Abstract:
Two proteins and their use as substrates for vaccines intended to initiate an immune response in a mammalian subject against infection with species of  Plasmodium  for use in the diagnosis of  Plasmodium  infection and for their use in the development of antimalarial drugs. This invention also relates to the diagnostic, isolation and purification assays based on these  Plasmodium  proteins. This invention further relates to immunological reagents, specifically antibodies directed against these  Plasmodium  proteins.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims priority to U.S. Provisional Application No. 60/361,282 filed Mar. 4, 2002. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    This invention relates specifically to two genes encoding  Plasmodium falciparum  proteins, methods for the detection of these and similar proteins located on the surface of  Plasmodium  infected mammalian cells, and vaccines for the protection against malaria in humans and non-human mammals. This invention further relates to the diagnostic, isolation and purification assays based on these  Plasmodium  proteins. This invention further relates to immunological reagents, specifically antibodies directed against these  Plasmodium  proteins.  
         DESCRIPTION OF THE PRIOR ART  
         [0003]    The disease Malaria is caused by infection with one of four species of  Plasmodium: P. falciparum, P. vivax, P. malariae  and  P. ovale. Plasmodium  parasites belong to the family  Apicomplexa  and are eukaryotic protozoan parasites that possess a complex life cycle which involves both an invertebrate host ( Anopheles  mosquito) and a mammalian host. The parasite life cycle includes direct inoculation into the mammalian host by the bite of an infected  Anopheles  mosquito which injects stages of the parasite known as “sporozoites”. The sporozoites rapidly invade cells of the liver by an active invasion process which is thought to involve attachment to the liver cells and which involves a cascade of processes which results in the parasite taken up residence inside a liver cell (hepatocyte) (Hollingdale, McCormick et al. 1998). The parasite undergoes asexual multiplication over a period of several days resulting in production of thousands of parasites which are released into the host circulation. These “merozoite” forms invade host cell erythrocytes (red blood cells) by an active process which involves attachment to the exterior surface of the erythrocyte, reorientation, and invagination (in folding) of the erythrocyte membrane until the parasite is completely enveloped by the erythrocyte (Preiser, Kaviratne et al. 2000). While inside the erythrocyte the parasite begins to grow using the erythrocyte hemoglobin as an energy source and divides into approximately one dozen additional parasites. During this growth phase, some of the  Plasmodium  proteins are exported to the surface of the erythrocyte and can be found associated with the erythrocyte membrane. Some of these proteins are thought to represent important targets for vaccine development as their location allows them exposure to the host immune system (Chen, Fernandez et al. 1998). Two models are often used to describe the development of immunity to malaria and as a tool for the development of new strategies for malaria vaccine development (Richie and Saul 2002):  
           [0004]    Irradiated Sporozoite Model  
           [0005]    Naturally Acquired Immunity (NAI)  
           [0006]    (a) The irradiated sporozoite model: This model involves immunizing volunteers via the bites of irradiated  Plasmodium -infected  Anopheles  mosquitoes. The parasites within the mosquitoes are damaged but not killed by the radiation, and thus constitute an attenuated whole organism vaccine. They are able to enter the blood stream of vaccinees while the mosquitoes feed, invade liver cells, and undergo limited development, but cannot progress to the pathogenic blood stages due to the attenuation caused by radiation. While undergoing development in the liver, these damaged parasites induce a strong protective immune response directed against liver stage parasites. As mentioned above, it appears that this strong protective immunity represents the sum of many immune responses directed at a variety of antigens derived from the whole organism attenuated sporozoite vaccine. When batches of irradiated, infected mosquitoes are allowed to feed on volunteers over a 6-month period, the level of immunity develops sufficiently to protect at least 95 percent of the human volunteers tested when subsequently challenged with intact parasites. The immunity lasts for at least 9 months and is not strain-specific (but does appear to be species-specific). If that level of immunity could be reproduced with a subunit vaccine, it would be considered very effective because all manifestations of disease would be prevented. Because this immunity is based on liver stage (pre-erythrocytic) immunity, it forms a model for pre-erythrocytic stage vaccines designed to completely prevent malaria infection.  
           [0007]    (b) The naturally acquired immunity (NAI) model: This model is based on studies of children and adults living in malaria-endemic areas. It has been noted that if children who live in malaria endemic areas survive and reach the age of 10, they remain susceptible to infection with malaria parasites, but do not develop severe disease or die of malaria. In other words, they are protected through acquired immunity against severe disease and death due to malaria infection. This immunity persists for the rest of their lives as long as they continue to live in the malarious area. They may continue to be re-infected with parasites, as shown in cleared-cohort studies, but their health will not be significantly affected by the parasites. NAI limits the number of parasites in the blood and reduces their clinical effect on the host. Because this immunity is based on blood stage antigens, it forms a model for erythrocytic stage vaccines designed to curtail disease and death, even if not preventing infection.  
           [0008]    It has been well established that protective immunity against malaria infection is mediated, in part, by circulating antibodies (Mohan and Stevenson 1998). Passive transfer of hyperimmune antibodies obtained from one geographical location can protect against malaria infection in other regions, indicating that the target antigens may be highly conserved among diverse parasite strains (McGregor 1963; McGregor and Wilson 1988). These conserved antigens, located on the surface of parasite-infected erythrocytes thus accessible to protective antibodies, are good vaccine candidates and yet to be identified. A conventional approach to identify surface antigens is to use hyperimmune sera from individuals living in endemic regions (Howard 1988; Fernaders 1998; Kyes 1999). Two surface antigens identified so far by this method, PfEMP1 proteins and Rifins, are highly variable and their roles in the humoral immune protection are still under investigation. In addition, the approach is limited to the identification of highly immunogenic or abundant molecules.  
           [0009]    The completion of  P. falciparum  genome sequencing project, combined with advanced proteomics technologies and bioinformatics tools, has allowed the profiling of expressed parasite proteins to be carried out in an unprecedented scale, with higher sensitivity and efficiency (Florens, Washburn et al. 2002). The advantage of MudPIT technology, a two-dimensional liquid chromatography coupled with tandem mass spectrometry, is its ability to analyze complex protein mixture, particularly, membrane protein mixture that is difficult resolve in other gel-based protein separation systems (Eng, McCormack et al. 1994; Washburn, Wolters et al. 2001).  
           [0010]    Development of vaccines against malaria is focused on the identification of parasite proteins found to be present at a particular stage of the parasites life cycle, the design and construction of a vaccine delivery system which is meant to stimulate the desired immune response against that identified protein and which is meant to eliminate, disable or interrupt the function of the parasite within the host(s).  
           [0011]    A key component of this vaccine strategy is the identification of proteins at particular stages of the parasite life cycle. Recently, an approach has been developed and applied to the identification of  Plasmodium  proteins from isolated stages of the parasite life cycle. This approach which employs microcapillary liquid chromatography coupled with tandem mass spectrometry has resulted in the identification of over 2,500  Plasmodium  proteins from several stages of the parasite life cycle (Florens, Washburn et al. 2002). Some of these proteins represent potential targets of new malaria vaccines.  
           [0012]    At present, there are no licensed vaccines against malaria. The most effective malaria vaccine that would result in sterilizing protective immunity would be directed toward eliminating the parasite while inside the liver cells. However, vaccines that are designed to reduce the number of circulating and sequestered parasites from the mammalian host blood stream would result in a substantial reduction in morbidity and mortality, especially in children and pregnant women living in areas of malaria transmission. This type of vaccine would mimic the naturally acquired immunity that develops over years of exposure to blood stage parasites living and circulating in the host blood stream. It would also be a vaccine which is directed toward parasite proteins expressed either by the circulating parasites before invasion into red blood cells, or to those parasite proteins expressed on the surface of the red blood cell. The most well characterized protein expressed on the surface of  P. falciparum  infected red blood cells, PfEMP1 (or variant surface antigen) has been shown actually to represent a large family of diverse proteins and has been shown to stimulate immune responses that can reduce parasite numbers in the circulation. The diversity of this protein within the parasite genome and its role in “antigenic switching” may limit its role in providing long-term protection against  P. falciparum.  There is a need to identify additional  Plasmodium  proteins on the surface of infected erythrocytes for the development of vaccines directed against these proteins. It is of further interest to develop diagnostic tests for the presence of  Plasmodium  infections in mammals. To date, the most reliable diagnostic test and the one that is the gold standard used in clinical laboratories is the examination of blood for the presence of parasites by Giemsa staining methods. This method, however, requires a skillful microscopist who has been trained in the identification of malaria parasites within red blood cells. In many areas of the world where malaria is highly endemic, there are an abundance of skilled microscopists who are adept at reading Giemsa stained blood films. However, in the US and other industrialized nations where malaria infection in humans is not abundant, misdiagnosis of malaria due to the absence of trained microscopists can result in a delay in providing adequate treatment and potential death in those infected. The development of a highly sensitive and reliable in vitro assay to detect the presence of  Plasmodium  in the blood would likely reduce the rate of misdiagnosis and likely result in prompt and appropriate treatment. The identification of parasite proteins expressed in the blood stage of  Plasmodium  would form the foundation for the development of a clinical assay for  Plasmodium  in humans and other mammals. Finally, development of new antimalarial drugs may be accelerated by the identification of  Plasmodium  parasite proteins and their association with biochemical and signal transduction pathways. Parasite proteins expressed at the surface of red blood cells may provide a link to parasite residing within to the external environment. These proteins may therefore represent components of a signal transduction pathway to which directed interruption either by drug or small molecule could result in the parasite receiving misinformation to its detriment and potential death.  
         SUMMARY OF THE INVENTION  
         [0013]    It is an object of this invention to identify two  Plasmodium falciparum  proteins expressed at the surface of infected erythrocytes in humans.  
           [0014]    It is another object of this invention to use these proteins singly or together as vaccines, either as native or recombinant proteins or peptides.  
           [0015]    It is another object of this invention to use the genes encoding these proteins as nucleic acid vaccines or in recombinant viruses, or other vaccine delivery systems whose intent is to generate an immune response in the recipient against these proteins.  
           [0016]    It is another object of this invention to use either the native or recombinant protein or peptide vaccines in combination with nucleic acid, recombinant viral vaccines or other delivery systems whose intent is to generate an immune response in the recipient against these proteins.  
           [0017]    It is another object of this invention to use these proteins or genes encoding these proteins to detect the presence of  Plasmodium  parasites in the blood or tissues of human or mammals. It is another object of this invention to use these proteins or genes encoding these proteins in the development of drugs or small molecule interventions designed to interrupt metabolic or signaling pathways in  Plasmodium.    
           [0018]    It is another object of this invention to identify the orthologous proteins or genes encoding these proteins from  Plasmodium  where the species is  P. vivax, P. ovale  or  P. malariae. These and additional objects of the invention are accomplished by identifying the presence of these proteins associated with the erythrocyte membrane in    Plasmodium  infected red blood cells or in the case of other species of  Plasmodium  ( P. vivax, P. ovale  or  P. malariae ) orthologous sequences based on sequence similarity comparisons using, for example, the computer program BLAST (Altschul SF et al) to identify proteins of similar primary amino acid sequence or genes of similar nucleic acid sequence. The detection of the proteins associated with the erythrocyte membrane is accomplished by the purification of erythrocyte membrane proteins from infected in vitro culture of  P. falciparum  using an affinity purification system and subjecting these purified proteins to liquid capillary/tandem mass spectrometry or multidimensional protein identification technology (MudPIT) to generate mass spectral patterns. These mass spectral patterns can be used to search computer databases for predicted mass spectral patterns of known or predicted proteins. When potential proteins are identified and represent  Plasmodium  proteins expressed in association with erythrocyte membrane, they are subjected to further verification of location by protein chemistry and immunological means. These means would include the production of protein-specific antisera in animals by immunization with native or recombinant protein, peptide, nucleic acid, recombinant virus or other means and the use of these antisera in immunolocalization by confocal microscopy, Immunofluorescence antibody testing, immunoelectron microscopy or other methods to localize the protein within or in association with the host cell. We have used these methods to identify two proteins from  Plasmodium falciparum  which are associated with the infected human erythrocyte. The proteins, designated PfSA1 for  Plasmodium falciparum  surface antigen 1 and PfSA2 for  Plasmodium falciparum  surface antigen 2 have been shown to be associated with the  P. falciparum  erythrocyte membrane but not from uninfected erythrocytes using antisera raised in mice to peptides derived from each protein by immunolocalization using confocal microscopy. We have further shown that these proteins are associated in part at the exterior surface of infected erythrocytes by demonstrating that exposure of whole infected erythrocytes to trypsin and chymotrypsin which digests proteins at the erythrocyte surface but not within the erythrocyte abolishes the reactivity of the mouse antisera to the infected erythrocytes and is further supported with the demonstration that inclusion of inhibitors to trypsin and chymotrypsin can prevent this abolished reactivity.  
           [0019]    It is also a feature and advantage of the inventive subject matter to provide potential new vaccine target antigens that would stimulate an immune response to  Plasmodium  infected erythrocytes and result in clearance from the body of these parasites, limit the parasite&#39;s ability to replicate inside the host and limit the clinical disease caused by the parasite or as the result of the parasite residing in the host and host cells.  
           [0020]    It is also a feature and advantage of the inventive subject matter to identify drugs or small molecules that would associate with or interact with these proteins causing an alteration in the parasite biological function and which would be deleterious to the survival of the parasite inside the host or interrupt the parasite life cycle.  
           [0021]    The foregoing and other features and advantages will become further apparent from the following detailed description of the presently preferred embodiments, when read in conjunction with the accompanying examples and made with reference to the accompanying drawings. It should be understood that the detailed description and examples are illustrative rather than limitative, the scope of the present invention being defined by the appended claims and equivalents thereof. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0022]    [0022]FIG. 1 is a cartoon diagram of the purification process of erythrocyte membranes using a combination of biotin and streptavidin and elution with guanidine.  
         [0023]    [0023]FIG. 2 is a figure demonstrating that the methods of purifying erythrocyte membranes are appropriate and will result in the proper identification of proteins previously demonstrated to be associated with the infected erythrocyte membrane.  
         [0024]    [0024]FIG. 3 is a figure demonstrating the specificity of the antisera raised against the PfSA1 and PfSA2 peptides.  
         [0025]    [0025]FIG. 4 is a figure of immunolocalization of PfSA1 and PfSA2 to the surface of  P. falciparum -infected erythrocytes by confocal microscopy in two of six strains of  P. falciparum  tested.  
         [0026]    [0026]FIG. 5 is a figure of immunolocalization of PfSA1 and PfSA2 to the surface of  P. falciparum -infected erythrocytes with  P. falciparum  Malayan Camp tested where the erythrocytes had been previously treated with trypsin and chymotrypsin and in another case where the erythrocytes has been treated with trypsin and chymotrypsin in the presence of an inhibitor of trypsin and chymotrypsin  
         [0027]    [0027]FIG. 6 is a sequence comparison of the protein sequence of PfSA1 from  P. falciparum  clone 3D7 against the PfSA1 sequences from three additional  P. falciparum  isolates (MC, R033 and 7G8).  
         [0028]    [0028]FIG. 7 is a sequence comparison of the protein sequence of PfSA2 from  P. falciparum  clone 3D7 against the PfSA2 sequences from three additional  P. falciparum  isolates (MC, R033 and 7G8). 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0029]    In accordance with the present invention, there is generally provided, two novel  Plasmodium falciparum  proteins that are expressed in association with infected human erythrocytes and these proteins are present in numerous additional strains of  P. falciparum  throughout the world. We have used the application of Multidimensional Protein Identification Technology (MudPIT) (Washburn et al) to analyze a mixture comprised of the  P. falciparum  parasitized red blood cell (PRBC) surface membrane proteins, and the identification and characterization of two novel conserved surface antigens, PfSA1 (SEQ ID NO:1) and PfSA2 (SEQ ID NO:2). In these experiments we first isolated and identified  P. falciparum  proteins from infected erythrocyte cultures, then raise antisera against peptide sequences from the resulting identified proteins, then confirmed the localization of the proteins near the infected erythrocyte surface, then demonstrated the protein localization on the surface of the infected erythrocytes and then determined the presence of these proteins and their variants in other  P. falciparum  isolates.  
         [0030]    In a first embodiment, the invention is directed to the production of a vaccine which contains the nucleic acid sequences (SEQ ID NO:3 and SEQ ID NO: 4) or amino acid sequences (SEQ ID NO:1 and SEQ ID NO:2) of either PfSA1 or PfSA2 or both.  
         [0031]    In a second, third and fourth embodiment this vaccine could be a recombinant protein, peptide vaccine, recombinant viral based vaccine or other vaccine delivery mechanism which when delivered by needle, needleless or ballistic injection into the body with or without adjuvants, excipients, carriers via intramuscular, intradermal, subcutaneous, intranasal, oral or other methods is designed to elicit a humoral immune response, cellular immune response or both in the human or animal in which the vaccine was administered.  
         [0032]    In a fifth embodiment of this invention, the vaccine could be a combination of two or more of the above vaccine delivery systems, for example the delivery of three doses of a PfSA1 DNA vaccine followed by a dose of a recombinant adenovirus expressing PfSA1. The immune response against these proteins delivery by any of the means listed above, would result in a decrease in the number of  Plasmodium  parasites in the body, the viability of  Plasmodium  parasites in the body and/or the clinical manifestations of  Plasmodium  parasite infection. The examples of vaccines listed here are illustrative and are not meant to be exclusive.  
         [0033]    In yet a sixth embodiment of this invention is the development of assays to detect  Plasmodium  parasites within the body. Antibodies are generated which react specifically with the PfSA1 or PfSA2 proteins and which would allow the development of an immunological detection assay. One example of how this would be accomplished would be to use these antibodies, alone or in combination, on biological samples taken from individuals who are suspected of being infected with  Plasmodium  parasites. These antibodies, for example, could be used in an Enzyme-Linked Immunosorbant Assay (ELISA) to detect the presence of PfSA1 or PfSA2 proteins in sera from patients, or in microscopic examination of blood films to detect parasites using a fluorescence-based readout. These examples are not meant to be comprehensive but only to illustrate potential uses of antibodies against PfSA1 and/or PfSA2.  
         [0034]    A seventh embodiment of this invention is directed to the development of assays to detect  Plasmodium  parasites within the body based on detection of nucleic acid sequences of PfSA1 and/or PfSA2. An example of this embodiment is the use of oligonucleotide primer sequences selected from the PfSA1 and/or PfSA2 gene sequence that if used in a polymerase chain reaction assay will amplify PfSA1 and/or PfSA2 DNA or cDNA and enable the detection of the parasites by the presence of this specific nucleic acid product by gel electrophoresis, hybridization methods, or other methods known to those of skill in the art.  
         [0035]    An eighth embodiment of this invention is directed to the identification of drugs or small molecules that can be used-as antimalarial compounds. An example of this would be the identification of a small molecule that is predicted to associate with the portion of either the PfSA1 or PfSA2 protein at the erythrocyte surface and interrupt the function of that protein with the result of causing a disruption in the  Plasmodium parasite function.    
         [0036]    The following examples are illustrative of preferred embodiments of the invention and are not to be construed as limiting the invention thereto.  
       EXAMPLE 1  
     Isolation of Proteins From  P. falciparum  Parasitized Erythrocytes  
       [0037]    In order to obtain ALL proteins on the surface of parasitized red blood cells (PRBCs), we developed a method to label the intact PRBCs with two non-permeable biotins, Sulfo-NHS-LC-Biotin and PEO maleimide activated Biotin, with binding specificity to lysine and cystine, respectively (FIG. 1). We chose the late trophozoite-early/schizonte stage (30-36 hours post invasion, named late trophozoite stage thereafter) for the labeling because 1) an extensive surface modification was observed at this developmental stage, 2) the PRBC membrane becomes more permeable at the later developmental stage (36-48 hours post invasion, named schizont stage thereafter), which would complicate data interpretation, and 3) though not accurately quantitative, our preliminary data indicated that the cells may shed surface proteins expressed earlier (FIG. 2). After extensive washes to remove the unbound biotin protein, cells were lysed and cell debris was washed again to remove soluble proteins. Subsequently, the cell membrane was dissolved and the dissolved proteins mixture was loaded onto a streptavidin column which retains labeled proteins via biotin. Hence, the mixture eluted from the streptavidin column was enriched with surface proteins and the complexity of the sample subject to MudPIT analysis was greatly reduced. Western blotting analysis using antibodies against known surface antigens was performed to verify the extraction method (FIG. 2). Recognition of PfEMP-1, Rifin, and CD36 by specific antibodies indicates that the method effectively extracted proteins on the surface of the PRBC. The use of late trophozoite for the MudPIT analysis was supported by the observations that 1) more protein is present in the preparation from late trophozoites (30-36h post invasion) than that from the schizonts; and 2) EBA-175, a component of microneme in merozoites expressed in mature schizonts/segments, was detected in schizont stage, indicating the alteration of the membrane permeability in schizont-infected erythrocytes. In addition, CD36 was only labeled by PEO-maleimide activated biotin, suggested the necessity in using two biotins with different specificities.  
       EXAMPLE 2  
     Identification of  P. falciparum  Proteins From the Purified Parasitized Red Cell Preparation  
       [0038]    The biotin-labeled fraction was digested with trypsin and endopeptidase C, and loaded onto biphasic microcapillary columns installed such as to spray directly into a ThermoFinnigan LCQ-Deca ion trap mass spectrometer equipped with a nano LC electrospray ionization source. Fully automated 12□step chromatography runs were carried out. SEQUEST was used to match MS/MS spectra to peptides in a sequence database combining  Plasmodium falciparum  and mammalian protein sequences (to account for contaminating host proteins). The validity of peptide/spectrum matches was assessed using the SEQUEST□defined parameters cross-correlation score (XCorr), Delta Cn valuer Sp rank and relative ion proportion. DTASelect (Eng, McCormack, et al 1994) was used to select and sort peptide/spectrum matches passing a conservative set of those parameters. Peptide hits from multiple runs were compared using CONTRAST (Eng, McCormack, et al 1994).  
         [0039]    Four surface protein samples, 2 labeled with lysine-specific Sulfo-NHS-biotin and 2 with cystine-specific PEO maleimide-activated biotin, were analyzed by MudPIT. Compiling peptide hits from those 4 independent samples, 623 unique proteins were confidently identified. Among those proteins, 371 were also found in the proteomic study of whole cell lysates from  P. falciparum  trophozoites-schizonts (Florens, Washburn, et al 2002). Differential analysis of the sequence coverage observed for those common proteins (i.e. number of peptides leading to protein identification) allowed us to distinguish between contaminating abundant trophozoite-schizont proteins and proteins specifically enriched in the biotin-labeled fractions.  
         [0040]    The proteins were selected for further characterization by the following criteria: 1) the presence of the signal peptide as predicted by SignalP; 2) the presence of transmembrane domain(s) as predicted by TAMP; 3) novel proteins whose function had never been characterized before; and 4) sequence conservation within multiple  P. falciparum  strains or/and cross  Plasmodium  ssp. More than 30 hypothetical proteins satisfied these criteria. Two proteins, denoted PfSA1 and PfSA2, from the 30 identified were selected for further characterization.  
       EXAMPLE 3  
     Bioinformatic Characterization of PfSA1 and PfSA2  
       [0041]    The informatics package contained within a suite of informatics computer programs on the website www.plasmodb.org were used to characterize the selected proteins. Gene model prediction used GlimmerM (Salzberg, Pertea et al. 1999). PfSA1 is a hypothetical acidic protein of 1297 amino acids with theoretical molecular weight (MW) of 154 kDa and isoelectricfocusing point (IP) of 5.14. It is encoded by a single copy gene 3885 nucleotides long, denoted PfC0435w, located on  P. falciparum  chromosome 3 (nucleotide positions 444174-448058) and has an orthologue in  P. knowlesi.    
         [0042]    PfSA2 is a hypothetical protein of 408 amino acids with theoretical MW of 49 kDa and IP 6.67. It is encoded by a single copy two exon gene near the telomeric region of chromosome 5 (nucleotide sequences 64605-64133 and 64332-65489). It does not have discernible orthologues in other organisms (BlastP cut-off E value of 10 −15 ). Both PfSA1 and PfSA2 are highly conserved in multiple strains of  P. falciparum  from various geographic locations (FIG. 6) suggesting their potential utility in vaccine construction.  
       EXAMPLE 4  
     Production of PfSA1- and PfSA2-Specific Antisera.  
       [0043]    Rabbit antisera were raised against synthetic peptides designed based on PfSA1 and PfSA2. The peptide sequence used for PfSA1 is NNSKFSKDGDNEDFNNKNDLYNPSDKLYNN (SEQ ID NO:5). The peptide sequence used for PfSA2 is YEIMHKEDESKESNQHNYKEGPSYEDKKNMYKE (SEQ ID NO:6). Two specific antibodies, denoted 108 and 112, recognized proteins corresponding to the theoretical MW of PfSA1 and PfSA2, respectively, in the whole cell lysate and the biotin-labeled fraction (FIG. 3).  
       EXAMPLE 5  
     Localizing the Expression of PfSA1 and PfSA2 to the Erythrocyte Membrane  
       [0044]    To confirm the surface location of the PfSA1 and PfSA2, we labeled the intact PRBC in suspension with purified IgG from antisera 108 and 112, followed by incubation with goat-anti-rabbit and chicken-anti-goat Alexa Fluor 488 as secondary and tertiary antibodies. Ethidium bromide was added to the incubation to stain the nuclei. The cells were allowed to adhere to cover slips pre-coated with polylysine, and examined by confocal microscopy. FIG. 4 demonstrates the localization of both antigens on the surface of PRBC. The antibody labels were abolished by pre-treating PRBCs with trypsin and chymotrypsin, confirming the surface location of the PfSA1 and PfSA2 (FIG. 5).  
       EXAMPLE 6  
     Further Characterizaiton of the Localization of PfSA1 and PfSA2 to Substructures on the Surface of PRBC.  
       [0045]    The pattern of the fluorescent label with both anti-PfSA1 and anti-PfSA2 prompted us to investigate whether the antigens were part of the knobs, a protruding structure on the PRBC surface. A knobless  P. falciparum  strain Malayan Camp was selected for the study. Whereas the strain was verified as knobless by using an anti-KaHRP, a marker for knob structure, both anti-PfSA1 and PfSA2 were localized on the surface of the parasite, indicating the antigens were not associated with the knobs (data not shown).  P. falciparum  strains Malayan Camp selected for resetting positive (MCR+), and rosetting-negative (MCR−) were also tested for reactivity with anti-PfSA1 and anti-PfSA2. The antigens were present on the surface of both strains, indicating the antigens are unlikely involved in the resetting process. Of all  P. falciparum  strains (3D7, R29, MCR+, MCR−, MCK−, T996) test for reactivity against anti-PfSA1 and anti-PfSA2, T996 was the only one shown negative toward both antibodies (data not shown). Since PCR with primers used for sequencing PfSA1 and PfSA2 in other  P. falciparum  strains (see below and FIG. 6) failed to amplify any sequences from the strain T996, it is likely that the genes were deleted form the strain, or it has diverged beyond recognition. This echoes the findings that a segment of chromosome 9 was also deleted from the strain T996 (Wu, unpublished data).  
       EXAMPLE 7  
     Characterization of PfSA1 and PfSA2 From Other Strains of  P. falciparum  Parasites With Diverse World Origins.  
       [0046]    To investigate the sequence conservation of PfSA1 and PfSA2, specific primers were designed to amplify and sequence the antigens from the selected  P. falciparum  isolates from various geographic location, 7G8 (South America), Malayan Camp (MC) (Southeast Asia), and R033 (Africa). As shown in FIGS. 6 and 7, both proteins are remarkably conserved with other  P. falciparum  strains, indicating both could be good vaccine candidates with broad specificity.  
         [0047]    This is the first study applying high throughput proteomics approach toward the identification of proteins on the surface of PRBCs. The method is highly efficient because, of two antigens selected for detailed characterization, both were confirmed to be on the surface of PRBCs. Further evaluation on immunogenicity of PfSAl and PfSA2 and efficacy of anti-PfSA1 and anti-PfSA2 will provide insight whether the antigens can be targets for antimalarial vaccines. Our findings also indicate that the surface composition of PRBC is more complex than we thought, as more candidates as result of our in silico analysis awaits to be analyzed and are also likely to be surface proteins. Some of these proteins might be account for the protective immunity, some might mediate cytoadherence, yet some might be channels responsible nutrient uptake.  
       PROPHETIC EXAMPLE 8  
     Development of a PfSA1 Malaria Vaccine  
       [0048]    In this example, a DNA vaccine encoding the full length of PfSA1 or PfSA2 is produced under GMP and is delivered in three doses intramuscularly at 5 milligrams per dose at monthly intervals, to be followed by a recombinant adenovirus vaccine which is designed to express PfSA1 or PfSA2 and which is delivered at dose of 10exp11 viral particles intramuscularly one month after the last dose of DNA vaccine. In another example, a recombinant adenovirus vaccine which is designed to express PfSA1 is delivered in two or three doses at one month intervals at a dose of l0expli viral particles per dose intramuscularly. In these examples, these vaccines could be used alone in a population of children living in SubSaharan Africa to reduce the number of circulating  Plasmodium  infected erythrocytes and would result in a decrease in morbidity and mortality associated with malaria. These vaccines could also be used in combination with other vaccines which are directed against the liver stages of the parasite to limit the risk of developing severe malaria in those individuals where the liver stage vaccines are less than 100% effective.  
       PROPHETIC EXAMPLE 9  
     Development of a Rapid Assay to Detect  Plasmodium  Infection in Humans  
       [0049]    In this example, polyclonal or monoclonal antibodies raised against polypeptide sequences from PfSA1 or PfSA2 can be used in an immunologic based assay to detect circulating PfSA1 and/or PfSA2 in serum, or to assist in the identification of parasite-infected erythrocytes in blood smears from patients suspected of being infected with  Plasmodium.  In these examples, the readout could be an enzyme linked immunosorbant assay, a fluorescence-based assay or a calorimetric based assay, though other means of assessing the detection of parasites using these antibodies may also be employed.  
       PROPHETIC EXAMPLE 10  
     Method for the Detection of Additional  Plasmodium  Proteins From the Surface of  Plasmodium -Infected Erythrocytes  
       [0050]    In this example, additional  Plasmodium  proteins that are located on the surface of infected erythrocytes are detected by a similar means as described above. These proteins would represent novel proteins for vaccine development as their location on the surface of infected-erythrocytes predicts that they will encounter cells of the immune system which will respond with the production of a humoral and/or cellular immune response against erythrocyte infected with  Plasmodium.  These additional proteins and the gene sequences encoding for these proteins can be used as vaccines delivered by DNA vaccine, recombinant protein, recombinant viral vaccine or other vaccine delivery systems.  
       PROPHETIC EXAMPLE 11  
     Development of a PfSA1 or PfSA2 recombinant protein malaria vaccine In this example, the DNA sequence of PfSA1 or PfSA2 is cloned into a bacterial expression system and a purified recombinant PfSA1 or PfSA2 protein is purified under cGMP and delivered at a dose of 50 micrograms intramuscularly at one month intervals for three months. In this example, antibodies against the PfSA1 or PfSA2 proteins will be produced will react with these proteins on the surface of the infected erythrocyte and result in the elimination of the infected erythrocyte from the circulation.  
     References  
       [0051]    Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. J Mol Biol Oct. 5, 1990;215(3):403-10  
         [0052]    Chen, Q., V. Fernandez, et al. (1998). “Developmental selection of var gene expression in  Plasmoodium falciparum.” Nature  394(6691): 392-5.  
         [0053]    Eng, J. K., A. L. McCormack, et al. (1994). “An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.”  Journal of the American Society for Mass Spectrometry  5: 976-989.  
         [0054]    Fernaders (1998).  J Exp Med  190: 1393-1404.  
         [0055]    Florens, L., M. P. Washburn, et al. (2002). “A proteomic view of the  Plasmodium falciparum  life cycle.”  Nature  419(6906): 520-6.  
         [0056]    Hollingdale, M. R., C. J. McCormick, et al. (1998). “Biology of malarial liver stages: implications for vaccine design.”  Ann. Trop. Med. Parasitol.  92: 411-417.  
         [0057]    Howard (1988).  Prog. Allergy  41: 98-147.  
         [0058]    Kyes (1999).  Proc natl Acad Sci USA  96: 9333-9338.  
         [0059]    McGregor (1963).  Trans. R. Soc. Tropo. Med. Hyg  57: 170-175.  
         [0060]    McGregor and Wilson (1988). Principles and Practices of Malaria.  Malaria.  Wensdorfer.  
         [0061]    Mohan, B. N. and Stevenson (1998). Pathogenesis and Protection.  Malaria Parasite Biology.  Sherman.  
         [0062]    Preiser, P., M. Kaviratne, et al. (2000). “The apical organelles of malaria merozoites: host cell selection, invasion, host immunity and immune evasion.”  Microbes Infect  2(12): 1461-77.  
         [0063]    Richie, T. L. and A. Saul (2002). “Progress and challenges for malaria vaccines.”  Nature  415(6872): 694-701.  
         [0064]    Salzberg, S. L., M. Pertea, et al. (1999). “Interpolated Markov models for eukaryotic gene finding.”  Genomics  59(1): 24-31.  
         [0065]    Washburn, M.  P., D. Wolters, et al. ( 2001). “Large-Scale Analysis of the Yeast Proteome via Multidimensional Protein Identification Technology.”  Nat Biotechnol.    
         [0066]    The inventive subject matter being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the inventive subject matter, and all such modifications are intended to be within the scope of the following claims.  
     
       
       
         1 
         
           
             6  
           
           
             1  
             1294  
             PRT  
             Plasmodium Falciparum  
           
            1 

Met Lys Val Gly Ile Ile Phe Phe Cys Leu Phe Phe Phe Val Val Leu 
1               5                   10                  15 

Gly Ala Cys Asn Asn Val Lys Glu Arg Ile Phe Lys Asn Ile Lys Lys 
            20                  25                  30 

Arg Thr Lys Phe Ile Ile Leu Asn Glu Pro Ile Val Asp Leu Ser Phe 
        35                  40                  45 

Ser Glu Asn Leu Phe His Thr Leu Leu Phe Asp Leu Asp Val Asp Lys 
    50                  55                  60 

Asn Leu Tyr Thr Leu Asp Glu Ser Leu Leu Asn Leu Glu Asn Leu Asn 
65                  70                  75                  80 

Tyr Ser Ser Ile Phe Arg Leu Leu Val Asp Thr Tyr Lys Asn Ile Lys 
                85                  90                  95 

Glu Asn Glu Asp Asp Asn Lys Asn Ile Arg Tyr Ile Phe Leu Gly Thr 
            100                 105                 110 

Ser Phe Ser Arg Ile His Pro Leu Asn Phe Glu Tyr Phe Leu Arg Lys 
        115                 120                 125 

Leu Asn Lys Tyr Ile Tyr Asn Gly Asn Ile Tyr Glu Lys Gly Asn Val 
    130                 135                 140 

Asp Ile Arg Gly Ile Leu Glu Glu Tyr Asn Lys Glu Ile Glu Glu Lys 
145                 150                 155                 160 

Lys Leu Glu Lys Gln Lys Leu Asn Lys Ile Lys Asp Lys Asn Asn Asn 
                165                 170                 175 

Asn Asn Asn Asn Asn Asn Ser Lys Phe Ser Lys Asp Gly Asp Asn Glu 
            180                 185                 190 

Asp Phe Asn Asn Lys Asn Asp Leu Tyr Asn Pro Ser Asp Lys Leu Tyr 
        195                 200                 205 

Asn Asn Asn Asp Asp Ile Asp Val His Glu Leu Leu Glu Glu Ile Ile 
    210                 215                 220 

Thr Lys Glu Lys Arg Phe Phe Leu Asn Asp Asp Asp Asp Asn Asp Ser 
225                 230                 235                 240 

Asn Asp Lys Tyr Ile Leu Lys Thr Asp Glu Val Asn Lys Tyr Lys Gly 
                245                 250                 255 

Phe Phe Ile Gly Tyr Gly Phe Asn Asp Asp Ile Pro Ser Val Ile His 
            260                 265                 270 

His Tyr Asn Phe Asp Lys Asn Phe Leu Phe Pro Ser Leu Asn Ser Gly 
        275                 280                 285 

Ile Ile Leu Asp Ile Thr Leu Leu Lys Asn Ile Tyr Glu Val Ser Asn 
    290                 295                 300 

Ile Leu Leu Ser Asn Asn Glu Lys Asp Gln Ser Ile His Ile Asp Tyr 
305                 310                 315                 320 

Ile Tyr Glu Val Thr Lys Tyr Ile Lys Glu Asn Leu Arg Val Arg Leu 
                325                 330                 335 

Thr His Ser Glu Asn Val Cys Leu Asn Glu Glu Gln Asn Ile His Leu 
            340                 345                 350 

Leu Asp Asn Asp Pro Asn Asn Phe Glu Ile Tyr Lys Tyr Tyr Gln Val 
        355                 360                 365 

Leu Asn Leu Phe Lys Asp Tyr Asn Lys Asn Thr Glu Glu Lys Gln Tyr 
    370                 375                 380 

Glu Lys Ile Gly His Glu Asn Val Arg His Glu Glu Thr Ser Ser Glu 
385                 390                 395                 400 

Gly Asn Glu Asn Leu Asn Arg Asn Thr Lys His Asn Asn Asp Asn Asn 
                405                 410                 415 

Asn Asp Asn Asn Asn Tyr Ser Glu Asp Ala Ile Ala Glu Leu Leu Leu 
            420                 425                 430 

Ser Tyr Phe Asn Val Phe Tyr Pro Ile Ser Thr Cys Met Cys Tyr Ser 
        435                 440                 445 

Ile Arg Ser Lys His Glu Ser Leu Met Asp Tyr Asp Lys Tyr His Met 
    450                 455                 460 

Ile Asn Leu Glu Asn Asp Ile Lys Leu Lys His Tyr Ile Lys Glu Thr 
465                 470                 475                 480 

Glu Glu Ile His Phe Asn Ser Ile Glu Glu Tyr Lys Met Lys Leu Asn 
                485                 490                 495 

Arg Ile Asn Tyr Lys Tyr Asp Thr Leu Leu Glu Glu His Glu Asn Leu 
            500                 505                 510 

Val Thr His Lys Asn Ile Leu Ile Gly Ile Lys Thr Ser Ile Asn Thr 
        515                 520                 525 

Glu Glu Glu Arg Ile Pro His Ile Lys Asn Thr Tyr Asp Asn Lys Glu 
    530                 535                 540 

Asn Thr Gln Ile Ile Phe Asn Thr Phe Asn Tyr Asp Asn Lys Leu Lys 
545                 550                 555                 560 

Glu Lys Asn Thr Phe Gly Phe Tyr Asn Asn Ser Leu Leu Gln Asn Ala 
                565                 570                 575 

Leu Glu Asn Asp Asn Ile Asp Leu Asp Ile Ile Tyr Met Ser Asp Lys 
            580                 585                 590 

Glu Ser Gln Lys Tyr Asp Asn Leu Tyr Phe Asn Ser Lys Val Thr Ser 
        595                 600                 605 

Lys Glu Gly Leu Cys Glu Lys Leu Lys His Met Ile Tyr Tyr Tyr Tyr 
    610                 615                 620 

Glu Glu Tyr Val Met Lys Asn Ser Glu Lys Lys Tyr Phe Phe Ile Ala 
625                 630                 635                 640 

Asp Asp Asp Thr Phe Val Asn Val Lys Asn Leu Ile Asp Val Thr Asn 
                645                 650                 655 

Leu Thr Leu Asn Thr Cys Ser His Ser Lys Lys Tyr Met Tyr Asp Lys 
            660                 665                 670 

Tyr Ile Lys Ser Tyr Asp Phe Val Lys Glu Asn Glu Ala Leu Phe Leu 
        675                 680                 685 

Gln Asn Phe Pro Lys Lys Thr Leu Phe Leu Tyr Ser Tyr Leu Lys Asp 
    690                 695                 700 

Thr Phe Ala Lys Thr Ile Gln Thr Leu Lys Lys Tyr Asp Tyr Val Pro 
705                 710                 715                 720 

Lys Tyr Cys Gln Gly Gly Ile Leu Ser Lys Lys His Lys Asn Asn Asp 
                725                 730                 735 

Ser Asp Asp Asp His Asp His His Val Gly Asn Lys Gln Asn Asn Asp 
            740                 745                 750 

Ser Thr Asn His Gln Asp Ile Glu Lys Asn Gln Val Asn Val Ile Asn 
        755                 760                 765 

Asn Asn Asn Asn Asn Asn Asn Asn Lys Ala Lys Ser Ile Pro Ile Tyr 
    770                 775                 780 

Leu Gly Arg Arg Tyr Ser Tyr Asn Thr Phe Ser Thr Asn Ser Asn Glu 
785                 790                 795                 800 

Tyr Phe Tyr Asp Tyr Leu Thr Gly Gly Ala Gly Ile Leu Ile Asn Asp 
                805                 810                 815 

Glu Thr Ala Lys Arg Ile Tyr Glu Cys Lys Glu Cys Thr Cys Pro Ser 
            820                 825                 830 

Thr Asn Ser Ser Met Asp Asp Met Ile Phe Gly Lys Trp Ala Lys Glu 
        835                 840                 845 

Leu Gly Ile Leu Ala Ile Asn Phe Glu Gly Tyr Phe Gln Asn Ser Pro 
    850                 855                 860 

Leu Asp Tyr Asn Lys Lys Tyr Ile Asn Thr Leu Val Pro Ile Thr Tyr 
865                 870                 875                 880 

His Arg Leu Asn Lys Asn Arg Thr Thr Lys Glu Ser Arg Asp Met Tyr 
                885                 890                 895 

Phe Asn Tyr Leu Val Asn Tyr Asn Arg Asn Asp Lys Glu Gln Asn Lys 
            900                 905                 910 

Asp Ile Tyr Val Asp Tyr Leu Asp Arg Asn His Lys Asn Met Ile Asp 
        915                 920                 925 

Asn Val Phe His Tyr Phe Phe Tyr Val Asn Met Tyr Asp Glu Lys Asn 
    930                 935                 940 

Lys Val Val Thr Lys Ile Glu His Asn Ala Asp Met Asn Ser Lys Lys 
945                 950                 955                 960 

Asn Lys Ser Lys Asn Pro Gln Lys Leu Asn Asn Thr Gln Gly Asp Lys 
                965                 970                 975 

Asn Val Asn Asp Asp Glu Asn Val Asn Asp Asp Glu Asn Val Lys Gly 
            980                 985                 990 

Asp Glu Asn Val Lys Gly Asp Glu  Asn Val Lys Gly Asp  Glu Tyr Met 
        995                 1000                 1005 

Lys Gly  Asp Glu Asn Val Lys  Gly Asp Glu Asn Val  Lys Asp Asp 
    1010                 1015                 1020 

Glu Asn  Val Lys Asp Asp Glu  Asn Ile Lys Gly Asp  Asp Asn Asn 
    1025                 1030                 1035 

Tyr Asn  Val Asp Asn Met Glu  Asn Ile Asp Asp Ile  Ile Asn Met 
    1040                 1045                 1050 

Val Glu  Ser Val Asp Asp Asp  Val Met Glu Arg Asn  Lys Lys Gly 
    1055                 1060                 1065 

Thr Gly  Lys Glu Lys Lys Asp  Asp Lys Asn His Asn  Asn Lys Glu 
    1070                 1075                 1080 

Lys Ala  Thr Asp Val Lys Lys  Ser Ser Val Pro Thr  Asn Asn Ile 
    1085                 1090                 1095 

Asp Lys  Asn Glu Asp Thr Thr  Lys Tyr Val Ile Lys  Met Asn Glu 
    1100                 1105                 1110 

Lys Ile  Tyr Asn Arg Met Gln  Glu Ser Gly Lys Tyr  Lys Gln Leu 
    1115                 1120                 1125 

Phe Asp  Ile Asn Lys Phe Phe  Lys Lys Glu Ile Glu  Gly His Pro 
    1130                 1135                 1140 

Tyr Phe  Gln Lys Ile Lys Lys  Lys Asn Glu Lys Ala  Lys Lys Glu 
    1145                 1150                 1155 

Lys Glu  Lys Met Asn Gln Leu  Lys Lys Gln Lys Asp  Tyr Thr Asn 
    1160                 1165                 1170 

Asn Tyr  Phe His Thr Ser Asn  Met Gln Gly Asn Phe  Asn Gln Gln 
    1175                 1180                 1185 

Lys Met  Gly Asn Tyr Gln Asn  Gln Glu Asn Glu Glu  Asn Asp Phe 
    1190                 1195                 1200 

Phe Asp  Gln Arg Pro Glu Ile  Glu Glu Asp Ala Ile  Asn Pro Met 
    1205                 1210                 1215 

Asp Tyr  Glu Glu Tyr Met Glu  Asn Leu Ser Asn Phe  Glu Asp Asp 
    1220                 1225                 1230 

Gly Glu  Pro Tyr Asp Glu Tyr  Asp Asp Tyr Asp Asp  Phe Val Asn 
    1235                 1240                 1245 

Thr Ile  Asn Ala Asp Lys Leu  Lys Ile Asn Asp Gln  Asn Lys His 
    1250                 1255                 1260 

Leu Tyr  Glu Gln Ile Lys Asp  Ile Ala Gln Pro Pro  Val Asn Phe 
    1265                 1270                 1275 

Gln Asn  Asp Gln Asn Ser Asn  Thr Phe Asp Phe Asp  Thr Asp Glu 
    1280                 1285                 1290 

Leu 

 
           
             2  
             408  
             PRT  
             Plasmodium Falciparum  
           
            2 

Met Leu Leu Phe Phe Ala Lys Leu Val Val Phe Thr Phe Phe Phe Trp 
1               5                   10                  15 

Leu Leu Lys Tyr Gly Lys Thr Arg Ser Tyr Pro Lys Ser Gly His Lys 
            20                  25                  30 

Gly His Thr Lys Leu Asn Gln Pro Val Val Arg Thr Leu Ala Asp Phe 
        35                  40                  45 

Asn Asp Met Phe Ala Asn Gln Lys Asn Thr Phe Asn Phe Leu Lys His 
    50                  55                  60 

Ile Asn His Tyr Lys Asn Glu Gln Asp Thr Asn Asn Thr His Thr Pro 
65                  70                  75                  80 

Asn His Asp Glu Tyr Ser His Asn Leu Pro Lys Asn His Glu Glu Ser 
                85                  90                  95 

Asn Ala Asn Met Asn Asn His Asn Ser Phe Asn Asp Lys Ser Val Asn 
            100                 105                 110 

Lys Lys Glu Ala Phe Asp Gln Phe Leu Gln Thr Leu Leu Asn Asn Tyr 
        115                 120                 125 

Glu Ile Met His Lys Glu Asp Glu Ser Lys Glu Ser Asn Gln His Asn 
    130                 135                 140 

Tyr Lys Glu Gly Pro Ser Tyr Glu Asp Lys Lys Asn Met Tyr Lys Glu 
145                 150                 155                 160 

Ile Leu Lys Gly Tyr Tyr Asn Val Phe Phe Glu Asn Tyr Ala Asn Asp 
                165                 170                 175 

Thr Glu Ser Asn Val His Asn Lys Pro Glu Glu Val His Lys His Glu 
            180                 185                 190 

Glu Ile His Lys His Arg Lys Leu His Lys His Glu Glu Val His Lys 
        195                 200                 205 

Pro Glu Glu Phe His Lys Pro Glu Glu Phe His Lys His Glu Lys Val 
    210                 215                 220 

His Lys His Glu Glu Val His Lys Pro Glu Glu Val His Lys His Glu 
225                 230                 235                 240 

Glu Asn His Lys His Glu Glu Asn His Lys Pro Gln Met Val Gly Gln 
                245                 250                 255 

Ala Pro Pro Glu Lys Glu Ile Arg Gln Glu Ser Arg Thr Leu Ile Leu 
            260                 265                 270 

Gly Ser Phe Pro Gln Ala Gly Glu Ile Leu Arg Glu Asp Leu Trp Asn 
        275                 280                 285 

Lys Glu Asp Asn Lys Phe Ser Tyr Ala Leu Asp Pro Asn Asp Tyr Ala 
    290                 295                 300 

Ser Ile Glu Asp Lys Leu Leu Gly Ser Ile Phe Gly Tyr Phe Lys Lys 
305                 310                 315                 320 

Asn His Asp Asn Leu Val Lys His Leu Leu Gln Gln Ile Asn Thr Tyr 
                325                 330                 335 

Lys His Lys Tyr Met Glu Leu Lys Glu Gln Tyr Ile Asn Glu Val Met 
            340                 345                 350 

Lys Leu Lys Lys Ile Tyr Asn Lys Ser Ile Met Val Ile Phe Ile Ala 
        355                 360                 365 

Ser Cys Ile Ser Ile Leu Gly Pro Val Met Leu His Met His Gln Asn 
    370                 375                 380 

Asn Pro Glu Glu Phe Phe Ala Thr Ile Leu Ser Phe Ser Ile Ser Leu 
385                 390                 395                 400 

Gly Leu His Asn Leu Leu Leu Thr 
                405 

 
           
             3  
             3885  
             DNA  
             Plasmodium Falciparum  
           
            3 

atgaaggttg gaattatatt tttttgttta tttttttttg tggttcttgg agcgtgtaac     60 

aatgtgaagg aaaggatttt taagaatatt aaaaaaagaa ccaaatttat tatattgaat    120 

gagcccatag tagatttaag ttttagtgag aatttatttc atactttatt atttgattta    180 

gatgtagata agaatttata tacattggat gagagtttat taaatcttga gaacttgaat    240 

tattcctcaa tatttcgttt acttgttgat acctataaga atataaaaga aaatgaagat    300 

gataataaaa atattcgata tatattttta ggtacatcgt tttcacgtat tcatccctta    360 

aattttgaat attttttgag aaagctgaac aaatatatat ataatgggaa catatatgaa    420 

aagggtaatg tggatatcag aggaatattg gaagaatata ataaggagat tgaagagaag    480 

aagctagaaa aacaaaaact gaacaagatc aaagataaga ataataataa taataataat    540 

aataatagta aattttctaa agatggtgat aatgaagact ttaataataa gaatgatttg    600 

tacaatccat cggataaatt atacaataat aatgatgata tcgatgtaca tgaactatta    660 

gaagagatta ttacaaaaga aaaaaggttt ttcttaaacg atgatgatga taatgatagt    720 

aatgataaat atatattaaa aactgacgag gttaataaat ataaaggatt ttttatagga    780 

tatggtttta atgatgatat accatcagta attcatcatt ataattttga taagaacttt    840 

ttatttcctt ctttaaatag tggtattata ttagatataa cattattaaa aaatatatat    900 

gaagtttcta atatattatt atcgaataat gaaaaggatc aatctattca tatagattat    960 

atttatgaag ttacaaaata tataaaagaa aatttaagag tacgtttaac acattccgaa   1020 

aatgtatgtt taaacgaaga acaaaatatt catttattag ataatgatcc taataatttc   1080 

gaaatatata aatattatca agtgctgaac ttatttaaag attataataa gaatacagaa   1140 

gaaaagcaat atgaaaaaat tggccatgaa aatgttagac atgaagaaac atcatctgaa   1200 

ggtaatgaaa accttaatag aaataccaaa cataataatg ataataataa tgataataat   1260 

aattatagtg aagatgcgat tgccgaatta cttctctcct attttaatgt gttctatcca   1320 

atatctacat gtatgtgcta ttcaataaga tcaaaacatg aatccctaat ggattatgat   1380 

aaatatcata tgatcaattt agaaaacgat ataaaattaa aacattatat aaaagaaaca   1440 

gaagaaatac attttaatag tattgaagaa tataaaatga aacttaatcg tattaattat   1500 

aaatatgata ctttattaga agaacatgaa aatttagtaa cacataaaaa tatactcata   1560 

ggtataaaaa caagtataaa tacagaagaa gaaagaattc cacatattaa aaatacatat   1620 

gataataaag aaaatacaca aataatattc aatacattca actatgataa taaattaaaa   1680 

gaaaaaaata catttggatt ttataataat tcccttttac aaaatgcttt agaaaatgat   1740 

aatatagatt tagatattat ctatatgtct gataaggaaa gccaaaaata tgataattta   1800 

tattttaatt ctaaagtaac atcaaaagaa ggcttatgtg aaaaattaaa acatatgata   1860 

tattattatt atgaagaata tgttatgaaa aattcagaaa aaaaatattt ctttattgca   1920 

gatgatgata cttttgttaa tgtaaaaaat ttaatagatg taacaaattt aacattaaat   1980 

acttgttcac attctaaaaa atatatgtat gataaatata tcaaatctta tgattttgtt   2040 

aaagaaaatg aagccttatt tcttcaaaat tttccaaaaa aaactttatt tctttattcc   2100 

tatttgaaag atacctttgc caaaactata caaaccttga agaaatatga ctatgttcct   2160 

aaatattgtc agggtggtat cctatcaaaa aaacataaaa ataatgatag tgatgatgat   2220 

catgatcatc acgtgggtaa taaacaaaat aatgatagta cgaatcatca agatattgaa   2280 

aaaaatcaag taaatgtaat aaataataat aataataata ataataataa agcaaaatcc   2340 

atacctatat acttaggaag aagatattca tataatacat tttctacaaa ttcaaatgaa   2400 

tatttttatg attatttaac tggaggtgct ggtattttaa ttaatgatga aacagctaaa   2460 

cgaatatatg aatgcaaaga atgcacatgc ccatcaacaa attcctcaat ggatgatatg   2520 

atatttggga aatgggctaa agaattagga attttagcca taaactttga aggatatttt   2580 

caaaactccc cacttgatta taacaaaaaa tatattaata ctcttgtacc tattacatat   2640 

catagattaa ataaaaatag aacaaccaaa gaatcaagag atatgtattt taattatcta   2700 

gtaaattata atagaaatga taaagaacaa aataaagaca tatatgttga ttatctagat   2760 

agaaatcata aaaatatgat agataatgta ttccattact ttttttatgt aaatatgtat   2820 

gatgaaaaaa ataaagtcgt caccaaaatt gagcacaatg ctgatatgaa cagtaaaaag   2880 

aataaatcaa agaacccaca aaaattaaat aatactcaag gggacaaaaa tgtaaatgat   2940 

gatgaaaatg taaatgatga tgaaaatgtg aaaggtgatg aaaatgtgaa aggtgatgaa   3000 

aatgtgaaag gtgatgaata tatgaaaggt gatgaaaatg tgaaaggtga tgaaaatgtg   3060 

aaagatgatg aaaatgtgaa agatgatgaa aatataaaag gtgatgataa taattacaat   3120 

gtggataata tggaaaacat agatgatatt attaatatgg ttgaaagcgt tgatgatgat   3180 

gttatggaac gtaacaaaaa aggaacgggt aaagaaaaaa aggatgataa gaatcataat   3240 

aataaagaaa aagctaccga tgtgaaaaaa tcaagtgtac ctactaataa tatagataaa   3300 

aatgaagaca ctacaaaata tgtcataaaa atgaatgaaa aaatttataa tagaatgcaa   3360 

gaaagtggta aatacaaaca attattcgat ataaataaat ttttcaaaaa agaaatcgaa   3420 

ggacatcctt attttcaaaa aataaaaaaa aagaatgaaa aggccaaaaa agaaaaagaa   3480 

aaaatgaatc aattaaaaaa acaaaaggat tatacaaata attatttcca tacatcaaat   3540 

atgcagggaa attttaatca acaaaaaatg ggaaactatc aaaatcaaga gaatgaagaa   3600 

aatgattttt ttgatcaacg tcctgaaata gaagaagatg caattaatcc aatggattat   3660 

gaagaatata tggaaaattt atcaaatttt gaagatgatg gcgaaccata tgacgaatat   3720 

gatgattatg atgatttcgt aaatacaatt aatgcagata aattaaaaat taatgatcaa   3780 

aataaacact tatatgaaca aatcaaagat atagcgcaac cacctgttaa tttccaaaat   3840 

gatcaaaatt caaatacttt tgattttgac acagatgagt tgtaa                   3885 

 
           
             4  
             1120  
             DNA  
             Plasmodium Falciparum  
           
            4 

atgttactct tttttgcaaa acttgtcgta tttacctttt tcttttggct tttaaaatat     60 

gggaaaacga ggtcatatcc caaatctggc cataagggac atacgaaatt aaatcaacca    120 

gtagttagaa cattagcaga ttttaatgac atgtttgcaa accaaaaaaa tacatttaat    180 

tttctaaaac atataaatca ttataaaaat gaacaagata caaataatac acacacgcca    240 

aatcatgatg aatattctca taatttgcca aaaaatcacg aagagtcaaa tgcaaatatg    300 

aacaatcata attctttcaa tgacaaatct gttaataaaa aagaagcttt cgatcaattt    360 

ttacaaacgt tattaaacaa ttatgaaata atgcataaag aagatgaaag taaagaatca    420 

aatcaacata actataaaga aggtccctca tatgaagata aaaaaaatat gtacaaagaa    480 

atattgaaag gatattataa tgtatttttt gaaaattatg caaacgacac agaatcaaat    540 

gtacataata aacctgagga agttcataaa catgaggaaa ttcataaaca taggaaactt    600 

cataaacatg aagaagttca taaacctgag gaatttcata aacctgagga atttcataaa    660 

catgagaaag ttcataaaca tgaagaagtt cataaacctg aggaagttca taaacatgag    720 

gaaaatcata aacatgagga aaatcataaa cctcaaatgg taggtcaagc acctccagaa    780 

aaagagatac gccaagaatc aagaactcta atacttggtt catttcccca agcaggtgaa    840 

atattaagag aggatttatg gaacaaagag gataacaaat ttagttacgc acttgaccct    900 

aatgattatg catctataga agataaactt ttaggatcta tatttggata ctttaaaaaa    960 

aatcatgaca atttggttaa acatttgtta caacaaatta atacttacaa acataaatat   1020 

atggaactta aagaacaata tattaatgaa gttatgaaac ttaaaaaaat atataacaaa   1080 

agcatcatgg tcatatttat agcatcttgt atttcaatat                         1120 

 
           
             5  
             30  
             PRT  
             Plasmodium Falciparum  
           
            5 

Asn Asn Ser Lys Phe Ser Lys Asp Gly Asp Asn Glu Asp Phe Asn Asn 
1               5                   10                  15 

Lys Asn Asp Leu Tyr Asn Pro Ser Asp Lys Leu Tyr Asn Asn 
            20                  25                  30 

 
           
             6  
             33  
             PRT  
             Plasmodium Falciparum  
           
            6 

Tyr Glu Ile Met His Lys Glu Asp Glu Ser Lys Glu Ser Asn Gln His 
1               5                   10                  15 

Asn Tyr Lys Glu Gly Pro Ser Tyr Glu Asp Lys Lys Asn Met Tyr Lys 
            20                  25                  30 

Glu