microprojectiles, a much lower dose of the vaccine may be effective for eliciting the desired immune response.
The dosage of vaccine and immunization protocol should be calibrated to obtain a beneficial response, which can be measured in a variety of ways, depending on the clinical setting, for example, by measuring anti-CETP antibody titer, change in lipoprotein profile (for example, increased HDLc level, decreased LDLc level, increased HDLc/LDLc ratio), serum CETP concentration, change (i.e., decrease) in CETP activity, etc. Methods and materials for assessing such titers, lipoprotein levels and CETP activity are well known in the art.
The following examples are provided in order to illustrate the invention described herein. These examples are not intended to in any way limit the scope of the invention.
A comprehensive screen for CETP B cell auto-epitopes was performed. Eleven New Zealand dWhite rabbits were immunized each with 0.2 mg of full-length human CETP (huCETP) in Complete Freund's Adjuvant (CFA). The huCETP was purified from conditioned media taken from cultures of a huCETP transformed CHO cell line (obtained from Dr. Alan Tall, Columbia University, New York). The rabbits received two booster immunizations (0.2 mg huCETP in Incomplete Freund's Adjuvant, or IFA), at 5 weeks and 10 weeks after initial vaccination. Serum samples collected 19 weeks after the initial injections were tested for the ability to recognize discrete segments of full-length rabbit CETP (SEQ ID NO:8): the full-length rabbit CETP amino acid sequence was divided into 31 peptides (see Table 1, SEQ ID NOs:10 to 40) comprised of thirty 21-mers and one C-terminal 16-mer, with N-terminal and C-terminal overlaps of five amino acids between adjacent peptides, spanning the entire rabbit CETP sequence. Table 1 (below) shows the amino acid sequence of each of the peptides 1-31 prepared for this experiment (SEQ ID NOs: 10-40).
Sera collected from rabbits immunized with human CETP were analyzed for their ability to recognize, i.e., show antibody binding to, each of the rabbit CETP peptides in the peptide array (Table 1) as a test for cross-reactivity of rabbit anti-human serum to similar rabbit and human CETP peptides. That is, as a test for the elicitation of autoreactive antibodies.
Table 1: Thirty-one overlapping rabbit CETP peptide fragments (cf. SEQ ID NO:8)
Each of the peptides was linked covalently via its N-terminus to an assigned spot on a modified polypropylene membrane (ResGen, Inc.); the full array of rabbit CETP peptides was presented on a single sheet of membrane. To account for non-specific binding and for binding be pre-existing antibodies, the peptides on the membrane were probed with sera taken from the rabbits one week prior to the prime injection (“pre-bleed” at “week −1” for baseline samples), while another membrane carrying an identical array of peptides was probed with post-vaccination sera. Dot-Blot techniques were employed for the probing of the solid-phase, bound peptides. Secondary antibodies (donkey anti-rabbit Ig) conjugated to horseradish peroxidase (HRP) were used to probe the bound rabbit antibodies, and development was carried out with chemiluminescence reagents (Boehringer Mannheim Corp.).
The results of this analysis are summarized in
The rabbits in
Based on these results, four peptides, i.e., peptide 1 (SEQ ID NO:10), peptide 22 (SEQ ID NO:31), peptide 30 (SEQ ID NO:39), and the 16-mer peptide 31 (SEQ ID NO:40), were chosen for further analysis of their potential to function as CETP B cell epitopes in CETP peptide vaccine constructs.
Because our primary interest is in developing CETP vaccines to elicit antibodies recognizing human CETP, the following experiments were carried out using sequences of human CETP that correspond to the rabbit peptides 1, 22, 30, and 31.
Based on the epitope usage results of Example I, peptides 1 (SEQ ID NO:10), 22 (SEQ ID NO:31), 30 (SEQ ID NO:39), and 31 (SEQ ID NO:40), were selected for further testing for the ability to function as CETP B cell epitopes in a vaccine peptide construct. Human sequences corresponding to those rabbit peptides were determined by comparison of the sequences:
Table 2: Human CETP peptides corresponding to rabbit peptides 1, 22, 30 and 31
Referring to
Referring to
Referring to
Referring to
The human CETP peptides 1, 22, 30, and 31 (Table 2) were conjugated to keyhole limpet hemocyanin (KLH), an immunogenic carrier protein. Peptides 1, 22, and 30 were conjugated through their C-terminus; peptide 31 was conjugated through its N-terminus. The peptide-KLH conjugates were administered to wild-type BALB/c mice with one priming and two booster subcutaneous injections. In the prime immunization each mouse received 0.1 mg of one of the peptides in 1:1 PBS/Complete Freund's Adjuvant (CFA) emulsion. Three weeks later the mice were given a booster injection of 0.1 mg peptide in 1:1 PBS/Incomplete Freund's Adjuvant (IFA) emulsion. Each of the peptide conjugates was used to immunize a group of 5 mice. A fifth group (control) of mice received only KLH (in a PBS/CFA or PBS/IFA emulsion).
Since mice, unlike humans, do not naturally express endogenous serum CETP, any CETP epitope presented to a wild-type mouse would be foreign to its immune system. Consequently, a wild-type mouse model does not mimic the conditions in a human patient where a CETP vaccine would be directed at an auto-antigen. To test the CETP vaccines in a mammalian system that would require overcoming the endogenous immune recognition of “self”, the subsequent experiments were carried out using transgenic mice expressing human CETP (Taconic, Germantown, N.Y.).
Human CETP transgenic mice were immunized by subcutaneous injection with KLH-conjugated human CETP peptides 1, 30, and 31, using the same protocol employed in Example II. The conjugates were injected (0.1 mg peptide in 0.2 ml PBS/CFA emulsion per rabbit per administration) into three groups of mice (one peptide conjugate/group; 9-10 mice/group); a fourth group received a mixture of all three peptide conjugates (0.1 mg of each peptide in 0.2 ml total of PBS/CFA emulsion per rabbit per administration) to test for potential additive responses. A booster vaccination (in IFA) was given 10 weeks later. A fifth group of mice served as negative controls, receiving PBS/CFA or PBS/IFA emulsion only. Serum samples were collected at 10 and 13 weeks post immunization and anti-CETP titers determined as described in Example II. The results are shown in
As seen in
In the group receiving a mixture of all three peptide conjugates, eight out of nine mice were positive for anti-huCETP titers. These titers were somewhat lower than when immunized with peptide-1-KLH alone, suggesting no additive immune responses and, moreover, implying interference in the immune reactivity to the epitopes presented by these peptides.
Sera collected in the Example III experiment was used to test effects of stimulating anti-CETP immune response with KLH-conjugated peptide vaccines on serum CETP activity. The method used to determine serum CETP activity was based on the assay described in C. Bisgaier et al., J. Lipid Res., 34:1625 (1993). CETP activity in a sample is measured as the degree of fluorescence de-quenching that results from CETP-mediated transfer of fluorescent-labeled cholesteryl ester from Donor to Accepter synthetic lipid micro-emulsions, which are added to the reaction mix at the onset of the reaction period. The synthetic Donor micro-emulsions are composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC, Avanti Polar-Lipids, Inc., Alabaster, Ala.), triolein, cholesteryl oleate and BODIPY-FL-cholesteryl (a fluorescent-labeled cholesteryl ester; Molecular Probes, Inc., Eugene, Oreg.). BODIPY-FL-cholesteryl is not included in the Acceptor micro-emulsions preparation, which are otherwise identical to that of Donor micro-emulsions. A mixture consisting of the test sample (whole serum), and Donor and Acceptor micro-emulsions, is incubated in a well of a black 96-well plate at 37° C. for 20 hours. At the end of the incubation period, a reading is taken with the aid of a fluorescence plate reader (CytoFluor™ II Microplate, Millipore, Inc., Billerica, Mass.; 485 nm excitation filter; 530 nm emission filter).
The results of the CETP activity analysis is shown in
Human CETP transgenic mice were immunized by subcutaneous injection with a CETP vaccine peptide according to the invention composed of human CETP peptide 1 (SEQ ID NO:51) covalently linked to a 14-mer universal (broad range) helper T cell epitope derived from tetanus toxin, QYIKANSKFIGITE (SEQ ID NO:1). To minimize masking of the functional epitope(s) in the CETP peptide, the tetanus toxin 14-mer was linked via its N-terminus to peptide 1, giving the vaccine peptide having the sequence: CSKGTSHEAGIVCRITKPALLQYIKANSKFIGITE (SEQ ID NO:4). This peptide was designated CETi-2.
For comparison, a positive control vaccine peptide was synthesized utilizing the C-terminal CETP peptide (i.e., human CETP peptide 31, SEQ ID NO:54) linked to the same tetanus toxin universal helper T cell epitope (here through the C-terminus of the tetanus toxin-derived peptide). This peptide was designated CETi-1 and had the following amino acid sequence: CQYIKANSKFIGITEFGFPEHLLVDFLQSLS (SEQ ID NO:41). The CETi-1 vaccine peptide was identical to vaccine peptide CETi-1 described in U.S. Pat. No. 6,410,022. This peptide carries an added (non-native) amino-terminal cysteine to aid in dimerization.
A further vaccine peptide according to the invention was synthesized which was identical CETi-2 described above, except that it lacked the native N-terminal cysteine residue. The N-terminal region of human CETP contains two cysteine residues (at positions 1 and 13) that can facilitate the formation of intra-molecular and inter-molecular disulfide bonds, and thus lead to the unintended creation of complex structures. We found that in the absence of the first cysteine, the peptide's tertiary structural complexity in solution is reduced significantly (data not shown). This peptide, comprising human CETP amino acids 2-21 linked to a C-terminal universal helper T cell epitope (SEQ ID NO:1) had the amino acid sequence: SKGTSHEAGIVCRITKPALLQYIKANSKFIGITE (SEQ ID NO:5) and was designated CETi-2.1.
Each of the three peptides CETi-1, CETi-2, and CETi-2.1 were used to immunize a group of 8-10 huCETP transgenic mice. A fourth group of mice was immunized with a mixture of CETi-1 and CETi-2. In the priming vaccination, each mouse received 0.1 mg of vaccine peptide in PBS/CFA emulsion; while in the two subsequent booster vaccinations (5 and 10 weeks later), IFA was substituted for CFA. A fifth group, serving as a negative control, received PBS/CFA emulsion or PBS/IFA emulsion injections only. All injections were subcutaneous injections at the base of the tail.
Serum samples were collected for evaluation three weeks after the third injection (second booster vaccination).
Serum CETP activity levels for each of the mice and their average values (bars) are represented in
Finally,
These results demonstrate the lack of a tight correlation between the levels of antibody titers raised by the anti-CETP vaccines peptides and their effectiveness in controlling serum CETP activity or HDLc levels. Thus, the site of interaction of the induced auto-antibodies with endogenous CETP may be more important. The very different effects obtained with CETi-2 and CETi-2.1 are most likely due to the differences in their secondary and tertiary structures, as their primary structures are practically identical. CETi-2, which has two Cys residues, at positions 1 and 13 of the peptide, can spontaneously encycle or polymerize by oxidation to form complex structures; whereas CETi-2.1, which is missing the Cys residue at position 1, would at most form vaccine peptide dimers when oxidized.
From the foregoing description it is evident that an autoantigenic vaccine peptide utilizing the N-terminal portion of cholesteryl ester transfer protein provides an unexpectedly effective autoimmunogen for eliciting an antibody response in a vaccinated individual that concomitantly controls (decreases) serum CETP activity and increases serum HDLc level.
Although a number of embodiments have been described above, it will be understood by those skilled in the art that modifications and variations of the described compositions and methods may be made without departing from the disclosure of the invention or the scope of the appended claims. The articles and publications cited above are hereby incorporated by reference.
