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Title:  Compositions and methods for the generation of protective immune responses against malaria
United States Patent: 
7,198,791
Issued: 
April 3, 2007

Inventors: 
Pluschke; Gerd (Bad Kronzing, DE), Markus; Mueller (Lorrach, DE), Robinson; John (Wermatswil, CH), Zurbriggen; Rinaldo (Schmitten, CH), Freund-Renard; Annabelle (Schonenwerd, CH)
Assignee: 
Pluschke, Gerd et al. (Bad Kronzing, DE)
Appl. No.: 
10/379,417
Filed: 
March 3, 2003


 

George Washington University's Healthcare MBA


Abstract

The invention presents vaccine formulations comprising highly antigenic epitopes identified within the semi-conserved loop-I of domain III that are capable of eliciting parasite growth inhibitory antibodies. The cyclized or linear peptides can be applied by known adjuvants or be encapsulated by or attached onto the surface of liposomes or virosomes (IRIVs) which serve as human compatible antigen delivery systems. Both cyclized and linear versions of the peptide antigens are surprisingly effective in eliciting immune responses that are cross-reactive with parasite-expressed AMA-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods for producing effective growth inhibitory immune responses against the blood stage of the malaria parasite. Aspects of the invention are based on the discovery that peptides comprising epitopes present in loop I of domain III of the merozoite protein AMA-1 are surprisingly effective in stimulating vigorous immune responses against AMA-1. The ectodomain of AMA1 comprises a region constituting 16 interspecies conserved cysteine residues which are crosslinked to form eight disulfide bridges. The three-dimensional structure of the AMA-1 protein is stabilized by these eight intramolecular disulfide bonds, which in turn divide the ectodomain into three subdomains (I, II, and III). Previous approaches using the whole AMA-1 ectodomain showed that the majority of antibodies raised against the ectodomain recognize epitopes in domain I, a highly variable region of the protein. However, efforts to design smaller peptide mimetics based on the loop structures in domain I failed to generate protective immune responses, possibly due to the inability of the peptides to accurately mimic the native tertiary structure of the protein (37).

Accordingly, the present invention provides immunostimulatory compositions based on the amino acid sequence of loop I of domain III of AMA-1. By immunostimulatory is meant the ability of the compositions to generate malaria parasite growth inhibitory immune responses. Such immune responses can be humoral or cell-mediated, or both, and their presence and effectiveness can readily be determined by the presence of antibodies that bind to and inhibit the growth of live malaria parasites. In preferred embodiments, the present invention provides epitopes identified in loop I of domain III that are useful for the design of immunostimulatory peptides capable of generating parasite cross-reactive, growth inhibitory immune responses against blood stage malaria merozoites. By epitope is meant that part of the AMA-1 molecule to which T-cell receptors respond or against which a growth inhibitory antibody will be produced and to which it will bind. Such epitopes may be linear, conformational, continuous or discontinuous.

In one preferred embodiment, the present invention provides immunostimulatory compositions comprising the epitopes of SEQ ID NO: 1, 2, 3, or 4, or any combination thereof. The epitopes identified by the present invention can be used by themselves as individual peptides to generate effective immune responses against AMA-1 expressing parasites, or they can be used in combination with each other. In more preferred embodiments, the epitopes form part of a longer peptide, such as that represented by SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. The epitopes of the present invention, when forming part of a longer peptide (or polypeptide), may be arranged in any orientation or order, including nested or overlapping arrangements, and the peptides containing the diverse arrangements of epitopes may be tested for immunogenicity and immunostimulatory effect by methods well known to persons of skill in the art and as further described herein.

The peptides of the present invention may be produced by chemical synthesis, or they may be of recombinant origin. In addition, their sequences may be modified as long as they retain their immunostimulatory effect, which can be measured by their ability to elicit parasite growth inhibitory immune responses. Thus, in a further preferred embodiment, the invention encompasses functional variants of the peptides of the invention. As used herein, a "functional variant" or "variant" of a peptide is a peptide which contains one or more modifications to the primary amino acid sequence of the peptides of the present invention while retaining the immunostimulatory effect disclosed herein. If a functional variant of a peptide of the present invention involves an amino acid substitution, conservative amino acid substitutions typically will be preferred, i.e., substitutions which retain a property of the original amino acid such as size, charge, hydrophobicity, conformation, etc. Examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (1) M, I, L, V; (2) F, Y, W; (3) K, R, H; (4) A, G; (5) S, T; (6) Q, N; and (7) E, D. Other suitable substitutions are easily established by the person of skill and may additionally be determined by reference to publications such as Voet, Biochemistry, Wiley, 1990; Stryer Biochemistry 4.sup.th Ed., Freeman N.Y., 1995; Peptide Chemistry. A Practical Textbook, 2nd ed., Miklos Bodanszky, Springer-Verlag, Berlin, 1993; Principles of Peptide Synthesis, 2nd ed., Miklos Bodanszky, Springer-Verlag, Berlin, 1993; Chemical Approaches to the Synthesis of Peptides and Proteins, P. Lloyd-Williams, F. Albericio, E. Giralt, CRC Press, Boca Raton, 1997; Bioorganic Chemistry: Peptides and Proteins, S. M. Hecht, Ed., Oxford Press, Oxford, 1998, Synthetic Peptides: A User's Guide, Gregory A. Grant (Editor), Oxford University Press, 2002, and the like, all of which are incorporated by reference herein.

Methods for identifying functional variants of immunostimulatory peptides of the present invention are provided according to another aspect of the invention. In a first aspect, functional variants of the peptides of the present invention, for example SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, can be identified by searching the publicly available literature or databases for protein and/or nucleotide sequences for AMA-1 of different Plasmodium strains or species. Once the divergent amino acids and their positions are determined, they can be substituted in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. For example, D448, K451, and K485 of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:7 may be substituted by any amino acid residue of choice, including D448 to N448, K451 to M451, and K485 to I485 to prepare variant peptides. In addition, K473 may be changed to E473 and D484 may be deleted altogether. The numbering of amino acid residues throughout this specification refers to their position in the peptides as depicted in FIG. 1. The ability of the variant peptides to generate or bind to merozoite growth-inhibitory antibodies is then determined, wherein binding to or generating parasite growth inhibitory antibodies comparable to that shown for the original peptides indicates that the variant peptide is a functional variant.

Modifications which generate functional variants of the peptides of the present invention may also comprise the addition of amino acids at either end of the peptides, i.e. at the N or C termini. Again, any well-known method for preparing modified or variant peptides can be employed, such as synthesis of the modified or variant peptide or its recombinant production using a mutated nucleic acid molecule. The peptides of the present invention may also be modified to be more resistant to hydrolysis by proteases, such as by containing D-amino acids or one or more non-hydrolyzable peptide bonds linking amino acids. Non-hydrolyzable peptide bonds are well-known in the art and may include -psi[CH.sub.2NH]-reduced amide peptide bonds, -psi[COCH.sub.2]-ketomethylene peptide bonds, -psi[CH(CN)NH]-(cyanomethylene)amino peptide bonds, -psi[CH.sub.2CH(OH)]-hydroxyethylene peptide bonds, -psi[CH.sub.2O] peptide bonds, and -psi[CH.sub.2S]-thiomethylene peptide bonds. The peptides of the present invention may also comprise unnatural and unusual amino acids and amino acid analogs, such as ornithine, norleucine, L-malonyltyrosine and others known to those of skill in the art. Alternatively, the peptides of the present invention and their functional variants may be rendered more resistant to degradation or their structural stability may be increased by the inclusion of nonpeptide moities. Preferably, the nonpeptide moieties permit the peptides to retain their natural conformation, or stabilize an optimized bioactive confirmation. Examples of suitable substitutions include D-isomer amino acids, N-methyl amino acids, L-isomer amino acids, modified L-isomer amino acids and cyclized derivatives. Such peptide mimetics can be tested in molecular or cell-based binding assays as described herein to assess the effect of the substitution(s) on conformation and/or activity. Procedures of medicinal chemistry may be applied by one skilled in the art using routine experimental methods of e.g. rational drug design, molecular modeling based on structural information from nuclear magnetic resonance or X-ray diffraction data, and other computational methods. Thus, the invention includes all of the foregoing modifications to the peptides.

In preferred embodiments, the peptides comprise amino acid residues located at or near the termini of the peptides with side chains suitable for the formation of an intramolecular crosslink for purposes of cyclizing the peptides. Suitable residues for crosslinking are well known to a person of skill in the art and may comprise disulfide (cysteine-cysteine), thioether (cysteine-electrophile, such as bromoacetyl, maleimidyl etc.) and other bonds. In a preferred embodiment, the crosslink for cyclization is provided by a disulfide bridge between cysteine residues located at or near the N and C termini of the peptides. In further preferred embodiments, terminal spacer residues are added to the peptides of the invention in order to ensure the spatial accessibility of the crosslinking residues, such as for the incorporation of the cyclic peptide into liposomes, virosomes, or other suitable delivery vehicles. For example, a GGC sequence may be added to the N-terminus and an additional glycine residue at the C-terminus, but many other spacer sequences known to those of skill in the art may be used for the purposes of the present invention, as long as the side chains of the spacer residues are small enough so as not to sterically interfere with the intramolecular crosslink. Examples of suitable spacer residues comprise amino acids such as alanine, serine, asparagine, glutamine, or glycine. Thus, in one preferred embodiment of the invention, the AMA-1 domain III loop I peptides are cyclized by the formation of an intramolecular crosslink. The cyclization of the peptides of the invention provides for the emulation of the native three-dimensional structure of AMA-1 loop I in domain III and is intended to further optimize growth inhibitory immune responses against the parasite. Again, internal crosslinks can be introduced via a number of residues, both natural and synthetic, which are well known in the art. In preferred embodiments terminally positioned (located at or near the N- and C-termini of the peptide) cysteine residues are used to cyclize the peptides through a disulfide bond. An example of such terminally positioned residues for crosslinking is found in SEQ ID NO:6 and SEQ ID NO: 7.

In a further preferred embodiment, the peptides of the present invention and functional variants thereof are cyclized by the use of a template, such as that depicted in FIG. 1. One advantage of using such widely available templates is their rigidity that may stabilize the three-dimensional conformation of the cyclized peptides more effectively than the use of internal crosslinks which typically introduce several rotatable bonds, thereby destabilizing the cyclized peptide structure. Suitable templates for the cyclization of peptide chains are well known in the art and may be tricyclic (Beeli et al., Helvetica Chimica Acta 79: 2235 2248, 1996), diketopiperazine-based (Bisang et al., Helvetica Chimica Acta 79: 1825 1842, 1996), bicyclic, such as a template derived from differentially substituted diaminoprolines (Pfeifer et al., Chem. Commun. 1977 78, 1998) or heterochiral diprolines (Favre et al. J. Am. Chem. Soc. 121: 2679 2685, 1999), to name only a few.

In another preferred embodiment, the present invention provides compositions comprising the AMA-1 domain III loop I peptides either cyclized (template-bound or internally crosslinked) or linear, including functional variants thereof, and a suitable delivery vehicle, such as a liposome or virosome (IRIV). IRIVs (immunostimulatory reconstituted influenza virosomes) are modified liposomes that contain reconstituted fusion-active viral envelope proteins anchored in the phospholipid bilayer. Both liposomes and virosomes are well known adjuvants used in vaccination protocols. Preparation and use of liposomes as adjuvants is standard in the art and described in The Theory and Practical Application of Adjuvants, D. E. S. Stewart-Tull (Ed.), Wiley, 1995 and Fries et al., PNAS 89: 358 362, 1992 among many other references. Virosomes can be prepared by detergent removal from a mixture of natural and synthetic phospholipids and influenza surface glycoproteins following protocols well known in the art (34).

The peptides of the invention can be attached to the surface of liposomes or they can be encapsulated by the liposomes using methods such as that described in Christodoulides et al., Microbiology 144: 3027 3037 (1998). Attaching of the peptides of the invention to the surface of delivery vehicles comprises incorporation into, crosslinking or adsorption to the surface of the delivery vehicles, all of which are procedures well known to those of skill in the art. Crosslinking of the peptides to the surface of liposomes, for example, may be performed by the use of amphiphilic PEG derivatives readily incorporated into liposomes and micelles via the PE residue that easily bind primary amino group-containing ligands via water-exposed pNP groups. Similarly, the peptides of the present invention may be encapsulated by virosomes (IRIV), by methods well known in the art and further described in Example 5. Attachment of the peptides to the surface of the virosomes may be performed by any method known to those of skill in the art. In one embodiment of the invention, the peptides of the present invention are attched to virosomes by conjugating the peptides through a succinate linker at the N-terminus to a regioisomer of phosphatidylethanolamine (PE). The PE-peptide conjugate is subsequently incorporated into IRIV as an antigen delivery system.

In a preferred embodiment, the invention provides kits comprising the aforementioned compositions which allow the skilled artisan to prepare a desired immunotherapeutic regimen. An example of a kit comprises the peptides of the invention, as well as their functional variants previously discussed. The kit may also comprise liposomes or virosomes loaded with the peptides of the invention and functional variants thereof, either by encapsulation or by surface-attachment, which includes crosslinking, conjugation and surface-association or -adsorption. In preferred embodiments, a kit provided by the present invention comprises mixtures of liposomes or virosomes encapsulating the peptides of the present invention, including functional variants thereof, with liposomes or virosomes that have the peptides of the present invention, including functional variants therof, attached to their surfaces. Such mixtures may be particularly effective in stimulating various branches of the immune system simultaneously. The kit preferably includes instructions for use of the compositions provided. Other components may be added to the kits, as desired.

Administration of the peptide-loaded virosomes is preferably preceded by one or more pre-immunizations with the empty virosomes or IRIV. Initial doses of the peptide-IRIV delivery system can be followed by booster doses, following immunization protocols standard in the art, and their effect may be potentiated by adjuvants or cytokines well known to those skilled in the art. Again, the peptides of the present invention, as well as functional variants thereof, may be encapsulated by or attached to the surface of the delivery vehicles in linear or cyclized form. The present invention also provides for the administration of the immunostimulatory AMA-1 peptides in a suitable pharmaceutical formulation. By administration or administering is meant providing one or more peptides or peptide-containing compositions of the invention to an individual in need of treatment or prevention of malaria. Such a composition which contains one or more of the peptides and/or peptide containing compositions of the present invention, including functional variants thereof, as the principal or member active ingredient, for use in the treatment or prevention of malaria, can be administered in a wide variety of therapeutic dosage forms in the conventional vehicles for topical, oral, systemic, local, and parenteral administration. Thus, the invention provides compositions for parenteral administration which comprise a solution of the peptides and their functional variants dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, among many others. Thus, a typical pharmaceutical composition for intradermal infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of peptide. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington: The Science and Practice of Pharmacy ("Remington's Pharmaceutical Sciences") Gennaro A R ed. 20th edition, 2000: Williams & Wilkins PA, USA, which is incorporated herein by reference.

The route and regimen of administration will vary depending upon the stage or severity of the malaria to be treated, and is to be determined by the skilled practitioner. For example, the immunostimulatory peptides, including their functional variants, and the peptide-containing compositions of the present invention can be administered in such oral dosage forms for example as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Similarly, they may also be administered in intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. In preferred embodiments, the peptides and peptide-containing compositions are administered intradermally or subcutaneously. All of these forms are well known to those of ordinary skill in the pharmaceutical arts.

The daily dose of the peptides and compositions of the invention may be varied over a range from 0.001 to 1,000 mg per adult per day. For oral administration, the compositions are preferably provided in the form of tables containing from 0.001 to 1,000 mg, preferably 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 10.0, 20.0, 50.0, 100.0 milligrams of active ingredient for the symptomatic adjustment of dosage according to signs and symptoms of the patient in the course of treatment. An effective amount of drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 50 mg/kg of body weight per day. The range is more particular from about 0.0001 mg/kg to 7 mg/kg of body weight per day.

In another preferred embodiment, the peptides of the invention and their functional variants can be administered by injection to a subject in the form of a peptide-based vaccine. Preferably, the peptides are injected intradermally or subcutaneously to allow for uptake by or exposure to antigen presenting cells located in the skin, epidermis or dermis, although other routes of administration known in the art may be equally suitable and are intended to be included in the present invention. In a more preferred embodiment, the peptides of the present invention are loaded, by encapsulation or surface attachment, onto virosomes or liposomes prior to administration to the subject, as described above. The peptide loaded delivery vehicles can then be injected into the subject via intradermal, subcutaneous or other suitable routes analogous to the administration of the peptides described previously. Advantageously, suitable formulations of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses for example of two, three, or four times daily. The peptides, including functional variants, and compositions of the present invention may be used to prepare a medicament or agent useful for the treatment of malaria. Furthermore, the immunostimulatory compositions of the present invention, particularly those containing virosomes or liposomes, can be administered in intranasal form, or via transdermal routes known to those of ordinary skill in the art.

For the treatment and prevention of malaria the peptides, including functional variants thereof, and compositions of the present invention may be used together with other agents known to be useful in treating malaria. Such agents may include chemotherapeutic drugs, such as quinine, quinidine, chloroquine, mefloquine, tetracycline, mefloxine, halofantrine, artemisinin and derivatives (artemether, artesunate), lumefantrine, doxycycline, proguanil, primaquine, atovaquone-proguanil, pyrimethamine-sulfadoxine etc. For combination treatment with more than one active agent, where the active agents can be administered concurrently, the active agents can be administered concurrently, or they can be administered separately at staggered times.

The dosage regimen utilizing the compositions of the present invention is selected in accordance with a variety of factors, including for example type, species, age, weight, sex and medical condition of the patient, the stage and severity of the malaria infection, and the particular compound employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the malaria infection. Optimal precision in achieving concentration of drug with the range that yields efficacy either without toxicity or with acceptable toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This process involves a consideration of the distribution, equilibrium, and elimination of the drug, and is within the ability of the skilled practitioner. Guidance as to indications, dosage and drug interactions can further be found in clinical manuals, including Harrison's Principles of Internal Medicine, 15.sup.th Ed. McGraw-Hill, 2001.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient and are typically administered in admixture with suitable pharmaceutical diluents or excipients suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups, and the like, and consistent with conventional pharmaceutical practices. For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, aga, bentonite, xanthan gum and the like.

The liquid forms may be suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl cellulose and the like. Other dispersing agents which may be employed are glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired. Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, for example, alcohols, aloe vera gel, allatoin, glycerine, vitamins A or E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, for example, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.

The immunostimulatory peptides including functional variants thereof and compositions of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihyrdo-pyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels. Generally, subjects can receive an intradermal injection of an effective amount of the peptides either in combination with delivery vectors, such as virosomes, or by themselves. The peptides of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilameller vesicles and multilamellar vesicles. Liposomes can be formed from a variety of compounds, including for example cholesterol, stearylamine, and various phosphatidylcholines.

Initial doses can be followed by booster doses, following immunization protocols standard in the art. The immunostimulatory effect of the compositions and methods of the instant invention can be further increased by combining any of the above-mentioned peptide compositions, including their combination with virosomes or liposomes, with an immune response potentiating compound. Immune response potentiating compounds are classified as either adjuvants or cytokines. Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes. Adjuvants of many kinds are well known in the art; specific examples include Freund's (complete and incomplete), mycobacteria such as BCG, M. Vaccae, or corynebacterium parvum, quil-saponin mixtures such as QS-21 (SmithKline Beecham), and various oil/water emulsions (e.g. IDEC-AF). Other adjuvants which may be used include, but are not limited to: mineral salts or mineral gels such as aluminum hydroxide, aluminum phosphate, and calcium phosphate; surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, keyhole limpet hemocyanins, and dinitrophenol; immunostimulatory molecules, such as saponins, muramyl dipeptides and tripeptide derivatives, CpG dinucleotides, CpG oligonucleotides, monophosphoryl Lipid A, and polyphosphazenes; particulate and microparticulate adjuvants, such as emulsions, liposomes, virosomes, cochleates; or immune stimulating complex mucosal adjuvants. Cytokines are also useful in vaccination protocols as a result of lymphocyte stimulatory properties. Many cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-2 (IL-2), IL-12, GM-CSF and many others.

When administered, the therapeutic compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents. The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. Generally, doses of immunogens ranging from one nanogram/kilogram to 100 miligrams/kilogram, depending upon the mode of administration, are considered effective. The preferred range is believed to be between 500 nanograms and 500 micrograms per kilogram. The absolute amount will depend upon a variety of factors, including the composition selected for administration, whether the administration is in single or multiple doses, and individual patient parameters including age, physical condition, size, weight, and the stage of the disease. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

In further preferred embodiments, the present invention provides methods of generating parasite growth inhibitory immune responses comprising administering to a subject the peptides, including functional variants thereof, and compositions of the invention. In the case of treating malaria, the desired response is inhibiting the progression of the infection, reducing the parasitic load, and/or clearance of the parasite from the subject. In the case of preventing malaria, the desired response is the induction of vigorous antibody- and/or cell mediated immune responses against AMA-1 expressing parasites. These desired responses can be monitored by routine methods or can be monitored according to diagnostic methods of the invention disclosed herein. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description, as well as from the examples. Such modifications are intended to fall within the scope of the appended claims.

Results

The present invention demonstrates for the first time that it is possible to elicit parasite growth inhibitory antibodies with formulations of peptides comprising epitopes found in loop I of domain III of P. falciparum AMA-1. The development of peptide based vaccines has been hampered both by the poor immunogenicity of many peptides, possibly due in part to a lack of conformational similarity between small linear peptides and the corresponding sequence in the native target protein. Accordingly, the present invention provides immunostimulatory peptides comprising newly identified immunogenic epitopes that can be combined with human compatible delivery systems, including virosomes (IRIVs) and/or liposomes. The peptides of the invention can be encapsulated by, or attached to, the delivery vehicles for the efficient generation of AMA-1 specific immune responses. IRIVs, or virosomes, are spherical, unilammelar vesicles, prepared by detergent removal from a mixture of natural and synthetic phospholipids and influenza surface glycoproteins. They have been shown to act as an efficient and highly effective means of enhancing the immune response to a variety of antigens, thus illustrating their broad suitability as a vaccine delivery system (14). The hemagglutinin membrane glycoprotein of influenza virus plays an important role in the mode of action of IRIVs. This major antigen of influenza virus is a fusion-inducing component, which facilitates antigen delivery to immunocompetent cells. In the case of the IRIV-based hepatitis A vaccine Epaxal-Bema, which is the first licensed vaccine in which IRIVs are used as a delivery system for a non-influenza antigen, the hepatitis A antigen spontaneously binds to the IRIVs, thus attaching to the delivery vehicles. Smaller antigens, including the peptides of the present invention can be linked to a phospolipid (PE) and the PE-antigen conjugates can be integrated into the virosomal membrane during the virosome reconstitution process (28, 34).

Compared to a virosome formulation loaded with a PE conjugate of a cyclic peptide mimotope of the repeat region of the P. falciparum circumsporozoite protein, an alum-adjuvanted mimotope-MAP (multiple antigenic peptide) construct, although capable of eliciting comparable levels of anti-mimotope antibody responses in mice, failed to produce antibodies that bind effectively to the parasites, indicating that PE-coupled antigens are located in a more native-like state on the surface of the virosomes. These results may indicate that intramolecular interactions lead to a correct folding of the linear peptide and that the anchoring to the surface of IRIVs has no deleterious effects on this process. The solution structure of the AMA-1 domain III, determined by NMR spectroscopy, consists of a disulfide-stabilized core region including all three disulfide bonds, but also contains significant regions of disorder (29). The epitope analysis of growth inhibitory anti-AMA49 MAbs using a library of 12-residue cyclic peptides covering the AMA.sup.444-489 sequence (FIG. 1) performed herein provides evidence that at least some of the MAbs may recognize discontinuous epitopes. Since discontinuous epitopes may contain short stretches of continuous sequences (1, 3, 38), analyses with sets of overlapping peptides are suitable to define both continuous linear epitopes and parts of discontinuous epitopes (15, 41). The analyses indicate that K.sup.459RIKLN.sup.464 (SEQ ID NO: 1) and D.sup.467DEGNKKII.sup.475 (SEQ ID NO: 2) represent sequence stretches of discontinuous epitopes recognized by inhibitory anti-AMA-1 MAbs.

AMA-1 lacks tandem repeat sequences found in many other P. falciparum antigens. However, a significant degree of sequence diversity is observed, which may reflect diversifying selection pressure. from naturally acquired immune responses (9, 13, 33, 42). Most of the polymorphic or dimorphic amino acid residues of the relatively conserved domain III are located far apart from each other in the primary sequence, but may cluster in the region of the disulphide core in the three-dimensional structure of the molecule (29). This is also the case for the three variable residues (D.sup.448, K.sup.451 and K.sup.485) present in the AMA-1.sup.446-490 sequence analyzed herein. The virosomal formulation of this peptide seems to focus the antibody response primarily to conserved loop structures away from this core region. Cross protection obtained with antibodies raised against recombinantly expressed AMA-1 has provided evidence for the existence of such common protective epitopes (17, 21). Taken together, these results indicate that the loop I sequence from domain III of AMA-1 represents a suitable component of an IRIV-based multi-antigen multi-stage synthetic peptide malaria vaccine candidate.

Accordingly, a sequence comprising 45 amino acid residues from loop-I in domain III of AMA-1, Y.sup.446KDEIKKEIERESKRIKLNDNDDEGNKKIIAPRIFISDDKDSLKC.sup.490 (SEQ ID NO: 5) with a GGC sequence added to the N-terminus and an additional glycine residue at the C-terminus was synthesized by standard solid phase peptide chemistry. In order to load the peptide onto virosomes, it was conjugated through a succinate linker at the N-terminus to a regioisomer of phosphatidylethanolamine (PE) and the PE-peptide conjugate designated AMA49-L1 (FIG. 1, L standing for the linear form) was incorporated into IRIV as antigen delivery system. After one pre-immunization with the influenza vaccine Inflexal, mice were immunized with AMA49-L1 loaded IRIV. All four mice immunized produced antibodies that reacted with AMA49-L1 in ELISA (FIG. 2).

The elicited antibodies were cross-reactive with blood stage parasites in IFA (FIG. 3), yielding a punctate staining pattern characteristic for AMA-1 (17, 27). Interaction of mouse anti-AMA49-L1 sera with parasite expressed AMA-1 was re-confirmed by Western blot analysis using total protein lysates of P. falciparum blood stage parasites (FIG. 4). The anti-AMA49-L1 antisera stained primarily proteins of an apparent molecular mass of approximately 83 and 66 kDa, which is the size of the full-length AMA-1 and of its major processed product, respectively (17, 30). Some of the anti-AMA49-L1 antisera stained an additional protein band, which may correspond to the previously described 46 kDa processing product of AMA-1 (18) that possesses the same N-terminus as the 66 kDa fragment.

In order to investigate whether cyclization of the peptide via the cysteine residues close to the C- and N-termini improves the yield of parasite binding antibodies, mice were immunized with IRIV loaded with a cyclized (AMA49-C1) and a linear (AMA49-L2) version of AMA49-L1 (FIG. 1). While AMA49-L1 contained free thiols, both thiol groups of the terminal cysteine residues were blocked by alkylation in the case of AMA49-L2. IFA titers obtained with the two constructs did not differ significantly and were comparable to those obtained with AMA49-L1 (data not shown).

For a detailed analysis of the humoral immune response, AMA49-C1 specific mouse B cell hybridomas were generated. All 11 hybridoma clones obtained secreted IgG:.kappa. (10 MAbs were IgG1, MAb DV3 was IgG2a) that reacted in ELISA both with the cyclic and the linear peptide with comparable efficacy. These 11 MAbs all stained blood stage parasites in IFA (data not shown). Competition ELISA experiments with a set of 4 overlapping linear peptides comprising the sequences AMA.sup.446-462, AMA.sup.452-472, AMA.sup.462-482, and AMA.sup.472-490 FIG. 1) were used for epitope mapping and differentiated between four groups of antibodies (Table 2).

Antigen binding of MAbs DV2 and DV6 was blocked by AMA.sup.446-462, MAb DV7 was blocked both by AMA.sup.446-462 and AMA.sup.452-472, MAbs DV5 and DV8 were blocked by AMA.sup.452-472 and MAbs DV1, DV3, DV4, DV9, DV10 and DV11 were blocked by AMA.sup.462-482. None of the antibodies was blocked by the C-terminal sequence AMA.sup.472-490. A library of 35 cyclic peptides each containing 12 residues scanning the AMA.sup.444-489 sequence each with an offset of one amino acid, was used for more detailed epitope mapping (see FIG. 1 and Table.2). These peptides were conformationally restrained by cyclization through linkage to a dipeptide template comprising a D-proline and an L-4-aminoproline conjugated to succPE. In ELISA, both MAbs DV2 and DV6 inhibited by AMA.sup.446-462 bound to none of the short cyclic peptides. MAb DV7 bound to the consecutive cyclic peptides comprising residues 450 461 through 455 466, which share the sequence E.sup.455RESKRI.sup.461 (SEQ ID NO: 3) present also in the overlap of the two inhibitory long peptides AMA.sup.446-462 and AMA.sup.452-472. MAbs DV5 and DV8 bound to cyclic peptides 453 464 through 459 470, which share the sequence K.sup.459RIKLN.sup.464 (SEQ ID NO: 1) located in the center of the inhibitory peptide AMA.sup.452-472. Additional binding of both MAbs to the non-consecutive cyclic peptide 477 488 and of MAb DV5 to cyclic peptide 474 485 is indicative of a discontinuous epitope. All six MAbs that were inhibited by AMA.sup.462-482 exhibited binding to the consecutive peptides 464 475 through 467 478 which share the sequence D.sup.467DEGNKKII.sup.475 (SEQ ID NO: 2) located in the centre of the inhibitory long peptide AMA.sup.462-482. MAb DV1 reacted in addition with the overlapping peptide 462 473. In the case of MAb DV3 the reactivity with peptide N.sup.466DDEGNKKIIAP.sup.477 (SEQ ID NO: 8) was outstanding. With the other four MAbs DV4, DV9, DV10 and DV11 reactivity patterns with non-overlapping peptides indicate recognition of a discontinuous epitope. All four MAbs showed reactivity with the peptide 456 67, which only overlaps at position D.sup.467 with the putative central D.sup.467DEGNKKII.sup.475 (SEQ ID NO: 2) recognition sequence. In addition, MAbs DV4 and DV9 also bound to the non-consecutive peptides 474 485 and 478 489 and to the peptides 462 473, 463 474 which share the sequence D.sup.467DEGNKK.sup.473 (SEQ ID NO: 9) with the central recognition sequence.

Mice were immunized with IRIV loaded with PE conjugates of each of the cyclic 12-mer peptides in order to analyze whether some of them can act as mimotopes of AMA-1 surface loops and elicit parasite-binding antibodies. While all 35 structures elicited significant antibody titers against the respective immunizing peptide sequence itself, only sera raised against AMA.sup.458-469 (containing the central recognition unit K.sup.459RIKLN.sup.464 (SEQ ID NO: 1) of MAbs DV5 and DV8) and AMA.sup.464-475 (containing the central recognition unit D.sup.467DEGNKKII.sup.475 (SEQ ID NO: 2) of MAbs DV1, DV3, DV4, DV9, DV10 and DV11) were weakly cross-reactive with blood stage parasites in IFA (data not shown).

When Mabs DV5 and DV11 representing the two major fine specificities were tested in a P. falciparum in vitro growth inhibition assay, both exhibited substantial inhibitory activity (FIG. 5). A 95.3% reduction of parasite growth was observed after addition of MAb DV5 at a final concentration of 300 .mu.g/ml (average of five independent sixtuplicated experiments). MAb DV11 also exhibited a significant, but lower growth inhibitory activity, while an isotype-matched control MAb had no effect. Taken together, these data clearly demonstrate that it is possible to elicit parasite binding and growth inhibitory antibodies by immunization with AMA-1.sup.446-490 in combination with IRIV as a human compatible antigen delivery system.

AMA-1 is a leading malaria blood-stage vaccine candidate, which has been shown to elicit protective immune responses when administered as a whole protein. Parasite invasion inhibitory activities of anti-AMA-1 antibodies indicate that the humoral arm of the immune response plays an important role in AMA-1 mediated immune protection. The ectodomain of AMA-1 is divided into three sub-domains defined by disulfide bonds (16). There is evidence that the majority of inhibitory antibodies in sera from malaria exposed humans and in rabbit antisera raised against the refolded AMA-1 ectodomain are directed against strain-specific epitopes in domain I (17). An analysis of tryptic fragments of P. chabaudi adami AMA-1 recently identified a loop-like structure within the putative domain I as a target of antibodies from hyperimmune mouse sera (37). A synthetic 45mer loop mimetic incorporating this element was found to elicit AMA-1 binding antibodies. However, these antibodies did not protect P. chabaudi adami challenged mice in passive immunization experiments. Since domain I is the most diverse region of AMA-1 (25, 46), an AMA-1 vaccine component which lacks this domain may be preferable in order to direct the immune response to region(s) that contain more conserved epitopes (17). Since production of large batches of clinical-grade recombinantly expressed AMA-1 has been notoriously difficult, the present invention is directed to the development of a synthetic peptide-based AMA-1 vaccine formulation. The results obtained by the present invention show that it is possible to elicit parasite growth inhibitory antibodies with a virosomal formulation of peptides comprising parts of the sequence of loop I from domain III of P. falciparum AMA-1.
 


Claim 1 of 24 Claims

1. An immunostimulatory composition consisting of the polypeptide of SEQ ID NO: 5 or functional variants thereof.
 

 

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