Background of the Invention It is the beginning of the 21St century and yet the threat of malaria looms large in tropical and sub-tropical countries especially among children. According to the World Health Organization (WHO) there are about 300 million cases of infection each year and 3 million fatalities (1-4). In addition to posing a health concern, malaria causes an economic damage due to the loss of productive hours when the disease incapacitates working men and women. Also the emergence of drug resistance and the lack of a successful vaccine cast a spell of apparent despair in the health care and medical research community. Fortunately, in the post-genomic era we have new information and technologies that can help us in combating malaria epidemic on a global scale. We now have key genomic and functional information on the parasite Plasmodium falciparum (4- 8), the Anopheles mosquito vector (9-14), and finally the two primary human target cells <BR> <BR> (i. e. , liver cells or hepatocytes and red blood cells or erythrocytes) (15-20). Of particular importance are the key P. falciparum proteins that support the life-cycle of the parasite inside the mosquito vector as well as inside the human host have been identified. Also, we know a great deal more about how P. falciparum exploits the molecular machinery inside mosquito midgut after its entry via blood meal ingestion. In order to develop effective malaria countermeasures, the appropriate therapeutic targets need to be identified.

Attempts to interrupt host-vector and vector-parasite interactions by insecticides that kill the mosquito vector and drugs that kill the parasite have been successful in the short term. Traditionally, these insecticides and drugs are small organic molecules and are often subject to several drawbacks. They often lead to emergence of vector and parasite resistance. Also, they can be potentially toxic. Finally, they are not long lasting (< 2 hours). In the earliest field studies dichlorodiphenyltrichloroethane (DDT) showed remarkable anti-mosquito repellant activity. However, the use of DDT had to be severely curtailed due to environmental concerns (35-37). N, N-diethyl-3-methyl-benzamide (DEET) is another repellant that is currently in use. DEET also poses a health concern

due to its association with the gulf war syndrome. Currently pyrethroid insecticides have been recommended for indoor use by the roll back malaria program in Africa. However, emergence and spread of pyrethroid resistance present yet another hurdle in malaria treatment (38). Recently, small organic solutes (e. g. , DL-Leucine/Methionine) that block cationic amino acid transporter channel 1 (CATCH1) have been shown to slow down the growth or cause death of the malaria parasite (39). It is quite likely that the malaria parasite may also develop resistance to such drugs through mutations in CAATCH1 (40).

The inherent drawbacks associated with organic insecticides and anti-malaria drugs call for alternative approaches.

Figure 1 shows the interaction between the malarial parasite, the mosquito vector and the human host. Figure 2 is a schematic description of the early stages of development inside the mosquito vector.

Summary of the Invention The present invention discloses novel countermeasures to block vector-host transmission of the parasite. For this, the mosquito vector or the parasite inside the vector is targeted. However, these developments represent a major paradigm shift from all previous or current approaches. Both vector-and parasite-killing agents are either endogenous to the mosquito vector or derived from plants and therefore, are longer lasting and less toxic than small organic molecules. These proteins target conserved pathways in malaria pathogenesis and therefore are less likely to be thwarted by vector or parasite resistance.

One embodiment involves the use of a cysteine protease that specifically destroys the mosquito vector, e. g., Anopheles gambiae, that transmits the malaria parasite Plasmodium falciparum to the human host during the blood meal. Useful cysteine proteases include bromelain, ananain, or papain, which shows mosquito killing activity.

Strategies using yeast display and flow cytometry can be used to develop superactive bromelain, ananain, or papain with enhanced mosquito killing function.

A second embodiment involves the use of two types of chimeric proteins that kill the parasite inside the mosquito. One chimeric protein contains two different moieties with two different functions: one is a chitinase inhibitor whereas the other is a defensin

(cecropin). The chitinase inhibitor (CI) is designed to block the breakage of the peritrophic matrix of the midgut, which is essential for the survival of the parasite inside the mosquito vector. While the CI ensures localization of the parasite outside the mosquito midgut, the defensin (cecropin) is designed to specifically target and kill the parasite before it can invade the mosquito midgut. The other chimeric protein contains a chitin binding protein (CBD) and a defensin (cecropin). The CBD is designed to sequester the parasite outside the peritrophic matrix of the midgut. The synergistic combination of these two functions will sequester the parasite outside the peritropic matrix and rapidly clear it. In both cases, synergistic use of parasite lytic function of defensin (cecropin) and parasite sequestering function of CI (or CBD) will make the chimera an effective countermeasure.

Brief Description of the Figures Figure 1 illustrates that malaria results from interplay among the parasite, the vector, and the human host. Disruption of host-vector and parasite-vector interactions is likely to stop the malaria infection at a very'early stage.

Figure 2 is a schematic description of how the proposed short-term countermeasures block the early stages of development inside the mosquito vector. The four (a-d) stages of development are described as follows. In stage a, female and male fertilized into a parasite zygote. In stage b, zygote moves into the peritropic matrix of the mosquito midgut and develops into ookinete. In stage c, ookinete develops into oocyst and is released from the basal lamina. Finally in stage d, sporozoites are released from oocysts and invade the hepatocytes in the salivary gland.

Figure 3A is a BLAST alignment of bromelain and papain sequences. Gaps and similarities are shown respectively as-and +.

Figure 3B illustrates the sequence and domain structure of bromelain. The enzyme consists of B-terminal signal peptide (underlined), the N-terminal Pro-peptide (italic), and the mature catalytic domain (bold).

Figure 3C illustrates the sequence and domain structure of ananain. The enzyme consists of B-terminal signal peptide (underlined), the N-terminal Pro-peptide (italic), and

the mature catalytic domain (bold). Bromelain and ananain share 88% sequence similarity.

Figure 4 illustrates the crystal structure of papain.

Figure 5 illustrates a process for development of superactive chimera or enzyme by yeast display.

Figure 6A illustrates a chimeric protein inhibitor including a defensin portion and a chitinase inhibitor portion to block the passage of ookinetes across the mosquito midgut PM.

Figure 6B illustrates a chimeric protein inhibitor including a defensin portion and chitin binding protein portion to block the passage of ookinetes across the mosquito midgut PM.

Figure 7 illustrates a chimeric inhibitor, chitinase-inhibitor/linker/defensin, designed to destroy the plasmodium in the mosquito midgut.

Description of the Invention The present invention relates to mosquito control reagents and process for treatment of mosquitoes and mosquito habitat. In accordance with one embodiment of the present invention, there is provided a chimeric molecule for malarial control, comprising a chitinase inhibitor or chitin binding protein portion connected by a linker to a defensin portion.

According to the present invention, the terms"chimera"or"chimeric"with regard to a protein refer to a protein that is composed of amino acid sequences derived from at <BR> <BR> least two distinct sources (i. e. , at least two heterologous amino acid sequences). As used herein, a chimeric protein is not a single naturally occurring protein, but rather, has been synthesized or genetically engineered. One type of chimeric protein is known in the art as a fusion protein. According to the present invention, the phrases"chimeric defensin protein"and"defensin-chitinase inhibitor/chitin binding protein chimera"or"chimeric protein"are used to refer to the same chimeric protein of the present invention, and thus can be used interchangeably.

Mosquitoes are insects of the order Diptera, Family Culicidae. Representative genera include Aedes, Anopheles, Wyeornyia, Uranoteania, Coquillettidia, Psorophora, Orthopodomydia, Culiseta, Toxorhynchites, Culex, Maillotia, Sabethes, Haemagoggus, and Deinocerites. Particularly preferred mosquitoes to treat with the present invention

are mosquitoes that are involved in human disease transmission, including malaria.

Preferable mosquito species to treat include genera and species known to be involved in the transmission of malaria, and include the genera Anopheles. Particularly preferred Anopheles species to treat include Anopheles gambiae, important in malarial transmission in Africa, and other species known to transmit malaria in Africa and elsewhere, particularly Anopheles quadrimaculatus, Anopheles freeborni, Anopheles hermsi, Anopheles punctipennis, Anopheles albicans, and Anopheles labranchiae.

The defensin portion of the chimeric molecule is derived from any defensin molecule, for example, an insect defensin molecule, a mosquito defensin molecule, and preferably, an Anopheles gambiae defensin molecule. Defensins are a class of cysteine- rich antimicrobial peptides that are widely distributed among insects and plants.

Defensins have a characteristic six cysteine/three disulfide bridge pattern. Insect defensins have the same cysteine pairing: Cysl-Cys 4, Cys2-Cys5 and Cys3-Cys6.

Insect defensins are 36 to 46 amino acids long with the exception of the 51 residue bee and bumblebee defensins. At least 40 members of insect defensins have been described.

Sequence comparisons between all insect defensins reveal that similarities range between 58% to 95% percent identity.

Preferred defensin portions are any portion of a defensin molecule which has defensin-like activity, particularly activity against the malarial parasite Plasmodium falciparum. Defensin biological activity is defined herein a measurable activity that is indicative of the biological activity of a naturally occurring defensin, as measured by an in vitro or in vivo assay. It is noted that modified forms of defensin, such as the chimeric defensin proteins described herein, may have different quantitative activity and specificity than a naturally occurring defensin protein, and such variations are intended to be encompassed by the present invention. Preferably the defensin portion is derived from an insect defensin molecule. A preferred insect defensin molecule is a mosquito defensin molecule. A preferred defensin molecule is a portion of an Anopheles gambiae defensin sequence. A portion may be defined as either the full-length defensin molecule or a less than full-length sequence thereof having defensin biological activity, such as lytic activity against Plasmodium falciparum. According to the present invention, reference to a

"defensin"can include any full-length defensin protein, truncated defensin protein, or any homologue of such an defensin protein. According to the present invention, a defensin homologue includes proteins in which at least one or a few, but not limited to one or a few, amino acids of full-length defensin have been accidentally or deliberately deleted <BR> <BR> (e. g. , a truncated version of the protein, such as a peptide), inserted, inverted, substituted<BR> and/or derivatized (e. g. , by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the defensin homologue has defensin biological activity as described previously herein. A defensin protein homologue can be identified as a protein having at least one epitope which elicits an immune response against a naturally occurring defensin protein.

In another embodiment, a homologue of a defensin protein is a protein having an amino acid sequence that is sufficiently similar to a naturally occurring defensin amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing <BR> <BR> under stringent conditions to (i. e. , with) a complement of a nucleic acid molecule encoding the naturally occurring defensin protein. A nucleic acid sequence complement of nucleic acid sequence encoding defensin refers to the nucleic acid sequence of the <BR> <BR> nucleic acid strand that is complementary to (i. e. , can form a complete double helix with) the strand which encodes defensin. It will be appreciated that a double stranded DNA which encodes a given amino acid sequence comprises a single strand DNA and its complementary strand, such complementary strand having a sequence that is a complement to the single strand DNA. As such, nucleic acid molecules which encode an defensin protein of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence of a defensin protein, and/or with the complement of the nucleic acid that encodes amino acid sequence of a defensin protein. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of defensin proteins included in the present invention.

A preferred defensin molecule has the sequence according to SEQ ID NO : 1.

Another preferred defensin molecule is a defensin molecule comprising an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, and at least about 95% homology with, SEQ ID NO : 1. As an alternative embodiment of chimeric molecules of the present invention, cecropin can be substituted for defensins. Insect cecropin molecules are known in the art.

In one embodiment, a chimeric protein of the present invention may include a chitinase inhibitor portion connected by a linker to a defensin portion. Chitinases, which hydrolyze linear polymers of (3- (1, 4) -linked N-acetylglucosamine (GIcNAc), have been implicated in infection of mosquitoes by the human malaria parasite Plasmodium falciparum. During development in the mosquito midgut, malarial parasites must traverse a chitin-containing peritrophic matrix (PM) that forms around the food bolus. It has been reported that the parasite secretes a protein with chitinase activity, and that parasite chitinase plays an important role in the parasite's egress from the blood meal. A preferred chitinase inhibitor portion is derived from the N-terminal portion of the precursor chitinase. Such peptides bind to the mature chitinase and inhibit its catalytic activity.

Preferred chitinase inhibitor portions are any portion of a chitinase inhibitor molecule which has chitinase inhibitor-like activity, particularly activity against the malarial parasite Plasmodium falciparum chitinase activity. Chitinase inhibitor biological activity is defined herein a measurable activity that is indicative of the biological activity of a naturally occurring chitinase inhibitor, as measured by an in vitro or in vivo assay. It is noted that modified forms of chitinase inhibitor, such as the chimeric proteins described herein, may have different quantitative activity and specificity than a naturally occurring chitinase inhibitor protein, and such variations are intended to be encompassed by the present invention. Preferably the chitinase inhibitor portion is derived from an insect chitinase inhibitor molecule. A preferred insect chitinase inhibitor molecule is a mosquito chitinase inhibitor molecule. A preferred chitinase inhibitor molecule is a portion of an Anopheles gambiae chitinase inhibitor

sequence. A portion may be defined as either the full-length chitinase inhibitor molecule or a less than full-length sequence thereof having chitinase inhibitor biological activity, such as inhibitor activity against Plasmodium falciparum chitinase. According to the present invention, reference to a"chitinase inhibitor"can include any full-length chitinase inhibitor protein, truncated chitinase inhibitor protein, or any homologue of such chitinase inhibitor protein. According to the present invention, a chitinase inhibitor homologue includes proteins in which at least one or a few, but not limited to one or a few, amino acids of full-length chitinase inhibitor have been accidentally or deliberately <BR> <BR> deleted (e. g. , a truncated version of the protein, such as a peptide), inserted, inverted,<BR> substituted and/or derivatized (e. g. , by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the chitinase inhibitor homologue has chitinase inhibitor biological activity as described previously herein. A chitinase inhibitor protein homologue can be identified as a protein having at least one epitope which elicits an immune response against a naturally occurring chitinase inhibitor protein. In another embodiment, a homologue of a chitinase inhibitor protein is a protein having an amino acid sequence that is sufficiently similar to a naturally occurring chitinase inhibitor amino acid sequence that a nucleic acid sequence encoding the homologue is capable of <BR> <BR> hybridizing under stringent conditions to (i. e. , with) a complement of a nucleic acid molecule encoding the naturally occurring chitinase inhibitor protein. A nucleic acid sequence complement of nucleic acid sequence encoding chitinase inhibitor refers to the <BR> <BR> nucleic acid sequence of the nucleic acid strand that is complementary to (i. e. , can form a complete double helix with) the strand which encodes chitinase inhibitor. As such, nucleic acid molecules which encode an chitinase inhibitor protein of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence of a chitinase inhibitor protein, and/or with the complement of the nucleic acid that encodes amino acid sequence of a chitinase inhibitor protein. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic

acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of chitinase inhibitor proteins included in the present invention.

A preferred chitinase inhibitor molecule has the sequence according to SEQ ID NO : 2. Another preferred chitinase inhibitor molecule is a chitinase inhibitor molecule comprising an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, and at least about 95% homology with, SEQ ID NO : 2.

Figure 6A is a two dimensional represention of a chimeric molecule of the present invention comprising a chitinase inhibitor portion. Without being bound by theory, it is believed that the killing power of a defensin is unexpectedly enhanced by a chimera combining a chitinase inhibitor, for the reason that the chitinase inhibitor targets P. falciparum chitinase, thus directing the defensin lytic activity towards P. falciparum. In this fashion, the lytic activity of the defensin towards P. falciparum is significantly enhanced. Figure 6A shows three separate chimeric molecules 500,502, and 504 in action. On chimeric molecule 504, defensin portion 508 binds to and lyses plasmodium membrane 506. Linker portion 518 joins chitinase inhibitor portion 510 to the defensin portion 508. Chitinase inhibitor portion 510 binds to and prevents the Plasmodium falciparum chitinase 512 from degrading the peritrophic matrix (not shown), localizing the parasite outside of the mosquito midgut.

In another embodiment, a chimeric protein of the present invention may include a chitin binding portion connected by a linker to a defensin portion. Chitinases, which hydrolyze linear polymers of ß-(1, 4)-linked N-acetylglucosamine (GlcNAc), have been implicated in infection of mosquitoes by the human malaria parasite Plasmodium falciparum. During development in the mosquito midgut, malarial parasites must traverse a chitin-containing peritrophic matrix (PM) that forms around the food bolus.

Preferred chitin binding protein portions are any portion of a chitin binding protein molecule which has chitin binding protein-like activity, particularly binding activity to mosquito chitin. Chitin binding protein biological activity is defined herein a measurable

activity that is indicative of the biological activity of a naturally occurring chitin binding protein, as measured by an in vitro or in vivo assay. It is noted that modified forms of chitin binding protein, such as the chimeric proteins described herein, may have different quantitative activity and specificity than a naturally occurring chitin binding protein, and such variations are intended to be encompassed by the present invention. Preferably the chitin binding protein portion is derived from an insect chitin binding protein molecule.

A preferred insect chitin binding protein molecule is a mosquito chitin binding protein molecule. A preferred chitin binding protein molecule is a portion of an Anopheles gambiae chitin binding protein sequence. A portion may be defined as either the full- length chitin binding protein molecule or a less than full-length sequence thereof having chitin binding protein biological activity, such as inhibitor activity against Plasmodium falciparum chitinase. According to the present invention, reference to a"chitin binding protein"can include any full-length chitin binding protein protein, truncated chitin binding protein protein, or any homologue of such chitin binding protein protein.

According to the present invention, a chitin binding protein homologue includes proteins in which at least one or a few, but not limited to one or a few, amino acids of full-length <BR> <BR> chitin binding protein have been accidentally or deliberately deleted (e. g. , a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized <BR> <BR> (e. g. , by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the chitin binding protein homologue has chitin binding protein biological activity as described previously herein. A chitin binding protein protein homologue can be identified as a protein having at least one epitope which elicits an immune response against a naturally occurring chitin binding protein protein. In another embodiment, a homologue of a chitin binding protein protein is a protein having an amino acid sequence that is sufficiently similar to a naturally occurring chitin binding protein amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing <BR> <BR> under stringent conditions to (i. e. , with) a complement of a nucleic acid molecule encoding the naturally occurring chitin binding protein protein. A nucleic acid sequence complement of nucleic acid sequence encoding chitin binding protein refers to the nucleic

acid sequence of the nucleic acid strand that is complementary to (i. e. , can form a complete double helix with) the strand which encodes chitin binding protein. As such, nucleic acid molecules which encode an chitin binding protein protein of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence of a chitin binding protein, and/or with the complement of the nucleic acid that encodes amino acid sequence of a chitin binding protein protein. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of chitin binding protein proteins included in the present invention.

A preferred chitin binding protein molecule has the sequence according to SEQ ID NO : 3. For example the chitin binding protein disclosed in A type I peritrophic matrix protein from the malaria vector Anopheles gambiae binds to chitin. Cloning, expression, and characterization, J Biol Chem. 1998 Jul 10; 273 (28): 17665-70 is a preferred example of a chitin binding protein. Another preferred chitin binding protein molecule is a chitin binding protein molecule comprising an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, and at least about 95% homology with, SEQ ID NO : 3.

Figure 6B is a two dimensional represention of a chimeric molecule of the present invention comprising a chitin binding protein portion. Without being bound by theory, it is the inventor's belief that the killing power of a defensin is unexpectedly enhanced by a chimera combining a chitin binding protein, for the reason that the chitin binding protein targets the mosquito midgut, thus directing the defensin lytic activity towards the location of P. falciparum. By this fashion, the lytic activity of the defensin towards P. falciparum is significantly enhanced. Figure 6B shows three separate chimeric molecules 520,522, and 524 in action. On chimeric molecule 524, defensin portion 528 binds to and lyses plasmodium membrane 526. Linker portion 538 joins chitin binding protein portion 530

to the defensin portion 528. Chitin binding portion 530 binds to and localizes chimera 524 onto the peritrophic matrix 534, localizing the parasite outside of the mosquito midgut and subjecting it to lytic activity.

Figure 7 shows the likely two dimensional structure of a chimeric molecule of the present invention. The chimeric molecule comprises at its N-terminus the P. falciparum chitinase inhibitor, and at its C-terminal end is joined to the linker. The linker is joined at its C-terminal end to the N-terminus of defensin.

Protein homologues of the present invention can be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.

In one embodiment of the present invention, defensin proteins suitable for use in the chimeric protein of the present invention include biologically active defensin proteins that are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to the complementary strand of a nucleic acid molecule encoding a defensin.

Preferred biologically active proteins for use in a chimeric protein of the present invention include defensin proteins comprising all or a biologically active fragment of a protein having an amino acid sequence SEQ ID NO : 1. As discussed above, suitable defensin proteins include biologically active fragments and homologues of any of the above identified defensin amino acid sequence and of any other defensin amino acid sequences. Additionally, the nucleic acid and amino acid sequences of many defensin proteins are known in the art, such information being publicly available, for example, on a database such as GenBank. It is noted that in the production of a chimeric protein of the present invention, modifications to the defensin sequences can be made to facilitate the production of the chimera. For example, the amino acid sequence of a defensin protein portion of the chimera may be modified to substitute a non-methionine residue for the initial methionine residue to avoid problems with multiple translation start sites in the nucleic acid molecule encoding the chimera. Such modifications are well within the ability of one of skill in the art.

In one embodiment of the present invention, chitinase inhibitor proteins suitable for use in the chimeric protein of the present invention include biologically active chitinase inhibitor proteins that are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to the complementary strand of a nucleic acid molecule encoding a chitinase inhibitor. Preferred biologically active proteins for use in a chimeric protein of the present invention include chitinase inhibitor proteins comprising all or a biologically active fragment of a protein having an amino acid sequence SEQ ID NO : 2. As discussed above, suitable chitinase inhibitor proteins include biologically active fragments and homologues of any of the above identified chitinase inhibitor amino acid sequence and of any other chitinase inhibitor amino acid sequences. Additionally, the nucleic acid and amino acid sequences of many chitinase inhibitor proteins are known in the art, such information being publicly available, for example, on a database such as GenBank. It is noted that in the production of a chimeric protein of the present invention, modifications to the chitinase inhibitor sequences can be made to facilitate the production of the chimera. For example, the amino acid sequence of an chitinase inhibitor protein portion of the chimera may be modified to substitute a non-methionine residue for the initial methionine residue to avoid problems with multiple translation start sites in the nucleic acid molecule encoding the chimera. Such modifications are well within the ability of one of skill in the art.

In one embodiment of the present invention, chitin binding proteins suitable for use in the chimeric protein of the present invention include biologically active chitin binding proteins that are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to the complementary strand of a nucleic acid molecule encoding a chitin binding protein. Preferred biologically active proteins for use in a chimeric protein of the present invention include chitin binding proteins comprising all or a biologically active fragment of a protein having an amino acid sequence SEQ ID NO : 3.

As discussed above, suitable chitin binding proteins include biologically active fragments and homologues of any of the above identified chitin binding protein amino acid sequence and of any other chitin binding protein amino acid sequences. Additionally, the nucleic acid and amino acid sequences of many chitin binding proteins are known in the

art, such information being publicly available, for example, on a database such as GenBank. It is noted that in the production of a chimeric protein of the present invention, modifications to the chitin binding protein sequences can be made to facilitate the production of the chimera. For example, thé) amino acid sequence of a chitin binding protein portion of the chimera may be modified to substitute a non-methionine residue for the initial methionine residue to avoid problems with multiple translation start sites in the nucleic acid molecule encoding the chimera. Such modifications are well within the ability of one of skill in the art.

As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid <BR> <BR> molecules. Such standard conditions are disclosed, for example, in Sambrook et al. , Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.

Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9. 62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of <BR> <BR> nucleotides are disclosed, for example, in Meinkoth et al. , 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

More particularly, stringent hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction, more particularly at least about 75%, and most particularly at least about 80%. Such conditions will vary, depending on whether DNA: RNA or DNA: DNA hybrids are being formed. Calculated melting temperatures for DNA: DNA hybrids are 10°C less than for DNA: RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA: DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between about 20°C and about 35°C, more preferably, between about 28°C and about 40°C, and even more preferably, between about 35°C and about 45°C. In particular embodiments, stringent hybridization conditions for DNA: RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between about 30°C and about

45°C, more preferably, between about 38°C and about 50°C, and even more preferably, between about 45°C and about 55°C. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%. Alternatively, Tm can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 TO 9.62.

Preferably, nucleic acid molecules encoding biologically active proteins suitable for use in a chimeric protein or other protein of the present invention have at least about 50%, at least about 60%, at least about 70%, more preferably, at least about 80%, at least about 90%, and most preferably, at least about 95% identity with a nucleic acid sequence encoding a naturally occurring protein. As used herein, reference to a percent (%) identity refers to a BLAST homology search with the default parameters identified in Table 1.

TABLE 1 BLAST Search Parameters HISTOGRAM Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).

DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page). See also EXPECT and CUTOFF.

ALIGNMENTS Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).

EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the

EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).

CUTOFF Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value.

Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.

MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).

The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN ; specifying the MATRIX directive in BLASTN requests returns an error response.

STRAND Restrict a TBLASTN search to just the top or bottom strand of the database sequences; or restrict BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.

FILTER Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993), or segments consisting of short-periodicity internal repeats, as determined by the SNU program of Claverie & States (Computers and Chemistry, 1993), or, for BLASTN, by the DUST program of Tatusov and Lipman (in preparation). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e. g. , hits against common acidic-, basic-or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted using the letter "N"in nucleotide sequence (e. g.,"NNNNNNNNNNNNN") and the letter"X"in protein sequences (e. g.,"XXXXXXXXX"). Users may turn off filtering by using the"Filter"option on the"Advanced options for the BLAST server"page.

Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, SNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.

To produce a chimeric molecule of the present invention, an amino acid sequence comprising a biologically active defensin as described above is linked to an amino acid sequence comprising a chitinase inhibitor and/or chitin binding protein as described above. The amino acid sequence of the chitinase inhibitor and/or chitin binding protein portion of the chimera can be linked to either the N-terminal end or the C-terminal end of the defensin portion, and preferably, is linked to the N-terminal end of the defensin portion. The entire amino acid sequence of the chimeric protein can contain additional amino acid residues other than those included in the defensin protein portion or the chitinase inhibitor and/or chitin binding protein portion. For example, a methionine residue is typically added to the N-terminal end of the entire amino acid sequence encoding the chimeric protein as a translation start codon, such a methionine residue.

Other"linker"amino acid residues may also be introduced to the amino acid construct of the chimera, such as between the defensin portion and the chitinase inhibitor and/or chitin binding protein portions.

An appropriate linker with which to join the defensin portion and the chitinase inhibitor and/or chitin binding protein portions is any linker which allows for joining of these portions while allowing the portions to retain their biological activity. A"linker," as used herein, is a molecule that is used to join two molecules. The linker is capable of forming covalent bonds or high-affinity non-covalent bonds to both molecules. Suitable linkers are well known to those of ordinary skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. The linkers may be joined to the constituent amino acids through their side

groups (e. g. , through a disulfide linkage to cysteine). A preferred linker is a peptide linker. A linker is chosen based on its ability to act as a scaffold to allow for proper folding of each portion of the chimera and the ability to preserve the biological activity of each portion such that the chimera retains the biological activities of each of its portions.

Any peptide linker providing for proper folding of each portion of the chimeric molecule and preservation the biological activity of each portion such that the chimera retains the biological activities of each of its portion may be used in the present invention. Many such linkers are known in the art. For example, two main types of linker include helical and non-helical. Helical linkers may act as rigid spacers separating the two portions of the chimera. Non-helical linkers are rich in prolines, which allow for structural rigidity and isolation of the linker from the attached domains. Both types of linkers are preferred.

A preferred linker has an amino acid sequence having the sequence SEQ ID NO: 4, derived from human mucin MUC1. Another preferred linker molecule is a linker molecule comprising an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, and at least about 95% homology with an amino acid sequence having the sequence SEQ ID NO: 4. Another preferred linker is the actual linker sequence between the chitin binding and catalytic domain of the Plasmodium falciparum chitinase.

Additional heterologous amino acid sequences may also be introduced into the chimera for the purposes which include, but are not limited to: confonnational arrangement of the peptides, facilitation of purification of the chimeric protein, as a marker to track the location of the protein, or for delivery of another substance into a cell.

The linkage of a first amino acid sequence comprising a biologically active defensin protein to a second amino acid sequence comprising the chitinase inhibitor and/or chitin binding portion, and/or to other flanking or intervening amino acid sequences to produce a chimeric molecule of the present invention to produce a molecule of interest can be accomplished by any means which produces a link between the components of the chimera and which is sufficiently stable to withstand the conditions used and which does not alter the desired properties of the chimera (e. g. , cytotoxic

activity against malarial parasites). Preferably, the link between the components of the chimera is covalent. For example, components of a chimeric molecule of the present invention can be linked recombinantly, by joining a nucleic acid molecule encoding an amino acid sequence comprising the chitinase inhibitor and/or chitin binding peptide to a nucleic acid molecule encoding an amino acid sequence comprising the defensin protein, and expressing the resulting recombinant nucleic acid molecule in a cell capable of expressing the construct. Alternatively, the two separate nucleic acid sequence can be expressed in a cell individually or the amino acid sequences can be synthesized chemically and subsequently joined, using known techniques. Alternatively, the chimeric molecule of the present invention can be synthesized chemically as a single amino acid sequence (i. e. , one in which all components are present) and, thus, joining is not needed.

Coupling of the components of the chimera, when they are produced separately by recombinant or chemical synthesis technology, can be accomplished via a coupling or conjugating agent. Numerous cross-linking agents are known and available in the art.

Such reagents include, for example, J-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or N, N'- (1, 3-phenylene) bismaleimide; N, N'-ethylene-bis- (iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges; 1, 5-difluoro-2,4- dinitrobenzene ; p, p'-difluoro-m, m'-dinitrodiphenylsulfone ; dimethyl adipimidate ; phenol- 1, 4-disulfonylchloride ; hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p- diisocyanate; glutaraldehyde and disdiazobenzidine. Such reagents and methods of using the same will be known to those of skill in the art.

Preferably, a chimeric molecule or other molecule of the present invention is made using recombinant technology. In one embodiment, a chimeric molecule or other molecule of the present invention is produced by expressing a recombinant nucleic acid molecule encoding the chimera, a component of the chimera, or other molecule under conditions effective to produce the protein. A preferred method to produce an encoded protein is by transfecting a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transfect include any bacterial, fungal (e. g., yeast), insect, plant or animal cell that can be transfected. Host cells can be either

untransfected cells or cells that are already transformed with at least one nucleic acid molecule.

In one embodiment, chimeric proteins of the present invention are produced by transfecting nucleic acid molecules encoding the proteins into mosquitoes to produce transgenic mosquitoes for expression by the mosquitoes to prevent the transmission of the malarial parasite. Such mosquitoes can then be introduced into the environment.

Methods for producing transgenic mosquitoes are known in the art.

In one embodiment, a chimeric protein of the present invention is produced by culturing a cell that expresses the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce a chimeric protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultant chimeric proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli ; or be retained on the outer surface of a cell or viral membrane.

The phrase"recovering the protein"refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography (e. g., heparin affinity chromatography), ion exchange chromatography, filtration,

electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in <BR> <BR> "substantially pure"form. As used herein, "substantially pure"refers to a purity that allows for the effective use of the protein as a research or therapeutic reagent.

According to the present invention, a recombinant molecule includes one or more isolated nucleic acid sequences as described herein operatively linked to one or more transcription control sequences. As used herein, the phrase"recombinant molecule" primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to a transcription control sequence, but can be used interchangeably with the phrase"nucleic acid molecule". According to the present invention, the phrase"operatively linked" refers to linking a nucleic acid molecule to a transcription control sequence in a manner such that the molecule is able to be expressed when transfected (i. e., transformed, transduced, transfected, conjugated or conduced) into a host cell. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells useful for expressing a chimeric molecule of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences <BR> <BR> include those which function in bacterial, fungal (e. g. , yeast), insect, plant or animal cells.

Recombinant molecules of the present invention, which can be either DNA or RNA, can also contain additional regulatory sequences, such as translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. In one embodiment, a recombinant molecule of the present invention also contains secretory signals (i. e. , signal segment nucleic acid sequences) to enable an expressed protein to be secreted from the cell that produces the protein.

Suitable signal segments include a signal segment that is naturally associated with a protein of the present invention or any heterologous signal segment capable of directing

the secretion of a protein according to the present invention. In another embodiment, a recombinant molecule of the present invention comprises a leader sequence to enable an expressed protein to be delivered to and inserted into the membrane of a host cell.

Suitable leader sequences include a leader sequence that is naturally associated with a protein of the present invention, or any heterologous leader sequence capable of directing the delivery and insertion of a protein to the membrane of a cell.

In accordance with the present invention, an isolated nucleic acid molecule or sequence that encodes an amino acid sequence comprising a biologically active defensin protein or an amino acid sequence comprising a the chitinase inhibitor and/or chitin binding peptide of the present invention is a nucleic acid molecule that has been removed <BR> <BR> from its natural milieu (i. e. , that has been subject to human manipulation) and can include<BR> DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated"does not reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e. g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Although the phrase"nucleic acid molecule"primarily refers to the physical nucleic acid molecule and the phrase"nucleic acid sequence"primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein.

Isolated nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a desired protein of the present invention or to <BR> <BR> form stable hybrids under stringent conditions with natural gene isolates (i. e. , a nucleic acid homologue). An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracies refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a defensin protein or a the chitinase inhibitor and/or chitin binding peptide of the present invention can vary due to degeneracies.

According to the method of the present invention, a host cell is transfected with mRNA encoding a protein of the present invention as described above. The mRNA includes the nucleic acid sequence encoding the protein to be expressed (i. e. , the coding region), and typically comprises a poly-A tail at the 3'terminus. Methods for producing mRNA encoding a given protein are known in the art and include in vitro transcription of an mRNA sequence from a DNA sequence (e. g. , a cDNA sequence encoding a desired protein). Briefly, a DNA fragment comprising the coding sequence of a desired protein can be isolated and amplified, if necessary. Preferably, capped mRNA encoding the desired protein is made using any in vitro transcription method. Any remaining DNA template is removed and the mRNA is preferably purified by any suitable method for purification of mRNA (e. g. , phenol: chloroform extraction) and/or filtration centrifugation. The resulting mRNA preferably has a A260/A2so ratio of at least about 1.8, and more preferably at least about 1.85, and more preferably at least about 1.9 and even more preferably at least about 1.95, with a A260/A280 ratio of 2.0 representing theoretically pure mRNA. Kits for performing in vitro transcription are commercially available (e. g., Message Machine kit (Ambion, Austin TX).

The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al. , Basic Methods in Molecular Biology (1986) ). The host cells containing one of polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, SF-9 cells, SF-21 cells and Hi-Fi cells, as well as prokaryotic host such as E. coli and B. subtilis. A preferred host is an insect cell, including SF-9 cells, SF-21 cells, or Hi-fi cells. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or

protein at low natural level. Mature proteins can be expressed in mammalian cells, insect cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N. Y.

(1989), the disclosure of which is hereby incorporated by reference.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127,3T3, CHO, HeLa and BHK cell tines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5'flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting- out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.

Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants,

HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast, insects or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, KluyveYOrnyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salynonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, a protein for malarial control comprises a cysteine protease. Preferred cysteine proteases are any portion of a cysteine protease molecule which has cysteine protease-like activity, particularly toxic activity against a mosquito species involved in the transmission of the malarial parasite Plasmodium falciparum. Cysteine protease biological activity is defined herein a measurable activity that is indicative of the biological activity against mosquitoes, as measured by an in vitro or in vivo assay. It is noted that modified forms of cysteine proteases, such as the cysteine proteases described herein, may have different quantitative activity and specificity than a naturally occurring cysteine protease protein, and such variations are intended to be encompassed by the present invention. A portion may be defined as either the full-length cysteine protease molecule or a less than full-length sequence thereof having cysteine protease biological activity, such as inhibitor activity against mosquitoes. According to the present invention, reference to a"cysteine protease"can include any full-length cysteine protease protein, truncated cysteine protease protein, or any homologue of such cysteine protease protein. According to the present invention, a cysteine protease homologue includes proteins in which at least one or a few, but not limited to one or a few, amino acids of full-length cysteine protease <BR> <BR> have been accidentally or deliberately deleted (e. g. , a truncated version of the protein,<BR> such as a peptide), inserted, inverted, substituted and/or derivatized (e. g. , by

glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the cysteine protease homologue has cysteine protease biological activity as described previously herein. A cysteine protease protein homologue can be identified as a protein having at least one epitope which elicits an immune response against a naturally occurring cysteine protease protein. In another embodiment, a homologue of a cysteine protease protein is a protein having an amino acid sequence that is sufficiently similar to a naturally occurring cysteine protease amino acid sequence that a nucleic acid sequence encoding the <BR> <BR> homologue is capable of hybridizing under stringent conditions to (i. e. , with) a complement of a nucleic acid molecule encoding the naturally occurring cysteine protease protein. A nucleic acid sequence complement of nucleic acid sequence 0 encoding cysteine protease refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i. e. , can form a complete double helix with) the strand which encodes cysteine protease. As such, nucleic acid molecules which encode a cysteine protease protein of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence of a cysteine protease protein, and/or with the complement of the nucleic acid that encodes amino acid sequence of a cysteine protease protein. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of cysteine protease proteins included in the present invention.

Preferred cysteine proteases of the present invention include papain, bromelain, and ananain. Figure 4 shows the crystal structure of papain. Crystal structure for bromelain and ananain are yet to be reported but are likely to adopt a similar tertiary fold.

While not being bound by theory, it is the inventor's belief that cysteine proteases act to kill mosquitoes by breaking down vital tissue structure on the mosquito. Figure 3A is a BLAST alignment of bromelain and papain sequences. Gaps and similarities are shown respectively as-and +. Figure 3B illustrates the sequence and domain structure of

bromelain. The enzyme consists of B-terminal signal peptide (underlined), the N- terminal Pro-peptide (italic), and the mature catalytic domain (bold). Figure 3C illustrates the sequence and domain structure of ananain. The enzyme consists of B- terminal signal peptide (underlined), the N-terminal Pro-peptide (italic), and the mature catalytic domain (bold). Bromelain and ananain share 88% sequence similarity.

In a preferred embodiment, a cysteine protease of the present invention comprises an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, at least about 95% homology with, an amino acid sequence comprising SEQ ID NO : 5.

In another preferred embodiment, a cysteine protease of the present invention comprises an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, at least about 95% homology with, an amino acid sequence comprising SEQ ID NO : 6.

In another preferred embodiment, a cysteine protease of the present invention comprises an amino acid sequence having at least about 50% homology with, at least about 60% homology with, at least about 70% homology with, at least about 80% homology with, at least about 90% homology with, at least about 95% homology with, an amino acid sequence comprising SEQ ID NO : 7.

Further embodiments of the present invention include methods to control malaria that include introducing one or more of the various active compounds described above (cysteine proteases and chimeric proteins) to a mosquito habitat to prevent the transmission of the malarial parasite from mosquitoes to humans. The active compounds of the present invention can be delivered by a variety of different delivery mechanisms.

For example, the active compounds can be formulated in aqueous solutions in a conventional manner for solutions of protein-based materials. These compounds can be formulated as liposome soaps or by fructose bases proteins. For example, the concentration of active compounds can be at nano-molar or micro-molar concentrations.

Such aqueous solutions can be applied to mosquito habitats, such as by aerosol spraying.

Mosquito habitats are well known to those of skill in the field and include running water, transient water, permanent water, and containers. Examples of transient water include woodland pools, fresh floodwater, and tidal floodwater. Examples of permanent water include freshwater swamps, acid water swamps, brackish water swamps, and polluted water. Examples of containers include small plants, tree holes, bamboo, and artificial containers. Compounds of the present invention can be applied at any time and in particular will be applied after rainy seasons when water has collected in containers, ponds, puddles, abandoned tires and other potential collection areas. This is the period in the tropics when the mosquitoes have hatched out.

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The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein should not, however, be construed as limited to the particular forms disclosed, as these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the present invention. Accordingly, the foregoing best mode of carrying out the invention should be considered exemplary in nature and not as limiting to the scope and spirit of the invention as set forth in the appended claims.