WO2005063805A1 - Antibodies against the amino terminus region of circumsporozoite protein prevent the onset of malaria infection - Google Patents

Antibodies against the amino terminus region of circumsporozoite protein prevent the onset of malaria infection Download PDF

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WO2005063805A1
WO2005063805A1 PCT/US2004/043269 US2004043269W WO2005063805A1 WO 2005063805 A1 WO2005063805 A1 WO 2005063805A1 US 2004043269 W US2004043269 W US 2004043269W WO 2005063805 A1 WO2005063805 A1 WO 2005063805A1
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polypeptide
protein
cs
antibodies
vaccine
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Dharmendar Rathore
Thomas F. Mccutchan
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445Plasmodium
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/408Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa
    • Y02A50/411Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa of the genus Plasmodium, i.e. Malaria
    • Y02A50/412Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa of the genus Plasmodium, i.e. Malaria the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera

Abstract

The present invention relates to a polypeptide comprising a sequence as shown as the Peptide P6-identified epitope domain from amino acid 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 - 113, 114, 115, 116, or 117 of the Circumsporozoite protein, or allelic variant, functional equivalent, derivative, homologue, or analogue thereof, that reacts with antibodies that inhibit liver cell invasion by P. falciparum, substantially in isolation from sequences naturally occurring adjacent thereto in the Circumsporozoite protein.

Description

ANTIBODIES AGAINST THE AMINO TERMINUS REGION OF CIRCUMSPOROZOITE PROTEIN PREVENT THE ONSET OF MALARIA INFECTION Related Applications This application claims the benefit of the U.S. Provisional Patent Application No.: 60/532,676, filed December 23, 2003, the entire disclosure of which is hereby expressly incorporated herein by reference. Field of the Invention The present invention relates to a polypeptide comprising a sequence as shown as the Peptide P6-identified epitope domain from amino acid 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 - 113, 114, 115, 116, or 117 of the Circumsporozoite protein, or allelic variant, functional equivalent, derivative, homologue, or analogue thereof, that reacts with antibodies that inhibit liver cell invasion by P. falciparum, substantially in isolation from sequences naturally occurring adjacent thereto in the Circumsporozoite protein. Background of the Invention Malaria is one of the parasitic diseases for which the development of a vaccine has been vigorously pursued. Each year 400-500 million cases of malaria are reported around the world. 1-2 million individuals, mostly young children in sub-Saharan Africa succumb to the disease. While treatments for malaria are being pursued with rigor all around the world, it is predicted that a licensed malaria vaccine will not be available for at least a decade (Long, C. A., and S. L. Hoffman. 2002 Science 297:345-7). Malaria infection starts with the bite of an infectious mosquito which introduces sporozoites into the microvasculature. The results of early epidemiological studies suggest that antibodies to sporozoites play an important role in naturally acquired immunity to malaria, hi Gambia, the sera from villagers living in endemic areas contain antibodies to the surface membrane of sporozoites of P. falciparum (Nardin, E. H. et al. 1979 Science 206:597-9). The level of antibodies increased with age in parallel with acquired immunity, and in several adults high levels of antibodies were detected in the absence of parasitemia and of a serologically detectable immune response to the blood stage of the parasites (Nardin, E. H. et al. 1979 Science 206:597-9). The sporozoites express Circumsporozoite protein, an immunodominant surface antigen, which it uses for the attachment and invasion of liver cells (Cerami, C. et al. 1992 Cell 70:1021-33; Rathore, D. et al. 2002 JBiol Chem 277:7092-8). CS protein has been widely investigated as a vaccine candidate and currently a CS-based subunit vaccine is undergoing Phase II clinical trials (Nardin, E. H. et al. 2000 J Infect Dis 182:1486-96; Stoute, J. A. et al. 1997 N Engl J Med 336:86-91; Wang, R. et al. 1998 Science 282:476- 80). The protein is constituted of three modules viz., the amino terminus, central repeat region and carboxyl terminus region, of roughly equal sizes. Most of the CS-based vaccine studies currently being performed include either the complete or selected regions of carboxyl terminus domain (Nardin, E. H. βt al. 2000 J Infect Dis 182:1486-96; Stoute, J. A. et al. 1997 N Engl J Med 336:86-91; Wang, R. et al. 1998 Science 282:476-80). Segue to the Invention Numerous groups have investigated the molecular mechanism of action of CSP across various stages of the parasite lifecycle (Frevert, U. et al. 1993 JExp Med 177:1287- 98; Rathore, D. βt al. 2002 J Biol Chem 277:7092-8; Sinnis, P. et al. 1994 J Exp Med 180:297-306). We have recently demonstrated that the amino terminus of the proteins plays a crucial role in the pathogenesis process by helping the sporozoite in the attachment and invasion of liver cells (Rathore, D. et al. 2002 JBiol Chem 277:7092-8). We have now investigated the immunological recognition of the amino terminus and demonstrate that antibodies against a 21 amino acid segment of the amino teraiinus can successfully prevent the invasion of liver cells by the sporozoites, which finding has important implications for malaria control. Summary of the Invention The present invention relates to a polypeptide comprising a sequence as shown as the Peptide P6-identified epitope domain from amino acid 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 - 113, 114, 115, 116, or 117 of the Circumsporozoite protein, or allelic variant, functional equivalent, derivative, homologue, or analogue thereof, that reacts with antibodies that inhibit liver cell invasion by P. falciparum, substantially in isolation from sequences naturally occurring adjacent thereto in the Circumsporozoite protein, and related polynucleotides, vectors, host cells, methods, vaccines, antibodies, molecular decoys and kits. Brief Description of the Drawings Figure 1. Schematic representation of CS protein and the synthesized peptides spanning the amino terminus region. Figure 2. Antibody titers against the amino terminus region of CS protein in mice. Figure 3. Antibodies against the amino terminus of CS protein can prevent invasion of liver cells by P. falciparum sporozoites. Pooled anti-CS serum was used for this experiment. Figure 4. Identification of immunogenic segments in the amino terminus region of CS protein. Equimolar amounts of each of the seven peptides were coated onto an ELIS A plate followed by the addition of anti-N terminus CS serum. A value shown here is the mean absorbance from individual animals. Figure 5. Epitope mapping within the 21 amino acid peptide (P6). Equimolar amounts of the truncated peptides were coated onto an ELISA plate followed by the addition of anti-amino terminus CS serum at a dilution of 1:100. Each panel represents serum from a single animal. Figure 6. SDS-PAGE Analysis of recombinantly expressed and purified CS protein and its mutant. Figure 7. Binding analysis of K104L and its comparison to umnutated P. falciparum CS protein. Filled Circles represent the unmutated protein. Open circles represent protein K104L.

Brief Description of the Sequences

Figure imgf000005_0001

Detailed Description of the Preferred Embodiment Introduction Circumsporozoite protein is a Plasmodium sporozoite surface antigen and a widely experimented malaria vaccine candidate. We have investigated the role of the amino terminus of this protein which is immunologically inert when presented to the immune system as part of the full protein. A P. falciparum CS polypeptide encoding the 93 amino acids (AA 25-117) representing the amino terminus was prepared recombinantly and used to immunize Balb/c mice. Antibodies were readily obtained and they completely prevented P. falciparum sporozoites from invading liver cells. Hence the antigen is somehow hidden or unreactive when presented as part of the full protein but reactive in isolation. The antibodies to the amino terminal region recognized native CS protein and blocked sporozoite invasion of human hepatoma cells. Subsequent epitope mapping experiments revealed a 21 amino acid configurational epitope that is recognized by the immune system and is easily synthesized by existing technology.

Results Recognition of N-terPfCSP by Balb/c mice: Six animals were immunized with the recombinantly expressed protein representing the anino terminus of P. falciparum Circumsporozoite protein (N-terPfCSP) (Figure 1). The serum samples were collected at regular intervals and screened for anti-CS immune responses. Antibodies against the N- terPfCSP could be detected after the first immunization. On subsequent boosting, anti- N- terPfCSP antibody titers improved significantly. Figure 2 depicts the recognition of N- terPfCSP in serum from individual animals. Semm from each animal recognized the antigen in a dose dependent manner. Potent anti- N-terPfCSP antibody titers were generated as the antigen was recognized even at 1:100,000 dilution of the serum. The recognition was specific for N-terPfCSP as (i) serum did not recognize a polypeptide representing the carboxyl tenninus of PfCSP (ii) normal mouse serum did not recognize N- terPfCSP. Anti-N-terPfCSP antibodies inhibited liver cell invasion by P. falciparum sporozoites. As antibodies efficiently recognized recombinantly expressed N-terPfCSP, so we investigated the potential of these antibodies in inhibiting the invasion of liver cells by live P. falciparum sporozoites. Anti-N-terPfCSP antibodies were successful in preventing the invasion of liver cells in a dose dependent manner (Figure 3). At the two dilutions

(1:100, 1:400) of anti-serum investigated, sporozoite invasion was inhibited by 90 and 80% respectively. This inliibitory activity was comparable to the inhibitory activity of anti-P. falciparum repeat region monoclonal antibody, which at two concentrations (100 μg and 25 μg/ml) also inhibited the invasion by 92 and 77% respectively. Normal mouse serum, used as control did not show any inhibition of invasion. Identification of the recognized epitope. To identify the epitope(s) recognized by antibodies, seven overlapping peptides spanning the entire N-terPfCSP were synthesized (Figure 1). These peptides were used as antigens in an ELISA to identify the peptide recognized by the anti-N-terPfCSP antibodies. A 21 amino acid Peptide P6 representing amino acids 93 to 113 was recognized by the antibodies. Peptide P7 (amino acids 99 to 117), which represents an overlap of Peptide P6 was also recognized though its recognition was reduced by more than 50% (Figure 4). Fine mapping of the recognized epitope. To identify the exact epitope, sub peptides of different sizes from 9 to 18 amino acids in length representing the peptide P6 were synthesized and used as an antigen in ELISA. Deletion of as little as three amino acids from either the amino terminus or the carboxyl terminus lead to a 50% decrease in the recognition of the epitope by the antibodies (Figure 5, Table I). Any subsequent shortening of the peptide lead to a near total loss of epitope recognition. This finding indicated that the epitope is located in the P6 peptide and the tertiary structure of the epitope is of importance for the recognition of the peptide by the antibodies. Discussion An effective vaccine against malaria is a long standing dream of malaria researchers and numerous efforts are currently underway towards that goal. Efforts to develop a sporozoite vaccine were started with the observation that mice immunized with radiation- attenuated P. berghei sporozoites were protected against challenge with infective, nonnal P. berghei sporozoites (Nussenzweig, R. S. et al. 1967 Nature 216:160-2). The subsequent demonstration that humans are biologically capable of generating a protective immune response to malarial sporozoites provided impetus for subsequent efforts in malaria vaccine development (Clyde, D. F. 1990 Bull World Health Organ 68:9-12). With the identification of Circumsporozoite protein (Dame, J. B. βt al. 1984 Science 225:593-9; Ozaki, L. S. et al. 1983 Cell 34:815-22), attention focused on the creation of specific synthetic or recombinant subimit molecules that would induce a potent immunity to sporozoites. Although it is currently unknown what immune mechanisms are required for sterile immunity in humans, a large body of evidence suggests that antibody-dependent immunity to sporozoites plays an important role in protection and high antibody titers may alone be sufficient for protection (Charoenvit, Y. et al. 1991 Science 251:668-71; Nardin, E. H. βt al. 2000 J Infect Dis 182:1486-96). Attempts to improve methods of inducing protective antibodies have therefore continued. In our quest to improve the current candidates we investigated the possibility of immunological targeting of amino terminus of Plasmodium falciparum CS protein to prevent the onset of malaria infection. We focused on the immunological recognition of the amino terminus due to our recent observation that this region contains the critical domain involved in binding and invasion of liver cells by the parasite (Rathore, D. et al. 2002 JBiol Chem 277:7092-8). Animals were immunized with a 93 amino acid peptide representing the N-terminus of CS protein and the antibody responses were investigated for their potential to recognize the antigen and their potential to inhibit the invasion of liver cells by P. falciparum sporozoites. All the immunized mice developed antibodies against the protein (Figure 2). The antibodies recognized native CS protein on the sporozoite surface and blocked sporozoite invasion of human hepatoma cells in vitro (Figure 3). The inhibitory activity was comparable to the inl ibition with anti-repeat monoclonal antibody and the full length protein, indicating that the anti-N-terPfCSP antibodies are equally effective in preventing an infection. Inliibition of sporozoite invasion is powerful evidence that these antibodies can protect against the natural infection. The antibody binding site was subsequently mapped to a 21 ar ino acid region of the protein from amino acids 93 to 113 (Figure 4). To identify the exact epitope, deletion peptides were synthesized and tested in an ELISA for their recognition by the antibodies. All 21 amino acids of peptide P6 turned out to be important for the recognition of the epitope as deletion of as little as tliree amino acids from either end led to a significant decrease in recognition of the peptide (Figure 5). Deletion of more than tliree amino acids from either end led to near complete loss of recognition of the epitope by the antibodies. This indicated that (i) the recognized epitope has a strong tertiary confirmation (ii) the residues recognized by the antibodies are likely to be distributed within this peptide (iii) this region is present on the surface of the sporozoite. Previously, Brown et al. prepared a repeat-less P. falciparum CS protein and investigated its recognition in Thai adults who had developed acute falciparum malaria. They demonstrated that the repeat-less regions of CS protein are also recognized by the host immune system (Brown, A. E. et al. 1992 Am J Trop Med Hyg 47:440-5). Further investigation using small overlapping peptides revealed a patchy recognition in a segment preceding Region I (White, K., et al. 1993 Vaccine 11:1341-6). Our study has identified a 21 amino acid segment and shown that its discontinuous and confϊgurational nature is important for its recognition. The absence of the highly immunogenic central repeat region in our study could have facilitated the recognition of this epitope. Previously, Anders et al. has proposed a "Smoke screen hypothesis" which suggests that central repeat regions are "immunological chaff for the host immune system to divert the immune response from epitopes cracial for parasite survival (Anders, R. F. et al. 1986 Ciba Found Symp 119:164-83). These ingenious immune escape strategies that allow malaria parasites to evade naturally induced immunity could be one of the reasons why the parasite has been so successful in evading the host immune system. An interesting finding was that among more than 100 P. falciparum CS protein sequences from various isolates reported in GenBank, the epitope shows a single polymorphic site at position 104, where a lysine residue has been changed to either asparagine or threonine. Previously, variations in the CS sequence have been attributed to iinmune selection pressure (De La Cmz, V. F. et al. 1989 J Immunol 142:3568-75). The variation seen in the epitope sequence indicates that this epitope is under immune selection pressure, thus confirming its impact on immunological recognition. Further Description hi one embodiment the invention provides a polypeptide comprising the sequence substantially as shown as the Peptide P6-identifιed epitope domain from amino acid 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 - 113, 114, 115, 116, or 117 of the Circumsporozoite (CS) protein, or functional equivalents thereof, substantially in isolation from sequences naturally occurring adjacent thereto in the Circumsporozoite (CS) protein. The Sequence Chart shows the amino acid sequence of AA 93-113 of the

Circumsporozoite protein that is known as Peptide P6 (SEQ ID NO: 6.). The peptides of the invention may include SEQ ID NO: 6, or may be as large as AA 25-117 of the Circumsporozoite protein (SEQ ID NO: 8) itself. The Sequence Chart shows the amino acid sequence of two allelic variants, at position 104, where a lysine residue has been changed to either asparagine or threonine (SEQ ID NOS: 15 and 16 respectively). It is clear to those skilled in the art that other minor alterations can be made to the sequence of the Peptide P6-identified epitope domain without significantly altering the biological properties thereof, so as to result in a functional equivalent. For example, as well as allelic variants, functional equivalents might include those in which there are one or more conserved amino acid substitutions (i.e., the substitution of an amino acid for one with similar properties). Other substitutions which could be made are those which change an amino acid from the Peptide P6-identified sequences to that from a sequence that substantially preserves the structure of the epitope domain as judged by the reaction with antibodies that inhibit liver cell invasion by P. falciparum. Alternatively, or in addition, minor additions, deletions or trancations of the Peptide P6- identified sequences could be made. Other obvious functional equivalents are those the Peptide P6-identified epitope domains present in the CS protein of other species of Plasmodium (such as the four species lαiown to infect humans, or the mouse pathogen P. yoelii). The sequences encoding the Peptide P6-identified epitope domains may be inserted into plasmids. The resulting plasmid constructs are able to direct the expression of the Peptide P6-identified epitope domains optionally as fusion proteins. Thus in another embodiment the invention provides a vector comprising the sequence encoding the Peptide P6-identified epitope domains substantially in isolation from sequences naturally occurring adjacent thereto in the CS protein. Generally the vectors defined above are capable of expressing the Peptide P6- identified epitope domain sequences. They may be expressed in such a way that the Peptide P6-identified epitope domain sequences substantially retain the confonnation of the epitope domain as judged by the reaction with antibodies that inhibit liver cell invasion by P. falciparum. hi another embodiment the invention provides a method of producing the Peptide P6-identified epitope domain sequences, the method comprising inserting the vector defined above capable of expressing the Peptide P6-identified epitope domain sequences in a suitable host cell, growing the host cell and isolating the Peptide P6-identified epitope domain sequences so produced. h a further embodiment the invention provides a nucleotide sequence comprising the sequence encoding the Peptide P6-identified epitope domains or functional equivalents thereof substantially in isolation from other CS protein-encoding nucleotide sequences. Such functional equivalents include those sequences which whilst possessing a different nucleotide sequence, by virtue of the degeneracy of the genetic code, encode the same amino acid sequence (or an amino acid sequence containing conserved substitutions or minor deletions, additions or tnmcations) and those nucleotide sequences which hybridize to the complement of the nucleotide sequence of the invention. The embodiment also encompasses use of the nucleotide sequences to inhibit expression of CS protein (antisense, ribozyme, and interfering RNA molecules). Optionally, the Peptide P6-identified epitope domains are expressed as fusion proteins. Preferably the fusion protein should be one that allows for ease of purification such as fusion with glutathione S-transferase. Other such readily-purified fusion proteins are known to those skilled in the art. Alternatively, the fusion protein should be one that self- assembles into particles, such as described in PCT/US01/25625 to Apovia or PCT/GB92/00589 or PCT/GB95/01618 for plant particles, in which the malaria epitope is expressed on the surface of the particles. Alternatively, the epitope is attached to self-assembled particles, such as described in PCT/IB99/01925 to Cytos. hi a further embodiment the invention provides a host cell transfonned with the vector defined above. The transfonned host cell may be of bacterial, plant, fungal, insect or animal origin. Expression of the Peptide P6-identified epitope domain sequences in a fonn that substantially reproduces the antigenicity of the epitope domain as judged by the reaction with antibodies that inliibit liver cell invasion by P. falciparum may allow for the use of recombinant DNA- or chemically synthetic-derived material as a vaccine to induce a protective immune response against malaria, which is supported by the data herein. Thus in another embodiment the invention provides a vaccine comprising the sequence substantially as shown as the Peptide P6-identified epitope domains or functional equivalents thereof substantially in isolation from sequences naturally occurring adjacent thereto in the Circumsporozoite protein. Conveniently the vaccine comprises the epitope domain in the confonnation it adopts in the CS protein and is generally administered with an appropriate adjuvant (e.g., alum). Typically the Peptide P6-identified epitope domains will be carried in a physiologically acceptable canier and/or be fused to another immunogen. The present invention further relates to a transmission blocking vaccine against malaria. A transmission blocking vaccine prevents the transmission of P. falciparum to mosquito vector. The vaccines of the present invention can include other malarial antigens. In another embodiment the invention provides a method of treating a human body by administering a vaccine comprising the sequence substantially as shown as the Peptide P6-identified epitope domains or functional equivalents thereof substantially in isolation from sequences naturally occuning adjacent thereto in the Circumsporozoite protein. The present invention also relates to methods of preventing transmission of malarial infections. When given as a series of administrations, inoculations subsequent to the initial administration are given to boost the immune response and may be refened to as booster inoculations. Additional Description Further to the above, the tenn "polypeptide" is used in a general sense to cover molecules ranging from a few amino acid residues to several amino acid residues. Furthennore, the term "peptide" is considered herein to cover a polypeptide. The peptides of the present invention may be refened to as "CS peptides" for peptides of Circumsporozoite protein and are those that cany a conformational epitope or portion thereof from CS protein. The CS peptides of the present invention may be recombinant, synthetic or fragment parts, derivatives, analogues or homologues of the naturally occurring CS protein and, hence, reference to "CS peptides" includes all such molecules. The conformational epitopes or parts thereof are said herein to be "carried" on the CS peptides of the present invention. This includes CS peptides having a particular amino acid sequence which constitutes a confonnational epitope or which contributes to a particular tertiary structure facilitating same. In any event, the presence of the conformational epitope of the peptide is readily determined as herein described. The CS peptides of the present invention may comprise an amino acid sequence exactly conesponding to a sequence in a region of the naturally occuning CS protein or may contain single or multiple amino acid substitutions, deletions and/or additions to the naturally occuning sequence. Conveniently, the CS peptides of the present invention are prepared by recombinant means and techniques for making mutations at predetermined sites in DNA having lαiown or partially lαiown sequence are well lαiown and include, for example, M13 mutagenesis. Other suitable techniques as described in Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY and include random mutagenesis. Another embodiment of the present invention, therefore, provides a nucleic acid isolate comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a CS peptide or precursor fonn thereof. The sequence of the nucleic acid may conespond substantially to the naturally occuning DNA sequence of P. falciparum or may contain single or multiple nucleotide substitutions, deletions and/or additions thereto. The nucleic acid may encode a single CS peptide or a repeated series of the same CS peptide for subsequent cleavage for into individual peptides. The nucleic acid may also encode a series of different CS peptides. By "precursor" is meant any polypeptide or peptide from which the CS peptides can be derived and include fusion proteins. Advantageously, the nucleic acid molecule is in a vector such as an expression vector capable of expression in a prokaryotic and/or eukaryotic host, i a particularly prefened embodiment, the nucleic acid isolate is in a naked DNA plasmid or attenuated live vector. Such a plasmid or vector is useful for delivery of a CS peptide in vivo. Advantageously, the CS peptides are a biologically pure preparation meaning that they have undergone some purification away from other proteins and/or non-proteinacous material. The purity of the preparation may be represented as at least 40% of the peptide, preferably at least 60%, more preferably at least 75%, even more preferably at least 85% and still more preferably at least 95% relative to non-CS peptide material as detennined by weight, activity, amino acid identity or similarity, antibody reactivity or other convenient means. A preparation of a CS peptide of the present invention which has undergone at least some purification is refened to herein as "isolated" or an "isolate". The CS peptides of the present invention are non-naturally occuning since they are non-full length molecules of the CS protein and/or have undergone some purification and/or are recombinant or synthetic. Amino acid insertional derivatives of the CS peptides of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table A. TABLE A Suitable residues for amino acid substitutions

Figure imgf000014_0001

Where a CS peptide is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-2 amino acid residues and deletions will range from about 1-2 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e., a deletion of two residues or insertion of two residues. The CS peptides contemplated herein may also be chemically synthesized such as by solid phase peptide synthesis or may be prepared by subjecting the CS protein to hydrolysis or other chemically disruptive processes to produce fragments of the molecule. Alternatively, the peptides are made by in vitro or in vivo recombinant DNA synthesis, hi this case, the peptides may need to be synthesized in combination with other proteins and then subsequently isolated by chemical cleavage or the peptides or polyvalent peptides may be synthesized in multiple repeat units. Furthermore, multiple antigen peptides or polyvalent peptides could also be prepared according to Tam et al. (1988 PNAS USA 85:5409-5413). The selection of a method of producing the subject peptides will depend on factors such as the required type, quantity and purity of the peptides as well as ease of production and convenience. The use of these CS peptides in vivo or in a vaccine may first require their chemical modification since the peptides themselves may not have a sufficiently long serum and/or tissue half-life. Such chemically modified CS peptides are refened to herein as "analogues". The tenn "analogues" extends to any functional chemical equivalent of the CS peptides of the present invention characterized by its possession of one or more conformational epitopes of the CS protein of P. falciparum. The tenn "analogue" is also used herein to extend to any amino acid derivative of the peptides as described above. Analogues of CS peptides contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers, and other methods which impose confonnational constraints on the peptides or their analogues. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzyl tion of amino groups with 2, 4, 6, trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 '-phosphate followed by reduction with NaBH4. The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a conesponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; perfonnic acid oxidation to cysteic acid; fomiation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsuιphonic acid, phenyl mercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, maybe altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-

3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. Crosslinkers can be used, for example, to stabilize 3D confonnations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n = 1 to n = 6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides could be conformationally constrained by, for example, incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Cα and Cβ atoms of amino acids and the fomiation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C tenninus. The present invention, therefore, extends to isolated, recombinant and/or synthetic peptides or polypeptides and amino acid and/or chemical analogues, homologues, derivatives, fragments and parts thereof conesponding to one or more regions of the CS protein acting as a conformational epitope. All such molecules are encompassed by the tenn "CS peptide". The prefened CS peptides of the present invention cover the Peptide P6-identified epitope domains substantially in isolation from sequences naturally occurring adjacent thereto in the Circumsporozoite protein, including any derivatives or analogues as contemplated above as well as multiple repeats and/or combinations of these sequences. The peptides of the invention may include AA 93-113 of the Circumsporozoite protein (SEQ! TD NO: 6), or may be as large as AA 25-117 of the Circumsporozoite protein (SEQ ID NO: 8) itself. As hereinbefore stated, however, the present invention extends to equivalent regions of other strains of Plasmodium. The present invention also extends to peptides and polypeptides having at least 40%, preferably at least 55%, more preferably at least 65%, still more preferably at least 75% and even more preferably at least 80-85% or at least 90-95% identity to the naturally occuning "equivalent" sequence. By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those tenninal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 95% identical to a reference amino acid sequence can be detennined conventionally using lαiown computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 5371 1). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in identity of up to 5% of the total number of amino acid residues in the reference sequence are allowed. The present invention further contemplates a method for vaccinating a human subject against P. falciparum infection comprising administering to said subject an immunogenically effective amount of a peptide comprising a conformational epitope of CS protein of P. falciparum or a part, fragment, derivative, homologue or analogue thereof for a time and under conditions sufficient for said immunity to develop or at least partially develop. Such a peptide has the same meaning as set forth above. Generally, this embodiment of the present invention can be accomplished by a vaccine composition. Accordingly, another embodiment of the present invention contemplates a vaccine useful in the development of immunity to P. falciparum and in particular for stimulating B- cell immunity to P. falciparum CS protein, said vaccine comprising a peptide can ing a confonnational epitope of CS protein of P. falciparum or a part, fragment, derivative, homologue or analogue thereof. The vaccine may also comprise a suitable adjuvant. Such a vaccine is particularly directed to the sporozoites phase of P. falciparum infection. Where the peptide is produced by recombinant means, the vaccine may be refened to as a "recombinant vaccine". If the peptide is produced by synthetic means, the vaccine may be refened to as a "synthetic vaccine". The vaccine may contain a single peptide type or a range of peptides covering different or similar B-cell epitopes. hi addition, or alternatively, a single polypeptide may be provided with multiple B-cell epitopes. The latter type of vaccine is refened to as a "polyvalent vaccine". The formation of vaccines is generally lαiown in the art and reference can conveniently be made to Remington's Phannaceutical Sciences, 17th ed., Mack Publishing Co., Easton,, Pennsylvania, USA. The present invention, therefore, contemplates a phannaceutical composition or vaccine comprising an immunity developing effective amount of CS peptide or its parts, fragments, derivatives, homologues or analogues and/or combinations thereof including other active molecules and one or more pharmaceutically acceptable earners and/or diluents. The vaccine may alternatively comprise a naked DNA plasmid or attenuated live vector encoding the CS peptide(s). The active ingredients of a phannaceutical composition comprising the CS peptide or a vector encoding same are contemplated to exhibit excellent therapeutic activity, for example, in the development of B-cell immunity to P. falciparum when administered in amount which depends on the particular case. For example, from about 0.5 μg to about 20 mg per patient or per kilogram of body weight of the patient per day, week, or month may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Depending on the patient or other conditions more prefened dosages comprise 10 μg to 10 mg, 20 μg to 5 mg or 100 μg to 1 mg per patient or per kilogram of body weight of the patient per administration. The active compound including naked DNA plasmid and attenuated live vector may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradennal or suppository routes or implanting (e.g., using slow release molecules). Depending on the route of administration, the active ingredients which comprise a CS peptide may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, due to the low lipophilicity of the CS peptides, these may potentially be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer CS peptides by other than parenteral administration, they may be coated by, or administered with, a material to prevent its inactivation. For example, CS peptides may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfuorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in- water CGF emulsions as well as conventional liposomes. The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative, to prevent the growth of microorganisms. The phannaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion, hi all cases the fonn must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The canier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof. When the CS peptides are suitably protected as described above, the active, compound may be orally administered, for example, with an inert diluent or with an assimilable edible canier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the fonn of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains an effective amount of CS peptide as hereinbefore described. Alternatively, where the active component is a naked DNA plasmid or attenuated live vector, a sufficient amount of CS peptide must be synthesized. The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cheny flavouring. When the dosage unit fonn is a capsule, it may contain, in addition to materials of the above type, a liquid canier. Various other materials may be present as coatings or to otherwise modify the physical fonn of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrap or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. As used herein "phannaceutically acceptable canier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well lαiown in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to fonnulate parenteral compositions in dosage unit fonn for ease of administration and uniformity of dosage. Dosage unit fonn as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical canier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail. The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable canier in dosage unit form as hereinbefore disclosed. A unit dosage fonn can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg includes 1.0 μg to 200 mg, 10 μg to 20 mg and 100 μg to 10 mg. hi the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and mamier of administration of the said ingredients. Still another embodiment of the present invention is directed to antibodies to the CS peptides. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occuning antibodies to the CS protein or may be specifically raised to the CS peptides. In the case of the latter, the peptides may need first to be associated with a canier molecule. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthennore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies and/or CS peptides of the present invention are particularly useful for immunotherapy and vaccination and may also be used as a diagnostic tool for malaria infection or for monitoring the progress of vaccination or therapeutic regima. For example, the CS peptides can be used to screen for naturally occuning antibodies to CS protein. Alternatively, specific antibodies can be used to screen for CS protein or CS peptides. The latter would be important, for example, as a means for screening or purifying CS peptides made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are lαiown in the art and include, for example, sandwich assays and ELISA. It is within the scope of this invention to include any second antibodies

(monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of CS protein but particularly those regions covered by a CS peptide. Both polyclonal and monoclonal antibodies are obtainable by immunization with the CS peptide and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less prefened but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of CS peptide, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent tecliniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favored because of the potential heterogeneity of the product. The use of monoclonal antibodies in an immunoassay is particularly prefened because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by tecliniques which are well lαiown to those who are skilled in the art (see, for example Douillard and Hoffman 1981 Basic Facts about Hybridomas, in Compendium of Immunology Vol H, ed. by Schwartz; Kohler G and Milstein C 1975 Nature 256: 495-497; Kohler G and Milstein C 1976. European Journal of Immunology 6: 511-519). The presence of a CS peptide or more commonly the presence of CS protein (indicative of infection) may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay tecliniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target. Sandwich assays are among the most useful and commonly used assays and are favored for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow fonnation of an antibody- antigen complex, a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing lαiown amounts of antigen. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well lαiown to those skilled in the art, including any minor variations as will be readily apparent, hi accordance with the present invention the sample is one which might contain antibodies and include serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fennentation fluid and supernatant fluid such as from a cell culture. hi the typical forward sandwich assay, a first antibody having specificity for the CS peptide or CS protein, or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking, covalently binding or physically adsorbing. The polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g., 2-40 minutes) and under suitable conditions (e.g., 25°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the antigen. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antigen. An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to fonn a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule. By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes) and chemiluminescent molecules. h the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the conesponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the cliromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody antigen complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody- antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like. Alternately, fluorescent compounds, such as fluorecein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody- antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest, hnmunofluorescence and EIA tecliniques are both very well established in the art and are particularly prefened for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bio luminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the above assays, for example, to screen for specific antibodies in a biological sample using CS peptides. All such variations are encompassed by the present invention. Furthermore, the CS peptides of the present invention may be packaged in kit form, for example, to conduct an antibody detection assay. The kit comprises a compartment adapted to contain one or more CS peptides and may further comprise in the same or different compartments the reagents for the antibody detection assay. hi an alternative embodiment, CS peptides are used to select monoclonal antibodies, for example, by using the phage display technology (See, Barbas, CF. et al. 1991 PNAS USA 88:7978-82). These can be human or humanized monoclonal antibodies. If administered by passive immunization, these antibodies to CS peptides would bind to the antigenic site of the CS protein (or sporozoites) and thus prevent liver invasion tlirough the hepatocyte receptor. Human chimeric and humanized antibodies of various predetermined specificities are engineered cunently (See, e.g., Presta, L.G. 1992 Curr. Op. Struct. Biol. 2:593-596; and Burton, D.R., 1992 Hospital Practice 27:67-74 and references cited in each; see also Barbas, CF. et al. 1991. PNAS USA 88:7978-82). The amount of monoclonal antibody administered should be sufficient to achieve a blood level ranging from about 1 to about 10 mg/ml. Yet another embodiment of the present invention is directed to molecular decoys. Molecular decoys are molecules that mimic a ligand binding site and compete in the animal's body with natural binding sites, thus acting as decoys. The present embodiment is applicable to CS peptides that inhibit the binding of the CS protein (and entire sporozoites) by competing for receptor sites on hepatocytes. Such CS peptides should possess substantial CS-binding inhibitory activity. Additionally, such CS peptides should be substantially non-inimunogenic in the system of the host. Such CS peptides are designed to be not substantially larger than the smallest size needed to retain the elements of the binding site of the CS protein for the hepatocyte receptor having an amino acid sequence selected from AA 25-117 of the Circumsporozoite protein of P. falciparum (SEQ HD NO: 8) or may be as large as AA 25-117 of the Circumsporozoite protein (SEQ ID NO: 8) itself. Such peptides need to be soluble in aqueous medium. Suitable CS peptides containing essential parts, or the entirety, of SEQ ID NO: 8 can be easily identified using one or more of the above-described assays and the overlapping peptide method, which is a peptide screening technique well-known in the art and no more than routine experimentation. With reference to SEQ HD NO: 8, peptides formed by omitting progressively one-by-one C-tenninal or N-terminal amino acids from AA 25-117 of the Circumsporozoite protein of Plasmodium and different malarial species can be tested for CS-binding inhibitory activity. It has already been determined that Peptide P6 is important (SEQ ID NO: 6). Stractural and chemically functional mimetics of the peptides above are also within the scope of the present invention. Methods of preparation of such mimetics are described, for example, in Yamazaki et al. 1991 Chirality 3:268-76; Wiley et al. 1993 Medicinal Research Reviews 13:327-84; Gurrath et al. 1992 Eur. J. Biochem 210:991-21; Yamazaki et al. 1991 Int. J. Peptide Protein Res. 37:364-81; Bach et al. 1991 Int. J. Peptide Protein Res. 38:314-23; Clark et al. 1989 J. Med. Chem. 32:2034-36; Portoghese 1991 J. Med. Chem. 34:1715-20; Zhou et al. 1992 J Immunol. 149:1763-69; Holzman et al. 1991 J. Protein Chem. 10:553-63; Masler et al. 1993 Arch. Insect Biochem. and Physiol. 22:87- 111; Saragovi et al. 1992 Biotechnology 10:773-78 ; Olmsted et al. 1993 J Med. Chem. 36: 179-80; Malin et al.1993 Peptides 14:47-51; and Kouns et al. 1992 Blood 80:2539-47. Such mimetics are typically non-peptide compositions that maintain the activity of the conesponding peptides because of stractural and/or chemical functionality similarities. Among the advantages of mimetics are their relative lack of antigenicity and their ability to withstand degradation to which peptides are susceptible. CS peptides or peptidomimetics can be administered to malaria susceptible subjects, for example intravenously, at sufficiently high concentrations to compete effectively with a challenge with sporozoites or to attenuate the severity of infection. These concentrations can be determined by means lαiown to one of ordinary skill in the art. For example, optimum concentrations can be established using serially diluted preparations of the peptide in connection with a suitable testing procedure. Prefened concentrations range from about 10 to about 100 mg in a human. Suitable vehicles for administration include, but are not limited to, isotonic saline. hi addition to the decoyants of the present invention, the pharmaceutical compositions may contain suitable phannaceutically acceptable earners comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used phannaceutically. Preferably, the preparations, particularly those which can be administered by injection, contain from about 0.1 to 99 percent, and preferably from about 25 to 85 percent by weight, of the active ingredient, together with the excipient. Any conventional route of administration may be used for the decoyants of the present invention. Although the prefened mode of administration is by injection, e.g., intravenously, intradermally, intraperitoneally, etc, they may also be administered orally, by suppository or by any other route. The pharmaceutical preparations of the present invention are manufactured in a manner which is itself lαiown, for example, by means of conventional mixing, dissolving, or lyophilizing processes. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble fonn. hi addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. The decoyants of the present invention may also be administered in the form of liposomes, phannaceutical compositions in which the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active ingredient may be present both in the aqueous layer and in the lipidic layer, or, in any event, in the non-homogeneous system generally lαiown as a liposomic suspension. EXAMPLE 1 Recombinant protein expression and purification. DNA encoding the 93 amino acids immediately following the N-tenninus signal peptide of Plasmodium falciparum Circumsporozoite protein (amino acids 25-117, Accession No. AAA29524) was cloned in a T7 promoter based E. coli expression vector. The construct was expressed in BL21 strain of Escherichia coli and the recombinant protein was secreted into the periplasm. The periplasmic fluid containing the recombinant protein was loaded onto a heparin-sepharose affinity column and the bound CS protein was eluted using a salt gradient. The fractions containing the recombinant protein were pooled and the protein was purified to apparent homogeneity using gel filtration chromatography. Peptide synthesis and purification. Peptides representing the amino tenninus region of CS protein were synthesized using FMOC (9-fluorenylmethyloxy-carbonyl group) chemistry on solid phase resins on chlorotrityl resins. Subsequent to synthesis the peptides were purified by reverse phase chromatography by a gradient of 0-80% acetonitrile in 0.1% TFA. Authentication was performed by Electro spray mass spectrometry using an Agilent 1100. Immunization of animals. Balb/c mice were intra-peritoneally immunized with 10 μg of recombinant CSP in complete freunds adjuvant. Three weeks after first immunization, the animals received a second dose (10 μg) of the protein in incomplete freunds adjuvant. The animals were bled two weeks after the last immunization and serum samples were collected. Sporozoite invasion assay. HepG2 (Human hepatoma) cells were collected, washed and resuspended in complete MEM. They were subsequently plated at a density of 50,000 cells /0.3 ml in ECL coated Glass Labtek glass slides and incubated overnight at 37°C in a C02 incubator. Plasmodium falciparum (strain NF54) sporozoites were obtained from the salivary glands of An. stephensi mosquitoes using a DE 52 cellulose column as described by Ozaki et al (1983 Cell 34:815-22). Next day, the medium was removed and 50 μl of diluted serum was added per well (in triplicates). This was immediately followed by the addition of 20,000 sporozoites in 50 μl of medium to each well. Final serum dilutions were 1:100 and 1:400. Two concentrations (100 μg/ml, 25 μg/ml) of a monoclonal antibody directed against the central repeat region of CS protein (NFS1), was used as control. The sporozoites were allowed to invade liver cells for tliree hours followed by the washing of cells with Phosphate buffered saline at pH 7.4. Subsequently, the cells were fixed with cold methanol. Sporozoites were visualized by immunostaining by using NFS1 as primary antibody and anti-mouse IgG-peroxidase conjugate. Diaminobenzidine was used as substrate. The slides were mounted with Paramount and intracellular sporozoites were identified and counted. Percentage inhibition of invasion was calculated with the following fonnula [(Control-test)/control] x 100. ELISA. Equimolar amounts of peptides in 10 mM carbonate-bicarbonate buffer

(pH 9.6 were coated onto the wells of Immunolon 4 microtiter plates and incubated at 37°C for two hours. Uncoated sites were blocked with 1% non-fat dry milk in 50 mM Tris- buffered saline pH 7.4. Different dilutions of anti-CS serum were added to the wells and the plate was incubated at 37°C for one hour. Unbound reagents were removed by washing the wells with 50 mM Tris-buffered saline pH 7.4, containing 0.1% Tween-20, followed by addition of anti-mouse alkaline phosphatase conjugate and incubation for 60 minutes at 37°C The wells were thoroughly washed to remove unbound conjugate and the bound antibodies were seen using p-nitrophenyl phosphate as substrate. The absorbance was measured at 405 nM.

Table I. Truncated P6 peptides and recognition by anti-N terminus CS antibodies

Peptide AA Sequence Absorbance Decrease (%) P6 21 DIORDGNNEDNEKLRKPKHKKL 0.781 + 0.11 - 6N3 18 DGNNEDNEKLRKPKHKKL 0.345 + 0.05 55.8 6N6 15 NEDNEKLRKPKHKKL 0.270 + 0.14 65.4 6N9 12 NEKLRKPKHKKL 0.020 + 0.01 97.4 6C3 18 DKRDGNNEDNEKLRKPKH 0.320 + 0.11 59.0 6C6 15 DKRDGNNEDNEKLRK 0.080 + 0.02 89.7 6C9 12 DKRDGNNEDNEK 0.030 + 0.01 96.1

EXAMPLE 2 Introduction Previously, we have shown that antibodies recognizing P6, a 21 amino acid peptide can block the entry of P. falciparum sporozoites into liver cells. This indicated that epitope could be serving as a binding ligand, which the parasite utilizes for liver cell invasion. We evaluated the sequence of this peptide for polymorphism by BLAST analysis against available P. falciparum CS sequences in the GenBanlc. A few isolates from Asia and Africa showed polymorphism, which was restricted to amino acid at position 104 encoded as lysine. Lysine residue was either changed to Asparagine (N) (Escalante, A.A et al. 2002 Mol Biochem Parasitol 125:83-90) or Threonine (T) (del Portillo, H.A. et al. 1987 Mol Biochem Parasitol 24:289-294). On hepatocytes, CSP interacts with heparan sulfate proteoglycans (HSPG) expressed on their surface. This interaction is electrostatic in nature and has been proposed to occur between positively charged (lysine, arginine) and polar residues of CSP and the negatively charged sulfate and carboxylate ions of heparan sulfate. If involved, lysinel04 and its substituents (asparagine and threonine), all polar amino acids, will retain their capacity to interact with the host receptor as all tliree residues have known heparin-interaction capabilities (Schlessinger, J. et al. 2000 Mol Cell 6:743-750). This selective substitution suggested that the parasite has a limited degree of freedom for replacing lysine 104 and the residue (and the peptide P6) could be playing an important role in the biology of the parasite. We investigated this possibility, by designing a mutant CS protein, where K104 was converted to leucine, by site directed mutagenesis. Materials and Methods Construction, Expression and Purification of K104L. Plasmid pCSl encoding the native CS protein sequence under the control of a T7 promoter was used as template to introduce a single amino acid change using QuikChange, a PCR-based site directed mutagenesis kit (Stratagene, CA)as previously described (Rathore, D. & McCutchan, T.F. 2000 Infect Immun 68:740-743; Rathore, D. & McCutchan, T.F. 2000 PNAS USA 97:8530- 8535). This gave rise to plasmid pK104L. The authenticity of the mutant constructs was verified by DNA sequencing. BL21 strain of E. coli was used for the expression of plasmid pK104L. The plasmid was transformed and the cells harboring the plasmid were selected on an ampicillin containing culture plate. The culture was grown in a shake flask and was induced with IPTG. Three hours after induction, the cells were harvested by centrifugation and the periplasmic fluid, containing the recombinant protein, was isolated. The periplasmic fluid was loaded onto a heparin sepharose column and the purified protein was isolated by a linear salt gradient. Relevant fractions, containing the recombinant protein, were pooled, concentrated using a centricon and the protein was purified to homogeneity by gel filtration chromatography. Binding activity of CS protein mutant. HepG2, a hepatoma human cell line was grown in MEM medium, supplemented with 2 mM glutamine, 10% heat-inactivated fetal bovine serum, penicillin (100 IU/ml) and streptomycin (100 μg/ml). The assay was performed as previously described (Rathore, D. et al. 2002 JBiol Chem 277:7092-7098). Briefly, cells at a density of 100,000 cells per well were plated in 96 well plate 36 hours before the experiment. The cells were fixed with 4% parafonnaldehyde followed by blocking with Tris-buffered saline containing 1% bovine seram albumin. Recombinantly expressed CSP mutant at various concentrations was incubated with the cells for 1 h followed by a 30 minute incubation with a monoclonal antibody that recognizes the central repeat region of the protein. Unbound material was removed followed by the addition of anti-mouse alkaline phosphatase-coupled conjugate for 30 min. One mM 4- methylumbelhferyl phosphate was used as substrate, and fluorescence was measured in a fluorometer with excitation at 350 nm and emission at 460 mn. Results Expression and Purification of protein K104L. The construct was expressed in E. coli and the protein was purified to homogeneity by a two step column chromatography. Figure 6 depicts the purified K104L protein along with its unmutated version. The purified protein was used for investigating its biological activity on HepG2 cells, an established cell line model for studying the role of parasite proteins in the infectivity process. Role of Lysine 104 in host-parasite interaction. Binding analysis of K104L was perfonned on HepG2 cells and was compared with the binding activity of the non-mutated protein. The analysis revealed that the substitution of lysine 104 to leucine leads to a >70% decrease in binding activity of the protein to host cells (Figure 7), indicating that the residue plays an important role in host-parasite interaction. Conclusions This data further confirms that by targeting the peptide region 25-117 of P. falciparum CS protein, we can stop the parasite from invading liver cells and control malaria infection. Specifically, this experiment clearly defines the role of peptide P6 in the biology of the parasite and indicates that it plays a critical role in host-parasite interaction.

By designing methods to disrupt P6-Host cell interaction, we can effectively control the start of malaria infection.

***** While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the trae scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, refened to above, are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS: I. A polypeptide comprising a sequence as shown as the Peptide P6-identified epitope domain from amino acid 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 - 113, 114, 115, 116, or 117 of the Circumsporozoite protein, or allelic variant, functional equivalent, derivative, homologue, or analogue thereof, that reacts with antibodies that inliibit liver cell invasion by P. falciparum, substantially in isolation from sequences naturally occuning adjacent thereto in the Circumsporozoite protein. 2. The polypeptide of Claim 1 comprising a sequence as shown as AA 93-113 of the Circumsporozoite protein (SEQ ID NO:6), SEQ HD NO:15, or SEQ ID NO:16. 3. The polypeptide of Claim 1 comprising a sequence as shown as AA 25-117 of the Circumsporozoite protein (SEQ ID NO:8). 4. A polynucleotide sequence encoding at least one polypeptide comprising the polypeptide of Claim 1, 2, or 3. 5. A vector comprising the nucleotide sequence of Claim 4. 6. The vector according to Claim 5, which when inserted into a suitable host cell allows for the expression of the polypeptide of Claim 1, 2, or 3. 7. The vector according to Claim 6, wherein said polypeptide is expressed as a fusion protein. 8. The vector according to Claim 7, wherein said fusion protein self-assembles into particles. 9. A particle comprising the polypeptide of Claim 1, 2, or 3. 10. A method of making the polypeptide of Claim 1, 2, or 3 comprising the steps of introducing the vector of any one of Claims 5, 6, 7, or 8 into a suitable host cell; growing said host cell; and isolating the polypeptide so produced. I I. A host cell transformed with a vector according to any one of claims 5, 6, 7, or 8. 12. A vaccine suitable for use in the prevention and/or treatment of malaria due to Plasmodium, said vaccine comprised of the polypeptide of Claim 1, 2, or 3, said vaccine further comprising a physiologically acceptable canier.
13. The vaccine according to Claim 12, wherein said polypeptide is present as comprising a particle. 14. A method of preventing and/or treating a human body for malaria due to Plasmodium, comprising administering an effective amount of a vaccine according to Claim 10 or 11. 15. A vaccine suitable for use in the prevention and/or treatment of malaria due to Plasmodium, said vaccine comprised of the polynucleotide of Claim 4, said vaccine further comprising a physiologically acceptable canier. 16. A method of preventing and/or treating a human body for malaria due to Plasmodium, comprising administering an effective amount of a vaccine according to
Claim 15. 17. A molecular decoy comprising a polypeptide not substantially larger than the smallest size needed to retain the elements of the binding site of the CS protein for the hepatocyte receptor having an amino acid sequence selected from the polypeptide of Claim 1, 2, or 3, or peptidomimetic thereof. 18. A method of preventing and/or treating a human body for malaria due to Plasmodium, comprising administering an effective amount of a molecular decoy according to Claim 17. 19. A kit for detecting naturally occuning antibodies to Circumsporozoite protein comprising in compartmental form a compartment adapted to contain the polypeptide of Claim 1, 2, or 3. 20. A method for detecting naturally occurring antibodies to Circumsporozoite protein, said method comprising subjecting a sample of antibodies to be tested to the polypeptide of Claim 1, 2, or 3 and screening for binding.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009071613A2 (en) * 2007-12-06 2009-06-11 Glaxosmithkline Biologicals S.A. Vaccine
US20130022612A1 (en) * 2004-08-11 2013-01-24 New York University Methods and compositions for malaria prophylaxis
WO2015085140A1 (en) * 2013-12-05 2015-06-11 Leidos, Inc. Anti-malarial compositions
US9169304B2 (en) 2012-05-01 2015-10-27 Pfenex Inc. Process for purifying recombinant Plasmodium falciparum circumsporozoite protein

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0278941A1 (en) * 1987-01-30 1988-08-17 Smithkline Biologicals S.A. Expression of the P. falciparum circumsporozoite protein by yeast

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0278941A1 (en) * 1987-01-30 1988-08-17 Smithkline Biologicals S.A. Expression of the P. falciparum circumsporozoite protein by yeast

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HEPPNER D G ET AL: "SAFETY, IMMUNOGENICITY, AND EFFICACY OF PLASMODIUM FALCIPARUM REPEATLESS CIRCUMSPOROZOITE PROTEIN VACCINE ENCAPSULATED IN LIPOSOMES", JOURNAL OF INFECTIOUS DISEASES, CHICAGO, IL, US, vol. 174, no. 2, August 1996 (1996-08-01), pages 361 - 366, XP002048585, ISSN: 0022-1899 *
RATHORE DHARMENDAR ET AL: "Binding and invasion of liver cells by Plasmodium falciparum sporozoites. Essential involvement of the amino terminus of circumsporozoite protein", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 9, 1 March 2002 (2002-03-01), pages 7092 - 7098, XP002324709, ISSN: 0021-9258 *
WANG RUOBING ET AL: "Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine", SCIENCE (WASHINGTON D C), vol. 282, no. 5388, 16 October 1998 (1998-10-16), pages 476 - 480, XP002324708, ISSN: 0036-8075 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130022612A1 (en) * 2004-08-11 2013-01-24 New York University Methods and compositions for malaria prophylaxis
WO2009071613A2 (en) * 2007-12-06 2009-06-11 Glaxosmithkline Biologicals S.A. Vaccine
WO2009071613A3 (en) * 2007-12-06 2009-08-13 Joseph D Cohen Vaccine
US9169304B2 (en) 2012-05-01 2015-10-27 Pfenex Inc. Process for purifying recombinant Plasmodium falciparum circumsporozoite protein
US9849177B2 (en) 2012-05-01 2017-12-26 Pfenex Inc. Process for purifying recombinant plasmodium falciparum circumsporozoite protein
WO2015085140A1 (en) * 2013-12-05 2015-06-11 Leidos, Inc. Anti-malarial compositions
US9321834B2 (en) 2013-12-05 2016-04-26 Leidos, Inc. Anti-malarial compositions

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