WO2008147826A1 - Malaria liver stage drugs - Google Patents

Abstract

The invention provides methods for inhibiting the growth of liver stage Plasmodium parasites using inhibitors of apicoplast fatty acid synthesis. The invention further provides methods for preventing malaria and methods for treating Plasmodium infections.

Description

MALARIA LIVER STAGE DRUGS

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/939,518, filed May 22, 2007, incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the use of inhibitors of enzymes fatty acid synthesis as anti-malarial drugs that target Plasmodium liver stages.

BACKGROUND OF THE INVENTION Malaria continues to kill between one and two million annually and is the world's most deadly parasitic disease. The global Roll Back Malaria initiative aims at halving the burden of malaria within the next five years (The World Health Report (1999) World Health Organization, Geneva) and The Millennium Development Goal's target is to halt the rising incidence of malaria by 2015 (Human Development Report (2003) Millennium Development Goals: A Compact among Nations to End Human Poverty, Oxford University Press, Oxford). Furthermore, the World Health Organization and the Bill and Melinda Gates Foundation have recently committed to eradicate malaria (Bill and Melinda Gates Foundation's Vision for Malaria (2007) In Malaria Forum: Collaboration I Innovation I Impact, Seattle). Malaria infection begins when the Anopheline female injects infective

Plasmodium sporozoites into the mammalian host. Sporozoites travel through different cells before settling into their final host hepatocyte. The sporozoite moves into a parasitophorous vacuole created by invagination of the hepatocyte plasma membrane. Inside this compartment the sporozoite transforms into a liver stage (LS). The LS grows rapidly and undergoes multiple rounds of nuclear division. The mature LS releases thousands of merozoites that will establish red blood cell infection.

In the continuing absence of fully effective malaria vaccines, preventing and treating malaria still relies heavily on chemoprophylaxis and chemotherapy. However, only a limited number of drugs have proved suitable for human use and several of these (including chloroquine and pyrimethamine/sulfadoxine) are now greatly compromised due to the spread of drug-resistant parasites (see Hyde (2007) FEBS J. 274:4688-98). Furthermore, most anti-malarials target the parasite's blood stage; currently primaquine and atovaquone are the only available anti-malarials that have been shown to have activity against the liver stage parasite (Rieckmann et al. (1968) Bull. World Health Organ. 38:625-32; Shapiro et al. (1999) Am. J. Trop. Med. Hyg. 60:831-6). The liver stages of Plasmodium are promising targets for prophylactic drugs because parasite numbers are limited and successful intervention during the asymptomatic liver stage infection prevents the clinically symptomatic blood stage infection. Thus, inhibition of LS provides true causal prophylaxis that prevents the clinical symptoms of malaria. Moreover, drugs that target LS may prevent the relapse of P. vivax and P. ovale infections by eradicating dormant LS (hypnozoites). Such prophylactic drugs would also be ideal for travelers and for containing seasonal and sudden outbreaks of malaria. Another advantage of drugs active against LS is that these would not be under prolonged selective pressure for resistance mutations. However, due to the inherent difficulty in studying the liver stage, little headway has been made in the discovery of new liver stage targets for rational drug design and the testing of already available drugs. New anti- malarial drugs are urgently needed (a) as alternatives to primaquine, the only available drug to effect a radical cure of P. vivax and P. ovale infections, (b) for use as single-drug treatments for mixed P. falciparum and P. vivax infections, (c) for intermittent preventative treatment in pregnancy, and (d) for chemoprophylaxis, e.g., for travelers. The present invention addresses these and other needs. SUMMARY OF THE INVENTION

One aspect of the invention provides methods for inhibiting the growth of Plasmodium liver stage parasites, comprising the step of contacting Plasmodium liver stage parasites with an effective amount of an inhibitor of apicoplast fatty acid synthesis. In some embodiments, the apicoplast fatty acidy synthesis inhibitors used in the methods of the invention are inhibitors that target and inhibit the activity of one or more enzymes of the FAS II pathway, which includes ACP, FabB/F, FabD, FabH, Fabl, FabG, and FabZ. In some embodiments, the invention provides methods for inhibiting the growth of P. falciparum liver stages. In some embodiments, the apicoplast fatty acid synthesis inhibitors used in the methods of the invention target and inhibit the activity of one of a PEP/phosphate translocator (e.g., PFiTPT and PfoTPT), ACCase, PDH El-alpha, PDH El-beta, PDH E2, and PDH E3. In some embodiments, the invention provides methods for inhibiting the growth of P. vivax or P. ovale hypnozoites. The Plasmodium liver stage parasites may be contacted with one or more apicoplast fatty acid synthesis inhibitors in vitro or in vivo.

Another aspect of the invention provide methods of inhibiting the growth of liver stage Plasmodium parasites in a vertebrate subject, comprising the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit the growth of Plasmodium liver stage parasites in the subject. In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit fatty acid synthesis of liver stage Plasmodium parasites in the subject. In some embodiments, the Plasmodium parasites are one of P. falciparum, P. vivax, and P. ovale parasites and the vertebrate subject is a human subject. The vertebrate subject may be infected with one or more species of Plasmodium parasites at the time of administration, e.g., the vertebrate subject may be may be suffering from a mixed P. falciparum and P. vivax infection. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered prophylactically to a vertebrate subject that has not been exposed to a Plasmodium parasite.

A further aspect of the invention provides methods for preventing malaria, comprising the step of administering an effective amount of an inhibitor of apicoplast fatty acid synthesis to a vertebrate subject in need thereof. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered prior to the appearance of blood stage Plasmodium parasites in an amount effective to inhibit fatty acid synthesis by liver stage Plasmodium parasites in the subject. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered prior to the appearance of blood stage Plasmodium parasites in an amount effective to inhibit the growth of liver stage Plasmodium parasites in the subject. In some embodiments, the methods of the invention prevent the relapse of P. vivax and P. ovale infections by eradicating hypnozoites.

Yet another aspect of the invention provides methods for treating a vertebrate subject suffering from a Plasmodium infection, comprising the step of administering an effective amount of an inhibitor of fatty acid synthesis to a vertebrate subject in need thereof. In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit the growth of Plasmodium liver stage parasites in the subject. In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit fatty acid synthesis of liver stage Plasmodium parasites in the subject. Further aspects of the invention include, but are not limited to, methods for eliciting protective immunity against malaria, comprising the step of administering an effective amount of an inhibitor of apicoplast fatty acid synthesis to a vertebrate subject suffering from a Plasmodium liver stage infection, and methods for screening test compounds for inhibitory activity against Plasmodium liver stages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention identifies fatty acid synthesis, including the type II fatty acid synthesis (FAS II) pathway, as essential for Plasmodium parasite liver stage (LS) development. Since the FAS II pathway is absent in the vertebrate host, this pathway is an attractive target for drugs to inhibit the growth of the liver stage (LS) of Plasmodium parasites.

The life of the protozoan Plasmodium parasite involves continuous cycling between the vertebrate host and the Anopheline mosquito vector. Within a host, the parasite must infect and propagate inside cells to produce the next generation of invasive stages that can go on to enter new target cells. All complex malaria pathologies are associated with erythrocyte infection and to date, most malaria research has focused on this part of the parasite's life cycle. However, initial transmission to a vertebrate host involves the inoculation of infective sporozoites that enter hepatocytes and develop further as liver stages (LS). This exo-erythrocytic life cycle stage is obligate for infection of the host and within a week, the liver stage parasite contained within the hepatocyte develops into approximately 30,000 merozoites. The release of merozoites from the liver into the bloodstream initiates the intraerythrocytic cycle of malaria infection with the invasion of red blood cells.

Liver stages can become dormant and persist for weeks, months or even years as hypnozoites in the human malaria parasites P. vivax and P. ovale. Currently, primaquine is the only effective anti-liver stage drug used to eliminate hypnozoites from the liver. The liver stage of the parasite is an attractive drug target as it is clinically silent and precedes the blood stage infection. Destroying the liver stage parasite would thus prevent the onset of disease. However, due to the historical difficulty in gaining experimental access to the liver stage of infection, little is known about it.

An enormous amount of lipid for membrane biogenesis is required for forming up to 30,000 merozoites within days of sporozoite invasion (two days in the rodent malaria parasites and six days in P. falciparum) of the hepatocyte. Yet, very little is known about the source of this lipid. It is likely that the Plasmodium liver stage utilizes host precursors for membrane synthesis in addition to synthesizing its own lipid. Fatty acids, one of the major lipid groups, are essential for the formation of phospholipids, the chief component of all membranes. The present invention is based on the finding that the enzymes involved in de novo fatty acid synthesis, including the enzymes of the type II fatty acid synthesis (FAS II) pathway, are highly expressed in Plasmodium liver stages. The enzymes of the FAS II pathway in Plasmodium are localized in a plastid organelle called the apicoplast (Ralph et al. (2004) Nat. Rev. Microbiol. 2:203-16). In order for de novo fatty acid synthesis to occur in the Plasmodium apicoplast, the building blocks of the initial condensation reaction, namely malonyl-ACP(CoA) and acetyl-CoA, must be present. Acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACCase) cannot be transported directly into organelles and thus must be generated inside the apicoplast. Within the apicoplast, acetyl-CoA is generated from pyruvate that is imported into the plastid as phosphoenolpyruvate (PEP) by a PEP/phosphate translocator (Fisher et al. (1997) Plant Cell 9:453-62), which is then converted to acetyl-CoA by a multi-subunit pyruvate dehydrogenase complex (Bao et al. (2002) Plant J. 22:39-50; Rawsthorne (2002) Prog. Lipid Res. 41:182:96). In P. falciparum, two translocator proteins, PfiTPT and PfoTPT are targeted to the two internal membranes of the apicoplast (Mullin et al. (2006) Proc. Natl. Acad. Sci. USA 103:9572-7). These transporters are proposed to facilitate the transport of phosphorylated C3 compounds like PEP into the stroma of the apicoplast, thereby providing the organelle the initial substrate for fatty acid synthesis. There is one P. falciparum pyruvate dehydrogenase complex, which is encoded by four genes (El- alpha, El -beta, E2 and E3) and is targeted to the apicoplast (Foth et al. (2005) MoI. Microbiol. 55:39-53).

The FAS II pathway is found in plants, prokaryotes, and Archaea, and is used for the synthesis of fatty acids that are required for membrane biogenesis (Ralph et al. (2004) Nat. Rev. Microbiol. 2:203-16). The four chemical reactions required to complete the elongation cycle of the FAS II pathway are catalyzed by distinct enzymes (FabB/F, FabG, Fabl, and FabZ), which are encoded by individual genes in bacteria and plants. In contrast, mammals have a type I fatty acid synthesis (FAS I) or "associative" fatty acid synthesis elongation pathway, where a multifunctional enzyme catalyzes all four steps in the elongation pathway. Therefore, the four key enzymes of the FAS II elongation pathway are ideal targets for rational anti-malarial drug design. FAS II inhibitors are already widely used as anti-bacterials and include hexachlorophene, which targets FabG (see, e.g., Wickramashinghe et al. (2006) Biochem. J. 393:447-57), triclosan, which targets Fabl (see, e.g., Chhibber et al. (2006) Bioorg. Med. Chem. 14:8068-98; Freundlich et al. (2007) J. Biol. Chem. 282:25436-44), and thiolactomycin, which targets FabB/F (see, e.g., Prigge et al. (2003) Biochem 42:1160-9).

Initiation of fatty acid synthesis starts with conversion of acetyl-CoA to Malonyl- CoA by ACCase. Malonyl-CoA is then transferred to acyl-carrier protein (also known as ACP) by malonyl-CoA transacylase (also known as FabD). Initiation is completed when a second acetyl-CoA moiety is added to the malonyl-ACP to form β-ketobutyryl-ACP by β-ketoacyl-ACP synthase III (FabH). The product of initiation β-ketobutyryl-ACP is reduced to β-hydroxyacyl-ACP by β-ketoacyl-ACP reductase (FabG), hydrolyzed to enoyl-ACP by β-hydroxyacyl-ACP dehydratase (FabZ), and further reduced to acyl-ACP by enoyl-ACP reductase (Fabl). Acyl-ACP is extended by the addition of malonyl-ACP in a condensation reaction catalyzed by β-ketoacyl-ACP synthase I/II (FabB/F), forming β-ketoacyl-ACP and starting the cycle again. Eventually fatty acyl-ACP moieties of between 8 and 12 carbons are produced (CIO/12/14 acyl:ACP).

A large body of in vitro work has analyzed the P. falciparum FAS II elongation pathway. The four P. falciparum enzymes involved in FAS II elongation as well as ACP, which is an essential cofactor for the elongation process, have been reconstituted in vitro using recombinant proteins expressed in E. coli (Sharma et al. (2007) Antimicrob. Agents Chemother. 51:2552-8). This in vitro system successfully catalyzed the formation of C10:0, C12:0, and C14:0 fatty acids from acetyl-CoA and malonyl-ACP, in agreement with previous studies carried out in blood stage P. falciparum (Surolia & Surolia (2001) Nat. Med. 7:167-73). Thus, there is strong evidence that P. falciparum possesses a functional and active FAS II pathway. P. falciparum Fabl has been crystallized bound to the anti-bacterial Fabl inhibitor triclosan (Muench et al. (2003) Acta Crystallogr. D. Biol. Crystallogr. 59:1246-8) and the recombinant enzyme has been shown to be inhibited by triclosan (Kapoor et al. (2001) Biochem. Biophys. Res. Commun. 289:832-7). P. falciparum FabG has also been recombinantly expressed and crystallized and is inhibited by the anti-bacterial FabG inhibitor hexachlorophene. Finally, recombinant P. falciparum FabB/F has been expressed in E. coli and is able to elongate carbon chains up to C14:0 fatty acids, and like its bacterial counterpart, is inhibited by thiolactomycin (Lack et al. (2006) J. Biol. Chem. 281:9538-46). Over-expression of recombinant P. falciparum FabZ allowed the discovery of novel inhibitors for this enzyme (Sharma et al. (2003) J. Biol. Chem. 278:45661-71) and crystallization of FabZ demonstrated the existence of an inactive dimer and an active hexamer (Swarnamukhi et al. (2006) FEBS Lett. 580:2653-60). These studies show that the P. falciparum FAS II elongation pathway enzymes behave in a similar manner to their bacterial counterparts. Some studies have indicated that treatment of P. falciparum blood stage cultures with the FabB/F inhibitor thiolactomycin (Waller et al. (1998) Proc. Natl. Acad. Sci. USA 95:12352-7) and the Fabl inhibitor triclosan (Surolia & Surolia (2001) Nat. Med. 7:167- 73; McLeod et al. (2001) Int. J. Parasitol. 31:109-113) inhibit blood stage parasite growth. However, these inhibitory effects were not shown to be a result of these inhibitors binding to their respective FAS II targets. In fact, several studies have reported a poor correlation between the potencies of inhibitors in binding to purified FAS II enzymes and the inhibition of growth of P. falciparum blood stages (Jones et al (2005) J. Med. Chem. 48:5932-41; Wickramashinghe et al. (2006) Biochem. J. 393:447-57). Moreover, studies on the developing intraerythrocytic Plasmodium have shown that the parasite is able to derive an array of lipid components from serum, including long-chain saturated and unsaturated fatty acids in association with serum albumin (Ofulla et al. (1993) Am. J. Trop. Med. Hyg. 49:35-40). This suggests that a de novo fatty acid synthesis pathway may not be necessary in blood stages. Palmitic (C16:0) and oleic (C18:l, n-9) acids in association with serum albumin have been identified as components of cell cycle progression and intraerythrocytic development of P. falciparum in vitro (Mitamura et al. (2000) Parasitol Int. 49:219-29). These fatty acids have been shown to be metabolized into various lipid species, including phospholipids as well as di- and tri- acylglycerols (Palacpac et al. (2004) J. Cell. Sci. 117:1469-80; Vial et al. (1982) J. Protozool. 29:258-63), which are major components of membranes and lipid bodies in the parasite. Some studies suggest that intraerythrocytic P. falciparum is able to desaturate and elongate fatty acids taken up from their surroundings (Mi-Ichi et al. (2006) Parasitol. 133:399-410) but this claim has been challenged by others (Krishnegowda & Gowda (2003) MoI. Biochem. Parasitol. 132:55-8). Moreover, recent evidence indicates that the mode of action of triclosan for reducing blood stage infections may not be through its inhibitory effect on the activity of enoyl-ACP reductase (Fabl) (Yu et al. (2007) Disruption of the enoyl-ACP reductase fatty acid enzyme results in reduced virulence of Plasmodium sporozoites, Abstract presented in Molecular Parasitology Meeting, Wood Hole, MA September 16-20, 2007). Specifically, Fabl gene knockout studies show that this enzyme is not essential for asexual blood stages in either P. falciparum or P. berghei. Moreover, triclosan had similar potency in both knockout and wild-type parasites suggesting a different mode of action for the drug in killing the blood stage parasite. Thus, the importance of the FAS II pathway in the blood stages of the complex Plasmodium life cycle has been unclear.

It has been shown that the different stages of the Plasmodium parasite differ in their drug susceptibilities. For example, liver stage parasites are not susceptible to blood stage antimalarials, such as chloroquine. Conversely, tazopsine, a semisynthetic derivative of a plant-derived compound is active against liver stages but inactive against the blood stages of the malaria parasite (Carraz et al. (2006) PLoS 3(12):e513). Moreover, young trophozoites are highly susceptible to triclosan, however merozoites and ring stages are refractory to the inhibitory effects of this drug (Surolia & Surolia (2001) Nat. Medicine 7(2): 167-73). These differences in drug susceptibilities of the different stages in the parasitic life cycle may be due to the distinct metabolisms of these stages and/or accessibility of the drug target.

As shown in EXAMPLE 1, below, expression of the enzymes involved in fatty acid synthesis, including the enzymes in the FAS II pathway, is highly upregulated in Plasmodium liver stages compared to all other life cycle stages. For example, one of the key enzymes in the FAS II pathway, Fabl, is highly expressed in the developing liver stage and localizes to the apicoplast, as described in EXAMPLES 1 and 2. Fabl expression was not detected in blood stages. In addition, de novo fatty acid synthesis is essential for Plasmodium liver stage development, but not required for blood stage replication. Specifically, targeted deletion of FabB/F, an enzyme in the FAS II pathway, resulted in a growth defect late in parasite liver stage development, but did not affect parasite blood stage replication or mosquito stage development, as shown in EXAMPLE 3. Similarly, Plasmodium parasites that have a targeted deletion of the El alpha subunit of pyruvate dehydrogenase (PDH El-alpha), which generates the acetyl-CoA needed for fatty acid synthesis, were unable to complete liver stage development, whereas blood stage replication was unaffected, as shown in EXAMPLE 4. These results, as well as the sheer magnitude of replication that occurs in the transformation of a single sporozoite to tens of thousands of merozoites, show that the Plasmodium de novo fatty acid synthesis, including the FAS II pathway, is essential for liver stage development. Moreover, inhibition of the FAS II pathway using fatty synthesis inhibitors leads to liver stage growth arrest, as shown in EXAMPLE 6.

Accordingly, one aspect of the invention provides methods for inhibiting the growth of liver stage Plasmodium parasites, comprising the step of contacting Plasmodium liver stage parasites with an effective amount of an inhibitor of apicoplast fatty acid synthesis. As used herein, the term Plasmodium refers to any member of the protozoan genus Plasmodium, including the four species that cause human malaria: P. vivax, P. malariae, P. falciparum, and P. ovale. In some embodiments, the invention provides methods for inhibiting the growth of P. falciparum LS. In some embodiments, the invention provides methods for inhibiting the growth of P. vivax or P. ovale hypnozoites.

The term "liver stage" or "LS" refers to the phase of the Plasmodium life cycle that occurs within hepatocytes, starting after the invasion of a hepatocyte by an infective sporozoite has invaded a hepatocyte and ending with the development of merozoites. As used herein, liver stages include the dormant LS (hypnozoites) of P. vivax and P. ovale. The term "inhibiting the growth of liver stage Plasmodium parasites" refers to preventing, slowing, suppressing or otherwise interfering with the growth of Plasmodium liver stage parasites, including, for example, by killing liver stage parasites. The term "inhibitor of apicoplast fatty acid synthesis" or " apicoplast fatty acid synthesis inhibitor" refers to a drug or compound that specifically targets and inhibits de novo fatty acid synthesis in the apicoplast of Plasmodium (such as in the apicoplast of Plasmodium LS). Exemplary enzymes targeted by inhibitors of apicoplast fatty acid synthesis according to the invention include, but are not limited to, the enzymes shown in Table 1.

Table 1. Fatty Acid Synthesis Pathways and Enzymes in Plasmodium

The term "FAS II inhibitor" or "type II fatty acid synthesis pathway inhibitor" refers to an inhibitor that targets and inhibits the activity of one or more enzymes of the FAS II pathway, which includes ACP, FabB/F, FabD, FabH, Fabl, FabG, and FabZ (see Table 1).

In some embodiments, the apicoplast fatty acid synthesis inhibitors used in the methods of the invention are FAS II inhibitors. FAS II inhibitors are known in the art and have been used as anti- microbials. Exemplary FAS II inhibitors useful in the practice of the invention that target the condensing enzymes FabH or FabB/F include, but are not limited to, thiolactomycin and analogs and derivatives thereof (see, e.g., Waller et al. (1998) Proc. Natl. Acad. Sci. USA 95:12352-7; Prigge (2003) Biochem. 42:1160-9; Waller et al. (2003) Antimicrob. Agents Chemother. 47:297-301; Lack et al. (2006) J. Biol. Chem. 281:9538-46; Jones et al, (2004) Bioorg. Med. Chem. 12:683-92; Jones et al. (2005) J. Med. Chem. 48:5932-41; Frederich et al. (2008) Trans. R. Soc. Trop, Med Hyg. 103(1): 11-9); and cerulenin and analogs or derivatives thereof (see, e.g., Price et al. (2001) J. Biol. Chem. 276(9):6551-9; Nomura et al. (1972) J. Antibiotics (Tokyo) 25:365-368; Bajsa et al (2007) Biol. Pharm. Bull.30: 1740- 1744); indole and analogs and derivatives thereof (see, e.g., Dianes et al. (2003) J. Med. Chem., 46(l):5-8); 4,5- dichloro-l,2-dithiole-3-one and analogs and derivatives thereof (see, e.g., He et al. (2004) Antimicrob. Agents Chemother. 48(8): 3093-102); benzoylaminobenzoic acid and analogs and derivatives thereof (see, e.g., Nie et at. (2005) J. Med. Chem. 48(5): 1596- 609); Platencin/Platensimycin and analogs or derivatives thereof (see, e.g., Wang et al. (2007) Proc. Natl. Acad. Sci. USA 104:7612-16); Phomallenic acid and analogs and derivatives thereof (see, e.g., Ondeyka et al. (2006) J. Nat Prod. 69:377-380); Bischloroanthrabenzoxocinone and analogs and derivatives thereof (see, e.g., Kodali et al. (2005) J. Biol.Chem. 280:1669-77); alkyl CoA disulfides analogs and derivatives thereof (see, e.g., Alhamadsheh et al. (2007) Chem. Biol. 14:513-24).

Exemplary FAS II inhibitors useful in the practice of the invention that target the reduction enzyme FabG include, but are not limited to, hexachlorophene and analogs and derivatives thereof (see, e.g., Wickramashinghe et al. (2006) Biochem. J. 393:447-57; Pillai et al. (2003) Biochem. Biophys. Res. Comm. 303(l):387-92).

Exemplary FAS II inhibitors useful in the practice of the invention that target FabZ include, but are not limited to, NAS-91 (4-Chloro-2-[5-chloroquinolin-8- yl)oxy]phenol) and NAS-21 (4,4,4- Trifluoro-l-(4-nitrophenyl) butane- 1,3-dione) and analogs and derivatives thereof (Sharma et al. (2003) J. Biol. Chem. 278:45661-71). Exemplary FAS II inhibitors useful in the practice of the invention that target reduction enzyme Fabl include, but are not limited to, triclosan and analogs and derivatives thereof (see, e.g., Kapoor et al. (2001) Biochem. Biophys. Res. Commun. 289:832-7; Surolia & Surolia (2001) Nat. Med. 7:167-73; McLeod et al. (2001) Int. J. Parasitol. 31:109-113; Chhibber et al. (2006) Bioorg. Med. Chem. 14:8068-98; Freundlich et al. (2007) J. Biol. Chem. 282:25436-44; Freundlich et al. (2006) Bioorg. Med. Chem. Lett. 16(8) :2163-9); rhodanine compounds and analogs and derivatives thereof (see, e.g., Kumar et al. (2007) J. Med. Chem. 50:2665-75; U.S. Patent Publication No. 2008/0051445); API and analogs and derivatives thereof (see, e.g., Karlowski et al. (2007) Antimicrob. Agents Chemother. 51:1580-81); diazoburine and analogs and derivatives thereof (see, e.g., Hogenauer et al (1981) Nature 293:662-64); secondary metabolites of Phlomis brunneogalaeata and analogs and derivatives thereof (see, e.g., Kirmizibekmez et al. (2004) Planta Med. 70(8):711-7); Indole-piperazine and pyrazole, and analogs and derivatives thereof (see, e.g., Kuo et al. (2003) J. Biol. Chem. 278:20851-9); catechins and other flavonoids, as well as analogs and derivative thereof (see, e.g., Sharma et al. (2007) J. Med. Chem. 50:765-775); and other Fabl inhibitors and analogs and derivatives thereof (see, e.g., Nicola et al. (2007) Biochem. Biophys. Res. Commun. 358(3):686-91; US Patent Publication No. 2007/0027190; US Patent Publication No. 2007/0135465; U.S. Patent No. 7,250,424; US Patent Publication No. 2006/0142265; WO 2008/009122).

Exemplary FAS II inhibitors useful in the practice of the invention that target ACP include, but are not limited to, pantothenamides and analogs and derivatives thereof (Zhang et al. (2004) J. Biol. Chem. 279:50969-75).

It is understood that some of the FAS II inhibitors described above may target more than one enzyme in the FASII pathway. Other exemplary FAS II inhibitors that may be used in the practice of the invention have been previously described (see, e.g., Lu et al. (2005) Combinatorial Chemistry & High Throughput Screening 8:15-26; Sharma et al. (2007) J. Med. Chem. 50:765-775; Ramya et al. (2005) IUBMB Life 57(4):371-3; Surolia et al. (2004) Biochem. J. 383:401-12; Surolia et al. (2002) BioEssays 24:192-6, Waller et al. (1998) Proc. Natl. Acad. Sci. USA 95:12352-7; Surolia & Surolia (2001) Nat. Medicine 7(2): 167-72; Sharma et al. (2007) Antimicrob. Agents Chemother. 51:2552-8; Sharma et al. (2003) J. Biol. Chem. 278(46):45661-71; and Tasdemir et al. (2006 J. Med. Chem. 49(11):3345-53).

In some embodiments, the apicoplast fatty acid synthesis inhibitors used in the methods of the invention target and inhibit the activity of one of a PEP/phosphate translocator (e.g., PFiTPT and PfoTPT), ACCase, PDH El-alpha, PDH El-beta, PDH E2, and PDH E3. Exemplary apicoplast fatty acid synthesis inhibitors useful in the practice of the invention that target ACCase include, but are not limited to, aryloxyphenoxypropionate herbicides and analogs and derivatives thereof (see, e.g., Zuther et al. (1999) Proc. Natl. Acad. Sci. USA 96:13387-92; Waller et al. (2003) Antimicrob. Agents Chemother. 47(l):297-301); CP-640186 and analogs and derivatives thereof (see, e.g., Harwood et al. (2003) J. Biol. Chem. 278(39): 37099-111); and Soraphen A and analogs and derivatives thereof (see, e.g., Shen et al. (2004) MoI. Cell 16(6):881-91).

Analogs and derivatives of the apicoplast fatty acid synthesis inhibitors described above are also within the scope of the invention. The design and synthesis of such analogs and derivatives in order to improve, for example, efficacy, solubility, penetration (e.g., into infected hepatocytes and into the apicoplast), stability, toxicity, pharmacokinetic properties, etc. is within the competence of one of ordinary skill in the art. Exemplary assays to test the inhibitory effects on Plasmodium LS in vitro and in vivo are described in EXAMPLE 6.

In some embodiments of the invention, the use of a combination of fatty acid synthesis inhibitors may have an additive or synergistic effect. Thus, the methods of the invention contemplate the use of more than one fatty acid synthesis inhibitor.

In some embodiments, the Plasmodium liver stage parasites are contacted with one or more apicoplast fatty acid synthesis inhibitors in vitro, as described in EXAMPLES 6 and 7. In some embodiments, the Plasmodium liver stage parasites are contacted with one or more apicoplast fatty acid synthesis inhibitors in vivo, as described in EXAMPLE 7. Accordingly, another aspect of the invention provide methods of inhibiting the growth of liver stage Plasmodium parasites in a vertebrate subject, comprising the step of administering an effective amount of an inhibitor of apicoplast fatty acid synthesis to a vertebrate subject in need thereof. The vertebrate subjects in need include those subjects already infected with Plasmodium parasites (such as liver stage parasites) and those who are at risk of being infected with Plasmodium parasites. In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit the growth of Plasmodium liver stage parasites in the subject. In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit fatty acid synthesis of liver stage Plasmodium parasites in the subject. The term "inhibiting fatty acid synthesis of liver stage Plasmodium parasites" refers to preventing, slowing, suppressing, reducing, or otherwise interfering with the synthesis of fatty acids Plasmodium liver stage parasites, for example, by interfering with the activity of one or more enzymes involved in fatty acid synthesis.

In some embodiments, the Plasmodium parasites are one of P. falciparum, P. vivax, and P. ovale parasites.

The term "vertebrate subjects" includes, but is not limited to, mammalian hosts that are susceptible to infection by Plasmodium parasites, including, but not limited to, humans, goats, rabbits, and mice. In some embodiments the vertebrate subject is a human subject. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered to a vertebrate subject that is infected with one or more species of Plasmodium parasites. For example, the vertebrate subject may be suffering from a mixed Plasmodium parasite infection, such as a mixed P. falciparum and P. vivax infection. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered prophylactically to a vertebrate subject that has not been exposed to a Plasmodium parasite.

The term "effective amount" for a therapeutic or prophylactic treatment refers to an amount or dosage of a composition sufficient to induce a desired response (e.g., inhibition of LS growth or prevention of malaria) in the individual to which it is administered. Preferably, the effective amount is sufficient to effect treatment, as defined below. The effective amount and method of administration of a particular therapeutic or prophylactic treatment may vary based on the individual patient and the stage of the disease, as well as other factors known to those of skill in the art. Therapeutic efficacy and toxicity of such compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosages for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the prevalence of Plasmodium in the geographical vicinity of the patient, the severity of the disease state of the patient, age, and weight of the patient, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. An appropriate effective amount may be readily determined using only routine experimentation. Several doses may be needed per individual in order to achieve a sufficient response to effect treatment. Accordingly, an appropriate effective amount may be readily determined using only routine experimentation. In some embodiments, an effective amount is between 1- 1,000 mg/kg daily, such as 5-500 mg/kg daily or 2-250 mg/kg daily, depending on the age, height, sex, general medical condition, previous medical history, as well as other factors know to those of skill in the art. In some embodiments, an effective amount is between 1-1,000 mg/kg weekly, such as 5-500 mg/kg weekly or 2-250 mg/kg weekly, depending on the age, height, sex, general medical condition, previous medical history, as well as other factors know to those of skill in the art.

Another aspect of the invention provides methods for preventing malaria, comprising the step of administering an effective amount of an inhibitor of apicoplast fatty acid synthesis to a vertebrate subject in need thereof. The vertebrate subjects in need include those subjects already infected with Plasmodium parasites (such as liver stage parasites) and those who are at risk of being infected with Plasmodium parasites. The term "preventing malaria" refers to the averting the clinical manifestations of blood stage malaria resulting from the infection of erythrocytes with merozoites. The liver stage of the Plasmodium parasite is clinically silent and precedes the blood stage infection. Destroying the liver stage parasite would thus prevent the onset of disease. In some embodiments, the methods of the invention prevent the relapse of P. vivax and P. ovale infections by eradicating hypnozoites. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered prior to the appearance of blood stage Plasmodium parasites in an amount effective to inhibit fatty acid synthesis by liver stage Plasmodium parasites in the subject. In some embodiments, the apicoplast fatty acid synthesis inhibitor is administered prior to the appearance of blood stage Plasmodium parasites in an amount effective to inhibit the growth of liver stage Plasmodium parasites in the subject. In some embodiments, the Plasmodium parasites are one of P. falciparum, P. vivax, and P. ovale parasites.

Yet another aspect of the invention provides methods for treating a vertebrate subject suffering from a Plasmodium infection, comprising the step of administering an effective amount of an inhibitor of fatty acid synthesis to a vertebrate subject in need thereof. The term "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disease. Those in need of treatment include those already with the disease as well as those prone to have the disease or those in whom the disease is to be prevented. Accordingly, in some embodiments, the subjects to be treated are human subjects suffering from malaria. In some embodiments, the subjects to be treated are human subjects at risk for contracting malaria. The subjects to be treated may or may not be infected by Plasmodium parasites. The methods of the invention also encompass treating mixed Plasmodium infection, such as a mixed P. falciparum and P. vivax infection. In some embodiments, the liver stage infection is a dormant infection, such as caused a dormant infection by P. vivax and P. ovale hypnozoites. In some embodiments, the methods of the invention prevent the relapse of P. vivax and P. ovale infections by eradicating hypnozoites. Intermittent preventative treatment in pregnancy is also within the scope of the invention.

In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit the growth of Plasmodium liver stage parasites in the subject. In some embodiments, the methods comprise the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit fatty acid synthesis of liver stage Plasmodium parasites in the subject. In some embodiments, the Plasmodium parasites are one of P. falciparum, P. vivax, and P. ovale parasites. A further aspect of the invention provides methods for eliciting protective immunity against malaria, comprising the step of administering an effective amount of an inhibitor of apicoplast fatty acid synthesis to a vertebrate subject suffering from a Plasmodium liver stage infection. Specifically, immunization with sporozoites that have a targeted deletions of enzymes involved in fatty acid synthesis in the Plasmodium apicoplast provide protective immunity against subsequent challenge with wild-type sporozoites, as shown in EXAMPLE 5. Thus, the presence of an inhibitor of apicoplast fatty acid synthesis at the same time as LS may result in the elimination of LS before the development and release of merozoites but after an immune response to LS is formed. Accordingly, the methods of the invention may be used to elicit protective immunity against subsequent Plasmodium infections, analogous to the protective immunity elicited by genetically attenuated sporozoites described, for example, in U.S. Patent Nos. 7,122,179 and 7,261,884. Yet another aspect of the invention provides methods for screening test compounds for inhibitory activity against Plasmodium liver stages, comprising the steps of (a) contacting Plasmodium liver stages with a test compound and (b) determining whether the test compound inhibits the growth of the Plasmodium liver stages. The Plasmodium liver stages may be contacted with the test compounds in vitro or in vivo. The availability of crystal structures for apicoplast fatty acid synthesis enzymes, including almost all enzymes in the FAS II pathway (see, e.g., Muench et al. (2003) Acta Crystallogr. D. Biol. Crystallogr. 59:1246-8; Swarnamukhi et al. (2006) FEBS Lett. 580:2653-60; Wickramasinghe et al. (2006) Biochem J. 393:447-57; Freundlich et al. (2007) J. Biol. Chem. 282(35):25336-444), allows rational drug design for development of inhibitors of this pathway, such as synthetic structure- activity screens, or in silico screens of existing databases (for example the National Cancer Institute diversity set or the ChemDB database at the University of California, Irvine) (see, e.g., Nicola et al. (2007) Biochem. Biophys. Res. Commun. 358:689-91). Exemplary assays to test the inhibitory effects of test compounds on Plasmodium LS in vitro and in vivo are described in EXAMPLE 7.

The following examples illustrate representative embodiments now contemplated for practicing the invention, but should not be construed to limit the invention.

EXAMPLE 1 This Example describes a comprehensive transcriptome analysis in combination with a proteomic survey of Plasmodium liver stages, demonstrating that Plasmodium liver stages show high levels of expression of enzymes involved in fatty acid synthesis (see also Tarun et al. (2008) Proc. Natl. Acad. Sci. USA 105(l):305-10, herein incorporated by reference). Due to the inherent difficulty in isolating sufficient numbers of liver stages for meaningful studies, we generated a green fluorescent protein-tagged P. yoelii (PyGFP) (Tarun et al. (2006) Int. J. Parasitol. 36:1283-93). This enabled us to efficiently isolate significant numbers of liver stage- infected hepatocytes from the rodent host by fluorescence-activated cell-sorting (FACS). The sorted cells were then processed and extracted RNA was amplified and used for genome-wide liver stage gene expression profiling and liver stage protein was used for proteomic analysis. For transcriptome analysis, RNA was extracted from each sample and subjected to two rounds of linear amplification. For each parasite stage analyzed, two to five independent biological replicates were obtained. Cy-3 and Cy-5 labeled RNA were hybridized to P. yoelii microarray slides spotted with 65-mer oligonucleotide probes. Transcriptome data were obtained at three time points post sporozoite infection, 24, 40 and 50 hours. To identify genes active in liver stages, we considered genes that showed >2-fold expression level changes and a false-discovery rate threshold of 0.01 during any of the three time points of liver stage development when compared with the other stages assayed. A total of 1448 genes were active in the 40- and 50-hour post infection liver stage transcriptome.

For proteome analysis, total proteins was extracted from sorted liver stage- infected hepatocytes and separated on SDS-PAGE. Gel bands were cut and in-gel trypsin digestion was performed. Mass spectra of the resulting peptides were obtained by nanoflow liquid chromatography tandem mass spectrometry (ID LC/MS/MS). The MS/MS data were searched against two sequence databases separately using the SEQUEST algorithm. One database contained annotated P. yoelii and Mus musculus protein sequences and the other contained the annotated P. berghei and M. musculus sequences. Peptide and protein identification were done using PeptideProphet and Protein-Prophet respectively (http://tools. proteomecenter.org/software.php). Proteome data were obtained at two time points post sporozoite infection, 40 and 50 hours. In P. yoelii at 24 hours the liver stage is in early schizogony, at 40 hours the liver stage is in late schizogony whilst at 50 hours one can visualize vigorous parasite movement and the expulsion of "merosomes" - membrane bound merozoites (Tarun et al. (2006) Int. J. Parasitol. 36:1283-93; Sturm et al. (2006) Science 3131:1287-1290). The data obtained were compared to other parasite life cycle stages. The proteomic analysis identified 816 liver stage proteins based on the mining of the P. yoelii and P. berghei databases. Of these 816 liver stage proteins, 712 were found in the P. yoelii database and 422 of these overlapped with the corresponding P. yoelii transcriptome data. A subset of the proteins appeared to be expressed only in the liver stage. The results strongly suggest the presence of parasite proteins that are only expressed during the liver stage. These proteins likely play a number of distinct roles in this life cycle stage of the parasite. One can envision subsets of proteins necessary for interacting with the highly metabolically active host cell in order to take up host cell nutrients. Also, proteins that interact with the host cell to prevent the inflammatory response associated with parasite invasion and proteins necessary for producing the enormous amount of lipids necessary for the membrane production of tens of thousands merozoites and their internal organelles - including the endoplasmic reticulum, apicoplast, rhoptries and mitochondria. A subset of proteins is also needed to enable the fully developed liver stage to burst from the hepatocyte and enter the bloodstream as erythrocyte-infectious merozoites.

Of particular interest, the data revealed that the liver stage, when compared to the other life cycle stages, has an increased transcription and expression of enzymes involved in fatty acid synthesis, including the FAS II pathway (Table 1, see also Tarun et al. (2008) Proc. Natl. Acad. Sci. USA 105(l):305-10). Specifically, the genes coding for ACP, FabB/F, Fabl FabG, and FabZ were present in the proteome and/or the transcriptome. Many of the enzymes involved in fatty acid synthesis are predicted to be found in the apicoplast (Ralph et al. (2004) Nat. Rev. Microbiol. 2:203-16) and, as well as the aforementioned enzymes involved in FAS II elongation, a host of other enzymes involved in fatty acid synthesis were present in the liver stage transcriptome and/or proteome. These include the phosphoenopyruvate (PEP)/phosphate translocator necessary for the import of PEP into the apicoplast. PEP is subsequently converted to pyruvate by pyruvate kinase and then to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl- CoA is the initial building block for fatty acid synthesis.

Table 2. Increased Ex ression of Fatt Acid S nthesis Enz mes in Liver Sta es

* upregulated in P. yoelii transcriptome * upregulated in P. yoelii proteome

These data clearly show that apicoplast fatty acid synthesis, including the FAS II pathway, is highly upregulated in liver stages when compared to other life cycle stages and indicates its importance in this phase of infection. It is likely that this up-regulation is necessary to provide the fatty acyl-CoAs necessary for construction of the phospholipid component of the membranes needed to surround the tens of thousands of merozoites that eventually burst from the liver stage-infected hepatocyte. Strikingly, expression of many of the genes and proteins in the FAS II pathway has not been detected at stages other than the liver stage in P. berghei and P. falciparum (Hall et al. (2005) Science 307:82-6; Le Roch et al. (2003) Science 301:1503-8).

To confirm the results from the transcriptome study, three of the genes in the FAS II elongation pathway, namely, ACP, FabZ and FabG, were analyzed by quantitative realtime PCR (qRT-PCR). RNA was extracted from P. yoelii at different life cycle stages and then used for qRT-PCR. Levels of transcription were normalized to two different house keeping genes, 18s RNA and the 14-3-3 protein. It was evident from the results that a profound increase in FAS II gene expression occurred in liver stages, both 24- and 40- hours after sporozoite infection when compared to blood stages.

When the liver stages are compared, expression at 40 hours after infection is significantly higher than at 24 hours, which is higher than in sporozoites suggesting that the FAS II enzymes are required more during later stage schizogony when growth is at its peak. This result confirms the global transcriptome analysis carried out above, suggesting that the FAS II pathway is crucial for the growth of liver stages. It also suggests that expression in blood stages may not be as significant. Indeed, transcriptional analysis of the eight genes in P. falciparum during the asexual intraerythrocytic life cycle using microarray data (Bozdeck et al. (2003) OLoS BIoI. I:e5; Le Roch et al. (2003) Science 301:1503-8; Llinas et al. (2006) Nucl. Acids. Res. 34:1166-73) available at PlasmoDB (Nucl. Acids Res. 31:212-5), suggests that their level of transcription is low.

Table 3. Quantitative Real-Time PCR Analysis of FAS II genes in P. yoelii life cycle

* using P. yoelii 18S rRNA as standard ** using P. yoelii PYO 1841 as standard

EXAMPLE 2 This Example describes the localization of expression of a Plasmodium FAS II enzyme. The enzyme (Fabl) was expressed throughout the liver stage until merozoite release, at which point expression was undetectable. The cellular expression pattern was consistent with apicoplast localization. Material and Methods

Generation of a Plasmodium yoelii transgenic parasite expressing Fabl-myc under Fabl's endogenous promoter (Py^{FabI myc}), χ₀ epitope tag Fabl, a quadruple (4x) myc tag sequence followed by a stop codon was introduced into the b3D.DT^ΛH.^ΛD vector (Catalog # MRA-80 in the MR4 Malaria Research and Reference Reagent Resource Center; http://www.malaria.mr4.org). Approximately one kilobase pairs (kb) of the 3' untranslated region of P. berghei dihydrofolate reductase thymidylate synthase (DHFR/TS) gene was added to the C-terminus of the 4x myc tag to ensure stability of the recombinant messenger RNA. The P. yoelii Fabl gene (PY03846), including approximately one kb of sequence upstream of the start codon was amplified from P. yoelii 17XNL genomic DNA and cloned in frame (without the stop codon) and upstream of the 4xmyc tag. The resulting plasmid was linearized with Bsal for integration into P. yoelii 17XNL blood stage schizonts using standard procedures (Labaied et al. (2007) Infect. Immun. 75:3758-68). Integration of the plasmid to create py^{FabI myc} g_{ave r}i_se to a parasite line that expressed two copies of Fabl, both with the endogenous promoter and one containing the 4x myc epitope tag.

In vitro analysis of py^{FabI myc} ϋ_ver stages. In vitro assays were conducted using the human hepatoma cell line HepG2 expressing the tetraspanin CD81 (HepG2 CD81) cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37⁰C and 5% CO₂. Infections were done by adding 5 x 10⁴ sporozoites to individual chambers of an eight well chamber slide (Lab-Tek Permanox eight- well chamber slide; Nalge Nunc International, Rochester, NY) which had been seeded with 10⁴ sub-confluent HepG2- CD81 cells the previous day. The slide was then centrifuged at 500 g for 2.5 minutes to aid sporozoite infection. Sporozoites that had failed to invade cells were removed after 2 hours and the media was replaced. For the liver stage development assay, infections were maintained for various time periods after the addition of salivary gland sporozoites. Subsequently the infected cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes then blocked and permeabilized in PBS with 2 % bovine serum albumin and 0.2 % Triton-X 100 (PBS/BSA/Triton) for immunofluorescence assay (IFA). IFA was carried out in PBS/BSA/Triton. The double staining was performed using a mouse anti-P. berghei HSP70 primary antibody, a parasite cytoplasmic marker and a rabbit anti-myc antibody, which recognizes the recombinant apicoplast-expressed Fabl-myc. Fluorescent staining was achieved with Alexa Fluor-conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA) specific to rabbit (Alexa Fluor 488, green) and mouse (Alexa Fluor 594, red) IgG. Cells were stained with 4', 6'-diamidino-2- phenylindole (DAPI) to visualize the DNA and mounted with FluoroGuard anti-fade reagent (Bio-Rad, Hercules, CA). Preparations were analyzed using a fluorescence inverted microscope (Eclipse TE2000-E; Nikon), and images were acquired using Olympus 1X70 Delta Vision deconvolution microscopy. Results

To further investigate the expression of FAS II in the parasite life cycle we generated a transgenic P. yoelii line expressing a myc epitope-tagged Fabl under control of the endogenous Fabl promoter (Py^{FabI myc}j, This allowed visualization of Fabl expression throughout the parasite life cycle using indirect immunofluorescence with anti-myc antibodies. Myc expression was absent in the developing mosquito midgut oocyst and also in oocyst sporozoites. However, myc expression was detected in salivary gland sporozoites and localized to a structure close to the nucleus, presumably the sporozoite apicoplast. To analyze Fabl expression during liver stage development, HepG2:CD81 hepatoma cells (Silvie et al. (2003) Nat. Med. 9:93-6) were infected with the py^{FabI myc} ii_ne At 7-hours post-infection (pi), apicoplast morphology as determined by Fabl-myc staining, was similar to that of salivary gland sporozoites. At 14 hours pi, the parasite nuclear division commenced and the apicoplast exhibited a dumbbell shape. By 24 hours pi, the apicoplast formed a branched lariat- shaped structure, which became more elaborate with advanced liver stage development at 30 hours pi. By 40 hours pi the apicoplast differentiated into hundreds of intertwining structures that appeared to be segregating. These results indicated that there is robust apicoplast- specific Fabl expression in the developing liver stage. Interestingly, the replication of the liver stage apicoplast bears striking similarities to that seen in the apicoplast of developing P. falciparum blood stages with reference to the changing appearance of the organelle (van Dooren et al. (2005) MoI. Microbiol. 57:405-19), but on a more expansive scale. Strikingly, by 48 hours pi, a time point when the liver stage schizont undergoes merozoite formation (Baer et al. (2007) PLoS Pathog. 3:el71), Fabl-myc expression was greatly reduced when compared to 40 hours pi. This strongly suggested that Fabl expression is down-regulated in hepatic (exo-erythrocytic) merozoites before their release into the blood stream. We next investigated whether Fabl is expressed during asexual blood stage replication but were unable to detect Fabl-myc expression in blood stages. The results show that between 2- and 40 hours pi, there is robust expression of

Fabl-myc in what appears to be the apicoplast. Since Fabl-myc expression is under its endogenous Fabl promoter, these results confirm the data from our transcriptome and proteome analysis of FAS II genes and demonstrate the robust expression of the FAS II elongation pathway in liver stages. Surprisingly, at 48 hours post invasion, very little myc staining was detected in most of the parasites visualized, even though highly nucleated bodies were present. This suggests that Fabl expression is turned off in the final stages of liver stage development, presumably because the FAS II pathway has served its purpose and it not needed in the invasive merozoites. Even more surprising was that we were unable to detect any myc staining in blood stages, which suggests that the FAS II pathway (or at least Fabl) is not expressed in blood stages or expressed at levels undetectable by our analysis. Taken as a whole, the expression data led us to hypothesize that FAS II is not essential for blood stage development but may be critical for liver stage development.

EXAMPLE 3 This Example describes that the FAS II pathway is only essential only for

Plasmodium liver stage development. Specifically, targeted deletion of FabB/F, an essential enzyme in fatty acid elongation, resulted in a growth defect late in parasite liver stage development, but did not affect parasite blood stage replication or mosquito stage development. Malaria parasites have two distinct replicating life cycle forms in the mammalian host. A linear replication occurs in the liver after inoculation of sporozoite stages by the bite of an infected mosquito and results in the release of tens of thousands infectious merozoites. These merozoites infect red blood cells and initiate the cyclic replication that leads to malaria disease. The discovery of the apicoplast, a relict plastid organelle in Plasmodium, gave great hope for new drug design to treat malaria. Since the apicoplast is of cyanobacterial origin, drugs that target the unique bacterial- like pathways contained within the organelle are unlikely to affect the mammalian host. One such apicoplast- targeted pathway is bacterial-like type II fatty acid synthesis (FAS II) (Waller et al. (1998) Proc. Natl. Acad. Sci. USA 95:12352-7), the de novo pathway by which Plasmodium synthesizes fatty acids from derivatives of acetate and malonate. The elongation step of FAS II involves four key enzymes - FabB/F, FabG, Fabl and FabZ - and the substrate/product of each reaction is covalently bound to the acyl carrier protein (ACP) co-factor. In contrast, the mammalian FAS I pathway utilizes a single enzyme complex. The four elongation enzymes of the parasite are thus promising drug targets. An early study identified a Plasmodium Fabl and showed that the Fabl inhibitor triclosan kills the blood stage parasites (Surolia & Surolia (2001) Nat. Med. 7:167-73) and subsequently a significant effort has been undertaken to develop blood stage FAS II inhibitors to treat malaria (Gornicki (2003) Int. J. Parasitol. 33:885-96; Sato & Wilson (2005) Curr. Top. Microbiol. Immunol. 295:251-73; Wiesner & Seeber (2005) Expert Opin. Ther. Targets 9:23-44). However, the expression of FAS II had not been studied throughout the parasites complex infection cycle. As described in EXAMPLE 1, we recently carried out a liver stage transcriptome and proteome analysis in the model rodent malaria parasite Plasmodium yoelii and observed that FAS II was up-regulated in the liver stages when compared to the blood stages (Tarun et al. (2008) Proc. Natl. Acad. Sci. USA 105(l):305-10). In addition, we used qRT-PCR to verify this result, also described in EXAMPLE 1. Transcript abundance was analyzed for two of the four FAS II genes involved in fatty acid elongation as well as ACP and was indeed highly increased in liver stages and not in blood stages, suggesting that FAS II is important for liver stage development. We also showed that one of the FAS II enzymes, Fabl is expressed during liver stage development but is greatly reduced at a time point when the liver stage schizont undergoes merozoites formation, as described in EXAMPLE 2.

Anti-malarial drug development efforts for FAS II have focused on parasite blood stages (Goodman & McFadden (2007) Cur. Drug Targets 8:15-30) since it is assumed that FAS II is essential for blood stage parasite replication. However, the relative importance of FAS II throughout the complex parasite life cycle remains unknown. In this Example we show that, in a rodent malaria model, the FAS II pathway enzymes are only critical for parasite liver stages. Targeted deletion of FabB/F did not affect parasite blood stage replication or mosquito stage development. However, after sporozoite transmission, FabB/F knockout parasites failed to emerge from the liver of mice and were unable to initiate blood stage infection. Strikingly, the growth defect was seen late in parasite liver stage development. Our unprecedented findings will refocus FAS II antimalarial drug development efforts to the parasite liver stage. Materials and Methods

Experimental animals. Female Swiss Webster (SW) mice were purchased from Harlan (Indianapolis, IN). Female (6 to 8 weeks old) BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animal handling was conducted according to institutional animal care and use committee-approved protocols. Generation of P. yoelii fabb/f(-) parasites. For the targeted deletion of the FabB/F

(PY04452) genomic locus, two DNA fragments containing approximately 0.8 kb of the 5'UTR and 3'UTR of the gene were amplified using P. yoelii 17XNL genomic DNA as a template. The two fragments were cloned into the b3D.DT^ΛH.^ΛD targeting vector between the Toxoplasma gondii DHFR/TS gene, which allows selection of recombination events with pyrimethamine. The plasmid was transfected into P. yoelii 17XNL blood stage schizonts using standard procedures (Labaied et al. (2007) Infect. Immun. 75:3758- 68). Two independent clones oϊfabb/f(-) parasites were obtained by limited dilution from independent transfection experiments.

Phenotypic analysis of blood stage P. yoelii fabb/f(-) parasites. To assay growth of non- lethal P. yoelii 17XNL wildtype (wt) and fabb/f(-) blood stages, blood was removed from infected SW mice when parasitemia was between 0.5% and 1.5%. The blood was diluted in RPMI- 1640 media (HyClone, Logan, UT) so that 100 μl contained either 10³ or 10⁶ parasites. SW mice (4 in each group) were then injected intravenously with 10³ or 10⁶ parasites (wt or fabb/f(-)). Percentage parasitemia was followed as often as daily until clearance, by assay of Giems a- stained blood smear.

Phenotypic analysis of P. yoelii fabb/fl-) parasites in the mosquito. Anopheles stephensi mosquitoes were infected with P. yoelii fabb/f(-) parasites and control wt parasites by blood feeding for 6 minutes on the first and second day on infected SW mice and subsequently maintained under a cycle of 12.5 hrs light/11.5 hrs dark and 70% humidity at 24.5⁰C. Gametocyte exflagellation capacity was evaluated microscopically before mosquito blood meal. Infected mosquitoes were dissected (at least 20 mosquitoes for each dissection) at days 10 and 14 (after the first infectious blood meal) to determine the presence of midgut oocyst sporozoites and the numbers of salivary gland sporozoites respectively.

In vivo analysis of P. yoelii fabb/f(-) liver stage development. To analyze in vivo sporozoite infection and liver stage development, Balb/c mice were injected intravenously (iv )with 10⁶ wt or fabb/f(-) sporozoites. For each parasite population, the livers were harvested from euthanized mice at several time points post infection (12-, 24-, 44- and 52-hours). Livers were perfused with PBS, washed extensively with PBS and then fixed in 4% paraformaldehyde. Liver lobes were cut into 50 μm sections were using a Vibratome apparatus (Ted Pella Inc., Redding, CA). For IFA, sections were permeabilized in Tris buffered saline (TBS) containing 3% H₂O₂ and 0.25% Triton X-IOO for 30 minutes at room temperature. Sections were then blocked in TBS containing 5% dried milk (TBS-M) at least one hour and incubated with primary antibody in TBS-M at 4°C overnight. Primary antibodies used were mouse anti-CSP, mouse anti-Hepl7 and rabbit anti-MSPl. After washing in TBS, secondary antibody was added in TBS-M for 2 hours at room temperature in a similar manner as above. After further washing, the section was incubated in 0.06% KMnO₄ for 10 minutes to quench background fluorescence. The section was then washed with TBS and cells were stained with DAPI to visualize the DNA and mounted with FluoroGuard anti-fade reagent (Bio-Rad, Hercules, CA). Preparations were analyzed as above for fluorescence with the addition of acquisition of a differential interference contrast image.

Comparison of wt and fabb/f(-) liver stage growth. To compare the sizes of parasite liver stages at 44 hours post-infection, liver sections labeled with Hep 17 (see above) were sequentially scanned using Nikon fluorescence microscopy. The greatest diameter for each liver stage detected was determined by adjusting the z plane of the liver section and the area of the parasite was subsequently determined. For the wildtype, 251 liver stages were assayed and for the fabb/f(-), 147. All parasite segments were less than 3000 mm² and the total number of parasites were divided into quartiles by area. Results To test whether FAS II is required for liver stage development, we deleted P. yoelii FabB/F, the initiating enzyme in the fatty acid elongation cycle, from the parasite genome using a double crossover recombination strategy (Menard & Janse (1997) Methods 13:148-57; Tarun et al. (2007) J. Infect. Dis. 196:608-16). PCR genotyping using specific primers pairs confirmed the recombination event and the deletion of the FabB/F gene in two cloned knockout parasite lines (fabb/f(-)) from two independent transfections. To assay the effect of FabB/F deletion on blood stage development mice were intravenously (iv) injected with IxIO³ or IxIO⁶ fabb/f(-) parasites and blood stage development was followed over time in comparison with wildtype (wt) parasites. There was no significant difference in the growth rate of fabb/f(-) parasites and wt parasites. Normal clearance of the parasite that is observed for this non-lethal P. yoelii strain occurred for both wt and knockout approximately 20 days after infection. This result demonstrates that the loss of FabB/F, and therefore the loss of fatty acid elongation, had no deleterious effect on P. yoelii asexual blood stage replication in vivo. Furthermore, the blood stage fabb/f(-) parasites showed normal gametocyte development and male gamete exflagellation.

After transmission to Anopheles stephensi mosquitoes, we observed normal development of mosquito midgut oocysts, formation of oocyst sporozoites and invasion of sporozoites into the salivary glands as indicated by the enumeration of salivary gland sporozoites in comparison to wt (wt (n=4): 10384 +/- 3803; fabb/f(-) (n=4): 8890 +/- 3565). Therefore, FAS II is not necessary for parasite development in the mosquito.

Next, salivary gland sporozoites were injected iv into Balb/c mice. Mice were injected with 10,000 (n=16) or 100,000 fabb/f(-) sporozoites (n=10). 10 mice were injected with 10,000 wt sporozoites as a control. After 3 days, all mice injected with the WT sporozoites exhibited patent blood stage parasitemia in Giems a- stained blood smears. Strikingly however, mice injected with fabb/fl-) sporozoites did not become blood stage patent (Table 4). This demonstrated that fabb/f(-) parasites are unable to infect the mammalian host via sporozoite inoculation.

Table 4. P. yoelii fabb/f(-) sporozoites are unable to elicit a blood stage infection

^a The mice injected with fabb/f(-) sporozoites were followed for 14 days post- infection and never became patent.

To further investigate the phenotype of fabb/f(-) parasites, Balb/c mice were iv- injected with IxIO⁶ sporozoites (wt or fabb/f(-)) and sacrificed at different time points of liver stage development. The infected livers were perfused with PBS, removed, sectioned and parasite load and development was assayed by immunofluorescence microscopy. At 12- and 24 hours pi, fabb/f(-) liver stages showed normal development that was indistinguishable from wt. However, by 44 hours pi the size of the fabb/f(-) liver stages was significantly less than that of wt. Furthermore, unlike wt, fabb/f(-) liver stages did not show expression of merozoite surface protein 1 (MSPl, Suhrbier et al. (1989) Am. J. Trop. Med. Hyg. 40:351-5). At 52 hours pi, most of the wt parasites had released merozoites or were in the final stages of merozoite formation but, although the fabb/f(-) liver stages had somewhat further increased in size, there was still no significant expression of MSPl and nuclear division was abnormal when compared to wt.

The sporozoite infectivity and liver stage developmental data therefore suggest thaifabb/f(-) parasites are normal in invasion and liver stage development up to a point in late liver stage schizogony. However, fabb/f(-) liver stages never reach maturity and are unable to form infectious merozoites, explaining the lack of onset of blood stage infection from the liver. This is an unprecedented phenotype in late liver stage differentiation. Previously, it was shown that depletion of UIS3 and UIS4 (Mueller et al. (2005) Nature 433:164-7; Mueller et al. (2005) Proc. Natl. Acad. Sci. USA 1102:3022-7), proteins that localize to the parasite-host membrane interface, as well as the sporozoite proteins P52 and P36 (Ishino et al. (2005) MoI. Microbiol. 58:1264-75; Labaied et al. (2007) Infect. Immun. 75:3758-68; van Dijk et al. (2005) Proc. Natl. Acad. Sci. USA 102:12194-9), cause early arrest in liver stage development. The phenotypic data of the fabb/f(-) parasites demonstrate the essential role of FAS II for liver stage development and strongly suggest that FAS II is dispensable for blood stage replication. This finding appears to contradict conclusions from previously published data suggesting that FAS II inhibitors are directly interacting with their apicoplast targets in Plasmodium blood stages (Surolia & Surolia (2001) Nat. Med. 7:167-73; Goodman et al. (2007) MoI. Biochem. Parasitol. 152:181-91; Jones et al. (2005) J. Med. Chem. 48:5932-41; Surolia et al. (2004) Biochem. J. 383:401-12; Tasdemir et al. (2006) J. Med. Chem. 49:3345-53), thereby inhibiting parasite growth. This however might be caused by FAS II drugs acting off- target, as has been previously shown for the effect of triclosan on Trypanosoma brucei (Paul et al. (2004) Eukaryot. Cell 3:855-61). We speculate that malaria parasite blood stages can fuel intra-erythrocytic replication solely by exogenous uptake of fatty acids. However, liver stages might additionally rely on endogenous FAS II synthesis to supply sufficient fatty acids for making the rather large amounts of membrane needed to form more than 10,000 merozoites per schizont.

Although our findings were generated with the rodent malaria parasite P. yoelii, the high conservation of FAS II among Plasmodium species (Carlton et al. (2002) Nature 419:512-9) suggests that FAS II is also essential for P. falciparum liver stage development. This might have consequences for the direction of anti-malaria FAS II inhibitor drug development. Rather than concentrating on the P. falciparum blood stage, research into FAS II inhibitors should concentrate on their efficacy against the clinically silent liver stage of the disease. This may significantly contribute to the goal of eradicating malaria.

EXAMPLE 4

This Example describes that targeted deletion of PDH El -alpha resulted in a growth defect late in parasite liver stage development, but did not affect parasite blood stage replication or mosquito stage development. Unlike most eukaryotes, which have two pyruvate dehydrogenase (PDH) complexes, Plasmodium has a single PDH localized to the apicoplast. Pyruvate dehydrogenase generates the acetyl-CoA needed for fatty acid photosynthesis from pyruvate (Foth et al. (2005) MoI Microbiol. 55:39-53). The PDH complex comprise the El, E2 and E3 subunits that catalyze successive parts of the overall reaction in the conversion of pyruvate into carbon dioxide and acetyl-CoA. To further test whether the de novo fatty acid synthesis is necessary in Plasmodium, we constructed a PDH El-alpha knockout by gene replacement of the PY00819 locus with a pyrimethamine resistant gene marker.

Our results indicate that targeted deletion of PDH El -alpha gene did not affect parasite blood stage replication or mosquito stage development. However, after sporozoite transmission, the PDH El -alpha knockout parasites failed to emerge from the liver of mice and were unable to initiate blood stage infection. The growth defect was seen late in parasite liver stage development and showed similar phenotypes as the FabB/F knockout parasite described in EXAMPLE 3. Materials and Methods

Experimental animals. Female Swiss Webster (SW) mice were purchased from Harlan (Indianapolis, IN). Female (6 to 8 weeks old) BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animal handling was conducted according to institutional animal care and use committee-approved protocols.

Generation of P. yoelii PDH el-alpha(-) parasites. For the targeted deletion of the

PDH El-alpha (PY00819) genomic locus, two DNA fragments containing approximately 0.5 kb of the 5'UTR and 3'UTR of the gene were amplified using P. yoelii 17XNL genomic DNA as a template. The two fragments were cloned into the b3D.DT^ΛH.^ΛD targeting vector between the Toxoplasma gondii DHFR/TS gene, which allows selection of recombination events with pyrimethamine. The plasmid was transfected into P. yoelii

17XNL blood stage schizonts using standard procedures (Labaied et al. (2007) Infect. Immun. 75:3758-68). Two independent clones of PDH el-alpha(-) parasites were obtained by limited dilution from independent transfection experiments.

Phenotypic analysis of blood stage PDH el-alpha(-) parasites. To assay growth of non- lethal P. yoelii 17XNL wildtype (wt) and PDH el-alpha(-) blood stages, blood was removed from infected SW mice when parasitemia was between 0.5% and 1.5%. The blood was diluted in RPMI- 1640 media (HyClone, Logan, UT) so that 100 μl contained either 10³ or 10⁶ parasites. SW mice (4 in each group) were then injected intravenously with 10³ or 10⁶ parasites (wt or PDH el-alpha(-). Percentage parasitemia was followed as often as daily until clearance, by assay of Giems a- stained blood smear.

Phenotypic analysis of P. yoelii PDH el-alpha(-) parasites in the mosquito. Anopheles stephensi mosquitoes were infected with P. yoelii PDH el-alpha(-) parasites and control wt parasites by blood feeding for 6 minutes on the first and second day on infected SW mice and subsequently maintained under a cycle of 12.5 hrs light/11.5 hrs dark and 70% humidity at 24.5⁰C. Gametocyte exflagellation capacity was evaluated microscopically before mosquito blood meal. Infected mosquitoes were dissected (at least 20 mosquitoes for each dissection) at days 10 and 14 (after the first infectious blood meal) to determine the presence of midgut oocyst sporozoites and the numbers of salivary gland sporozoites respectively. In vivo analysis of P. yoelii PDH el-alpha(-) liver stage development. To analyze in vivo sporozoite infection and liver stage development, Balb/c mice were injected iv with 10⁶ wt or PDH el-alph (-) sporozoites. For each parasite population, the livers were harvested from euthanized mice at several time points post infection (12-, 24-, 44- and 52-hours). Livers were perfused with PBS, washed extensively with PBS and then fixed in 4% paraformaldehyde. Liver lobes were cut into 50 μm sections were using a Vibratome apparatus (Ted Pella Inc., Redding, CA). For IFA, sections were permeabilized in Tris buffered saline (TBS) containing 3% H₂O₂ and 0.25% Triton X-IOO for 30 minutes at room temperature. Sections were then blocked in TBS containing 5% dried milk (TBS-M) at least one hour and incubated with primary antibody in TBS-M at 4°C overnight. Primary antibodies used were mouse anti-CSP, mouse anti-Hepl7 and rabbit anti-MSPl. After washing in TBS, secondary antibody was added in TBS-M for 2 hours at room temperature in a similar manner as above. After further washing, the section was incubated in 0.06% KMnO₄ for 10 minutes to quench background fluorescence. The section was then washed with TBS and cells were stained with DAPI to visualize the DNA and mounted with FluoroGuard anti-fade reagent (Bio-Rad, Hercules, CA). Preparations were analyzed as above for fluorescence with the addition of acquisition of a differential interference contrast image. Results To test whether apicoplast fatty acid synthesis is required for liver stage development, we deleted P. yoelii PDH el-alpha(-), a subunit of the PDH complex that generates the acetyl-CoA precursor for fatty acid biosynthesis, from the parasite genome using a double crossover recombination strategy (Menard & Janse (1997) Methods 13:148-57). PCR genotyping using specific primers pairs confirmed the recombination event and the deletion of the PDH el -alpha gene in two cloned knockout parasite lines. To assay the effect of PDH el-alpha(-) deletion on blood stage development mice were intravenously (iv) injected with IxIO³ or IxIO⁶ PDH el-alpha(-) parasites and blood stage development was followed over time in comparison with wildtype (wt) parasites. There was no significant difference in the growth rate of PDH el-alpha(-) parasites and wt parasites. This demonstrates that the loss of PDH el -alpha, and therefore the loss of the acetyl CoA precursor, had no deleterious effect on P. yoelii asexual blood stage replication in vivo. Furthermore, the blood stage PDH el-alpha(-) parasites showed normal gametocyte development and male gamete exflagellation.

We observed normal development of PDH el-alpha(-) parasite in Anopheles stephensi mosquitoes. The midgut oocysts, formation of oocyst sporozoites and invasion of sporozoites into the salivary glands is similar to wildtype (data not shown). Thus, acetyl CoA production, and therefore de novo fatty acid synthesis, is not necessary for parasite development in the mosquito.

Next, salivary gland sporozoites were injected iv into Balb/c mice. Mice were injected with 10,000 (n=20), 50,000 (n=15), or 1,000,000 PDH el-alpha(-) sporozoites (n=2). 10 mice were injected with 10,000 wt sporozoites as a control. After 3 days, all mice injected with the WT sporozoites exhibited patent blood stage parasitemia in Giems a- stained blood smears. Strikingly however, mice injected with PDH el-alpha(-) sporozoites did not became blood stage patent even when injected with 1 million sporozoites (Table 5). This demonstrated that PDH el-alpha(-) parasites are unable to infect the mammalian host via sporozoite inoculation.

Table 5. P. yoelii PDH el-alpha(-) sporozoites are unable to elicit a blood stage infection

a The mice injected with PDH el-alpha(-) sporozoites were followed for 14 days postinfection and never became patent.

To further investigate the phenotype of PDH el-alpha(-) parasites, Balb/c mice were iv injected with IxIO⁶ sporozoites (wt or PDH el-alpha(-)) and sacrificed at different time points of liver stage development. The infected livers were perfused with PBS, removed, sectioned and parasite load and development was assayed by immunofluorescence microscopy. The in vivo liver stage development of PDH el- alpha(-) parasites was similar to fabb/f(-) (see EXAMPLE 3). At 12- and 24 hours pi, PDH el -alpha (-) liver stages showed normal development that was indistinguishable from wt. However, by 44 hours pi the size of the PDH el-alpha(-) liver stages was significantly less than that of wt.

The sporozoite infectivity and liver stage developmental data therefore suggest that PDH el-alpha(-) parasites are normal in invasion and liver stage development up to a point in late liver stage schizogony. Like fabb/f(-), PDH el-alpha(-) liver stages never reach maturity and are unable to form infectious merozoites, explaining the lack of onset of blood stage infection from the liver. This is an unprecedented phenotype in late liver stage differentiation. The phenotypic data of the PDH el-alpha(-) parasites demonstrate the essential role of acetyl CoA, which is required in the FAS II pathway, for liver stage development and strongly suggest that FAS II is dispensable for blood stage replication.

EXAMPLE 5 This Example describes that Plasmodium sporozoites with targeted deletions of

FabB/F or PDH El -alpha confer sterile protection against subsequent challenge with wildtype sporozoites.

We tested whether Pyfabb/β-) or PyPDH el-alpha(-) salivary gland sporozoite immunization of mice can induce sterile protection against wildtype (wt) P. yoelii sporozoite challenge. The generation of Pyfabb/β-) and PyPDH el-alpha(-) parasites is described in EXAMPLES 3 and 4, respectively. For challenge and immunizations, BALB/c mice were intravenously (iv) injected with sporozoites resuspended in incomplete DMEM-F12 medium. Blood stage patency was monitored daily by evaluation of Giems a- stained blood smears from day 3 to day 14 post sporozoite infection. One group of BALB/c mice was immunized iv with three doses of 10,000

Pyfabb/β-) or PyPDH el-alpha(-) salivary gland sporozoites, in two week intervals, then challenged with wt sporozoites 7 days after the last boost (Group I Table 6). One group of BALB/c mice was immunized iv with three doses of 10,000 Pyfabb/β-) or PyPDH el- alpha(-) salivary gland sporozoites, in two week intervals, then challenged with wt sporozoites 30 days after the last boost (Group II Table 6). One group of BALB/c mice each was immunized iv with a single does of 10,000 Pyfabb/f(-) or PyPDH el-alpha(-) salivary gland sporozoites without any booster shots, then challenged with wt sporozoites 7 days after immunization (Group III Table 6). One group of BALB/c mice was immunized with a single dose of 50,000 PyPDH el-alpha(-) salivary gland sporozoites without any booster shots, then challenged with wt sporozoites 7 days after immunization (Group IV, Table 6).

Table 6. Immunization with Pyfabb/f(-) and PyPDH el-alpha(-) sporozoites confers sterile protection against wildtype sporozoite challenge

* days after last boost

** naϊve mice controls became blood stage patent on day 3 after wildtype sporozoite challenge

All mice were protected when challenged with wt sporozoites and did not develop any blood stage infection (Table 6). The data demonstrate that PyfabbZf(-) or PyPDH el- alpha(-) salivary gland sporozoite immunizations induce sterile immunity against subsequent .PyWT sporozoite infection, even with a single dose of 10,000 sporozoites. EXAMPLE 6

This Example describes the inhibition of growth of Plasmodium liver stages by FAS II inhibitors (see also Tarun et al. (2008) Proc. Natl. Acad. Sci. USA 105(l):305-10, herein incorporated by reference). Materials and Methods

Drug inhibition studies were carried out in vitro by using HepG2:CD81 hepatoma cells infected with PyGFP parasites. The HepG2:CD81 cells were plated on a 48-well plates (7.5 x 10⁴ cells per well; Corning) coated with a 0.03 mg/ml ECL Cell Attachment Matrix (Millipore) solution. Cells were grown in a closed incubator at 37⁰C in 5% CO₂. Using a 48-well plate, each well was infected with 5.0 x 10⁴ sporozoites isolated from salivary gland dissections of PyGFP infected mosquitoes. The plate was centrifuged at 1,000 RPM for 3 min and left to incubate at 37⁰C for 2 h. The cells were carefully washed twice with incomplete DMEM/F12 to remove mosquito debris and sporozoites that had not invaded the cells and afterward replaced with 750 ml of complete DMEM/F12 medium containing various concentrations of hexachlorophene, rifampicin, or tetracycline prepared from stock concentrations made in DMSO. PyGFP parasites were grown in HepG2:CD81 culture at 37⁰C with daily media changes containing the corresponding drugs to ensure the drug concentrations remained constant for 40 h.

After 40 h, the infected hepatoma cells were detached from the flasks using TrypLE (Invitrogen). For each sample, the detached cells were transferred to a separate 12 x 75-mm polypropylene tube containing 300 ml of complete DMEM/F12 medium. The tubes were briefly centrifuged at 1,000 RPM for 1 min, and the supernatant was discarded. Each sample cell pellet was resuspended in 200 ml of complete DMEM/F12 medium containing the cell viability marker 7-aminoactinomycin D (7AAD; Invitrogen). PyGFP liver stage infection and host cell viability were simultaneously determined using flow cytometry analysis using the Cytopeia flow cytometer using the Spigot Operating Software Version 5.0.3.1 (Cytopeia). The Influx Sorter was equipped with a 488 nM Coherent Sapphire 488 nM, 200 mWa CDRH Laser. Analyses of data were performed using the flow cytometry analysis programs FlowJo Version 7.0.3 (TreeStar). Measurements were normalized against an untreated PyGFP culture. IC₅₀, the inhibitor concentration at which the LS parasite growth was reduced by 50% compared to untreated LS infections, was estimated from four independent experiments and calculated by nonlinear regression using the Prism 4 program (GraphPad).

Results

We first tested the effects on LS parasite growth of hexachlorophene, a FAS II inhibitor that inhibits beta-oxoacyl-ACP reductase (FabG) (Wickramasinghe et al. (2006) Biochem J. 393:447-57) and triclosan, a Fabl inhibitor (Sivaraman et al. (2004) J. Med. Chem. 47:509-18). Tables 5 and 6 show the inhibition of LS parasite growth by triclosan and hexachlorophene, respectively. The results show that both triclosan and hexachlorophene inhibited liver stage development with IC₅₀ values of 32 μM and 5.0 μM, respectively. For comparison, the IC₅₀ for primaquine on P. yoelii liver stages has been reported to be 0.64 μM (Carraz et al. (2006) PLoS 3(12):e513).

Table 7. Inhibition of f, yoelii liver stage growth by triclosan

Table 8. Inhibiton of f, yoelii liver stage growth by hexachlorophene

We next tested the effects on LS parasite growth of cerulenin and thiolactomycin.

The IC₅₀ values for thiloactomycin and cerulenin were determined to be more than 200 μM. EXAMPLE 7

This Example describes in vitro and in vivo growth inhibition assays for Plasmodium liver stages.

Assay using transgenic P. yoelii parasites expressing luciferase. Our experiments using antibodies and the Odyssey phosphorimager to detect and count LS parasites in microtiter plates shows that an in vitro drug screening assay is feasible. In order to increase throughput and reduce costs, in vitro LS drug inhibition assays using transgenic

P. yoelii parasites constitutively expressing luciferase (PyLuc) or GFP (PyGFP) are used.

Luciferase routinely provides very high sensitivity (a critical factor when there are only 100 LS parasites/well), rapid readout, and is amenable to high throughput approaches. In vitro LS drug inhibition assays using GFP-transgenic parasites (Tarun et al. (2006) Int. J.

Parasitol. 36:1283-93) are described in EXAMPLE 6.

Transgenic PyLuc parasites that express luciferase during the LS are prepared by integrating a plasmid expressing firefly luciferase under the control of the constitutive EF-Ia promoter into the rRNA locus.

The LS growth inhibition assay is performed by infecting HepG2:CD81 cells in 96- well plates with PyLuc or PyGFP sporozoites. Uninvaded sporozoites are removed by washing with complete medium 3 hours post- infection. LS-infected hepatoma cells are exposed to serial dilutions of test compounds, in triplicate. Atovaquone (Srivastava et al. (1997) J. Biol. Chem. 272(7):3961-6) is used as the positive control; negative controls are untreated infected cells and untreated, uninfected cells. The plates are incubated at 37°C with daily changes of media with drug to ensure that the drug concentration remains stable. Forty hours post-infection, the luciferase activity in each well is determined using commercially available reagents and a plate luminometer. Fluorescence of PyGFP LS is assayed using a plate fluorimeter. IC₅₀ and IC₉₀ values are calculated by non-linear regression. Assay parameters such as the sporozoite inoculum and incubation times are optimized.

Drugs with known activity profiles are tested against LS and blood stage parasites and the IC₅₀ values determined via the new assays are compared with those determined by qRT-PCR of parasite 18S rRNA or manual counting of LS parasites. In addition, drug combinations are tested to identify synergistic or antagonistic effects. Assays using the BD LSRII Multiwell AutoSampler flow cytometer. In parallel to the assay described above, a high throughput flow-based in vitro assay to test the anti- LS drugs using HepG2:CD81 hepatoma cells infected with PyGFP parasites is used. The HepG2:CD81 cells are plated on 96-well flat bottomed ECL-coated tissue culture plates (50,000 cells/well) and grown in a closed incubator at 37°C and 5% CO2. Each well is infected with 25,000 PyGFP sporozoites and the infected cells are treated with increasing concentrations of the candidate drugs. The plates are incubated at 37°C with daily media changes containing the drug to ensure its concentration remains stable. After 40 hrs, the cells are trypsinized and transferred to 96-well v-bottom plates using a multi-channel pipet. After the cells are washed with PBS, the cell pellet will be resuspended in 200 μl media containing 7-AAD live-dead stain. The plate is loaded onto the BD Multiwell Autosampler and both GFP (to assess PyGFP LS infection) and 7-AAD (host cell viability) fluorescence are measured using the BD FACS LSRII instrument and the FACS DIVA and FLOW-JO software. The BD HTS automatically introduces samples into the LSRII; data acquisition is <15 or 44 minutes per plate when sample volumes under 20 or 200 μl per well are analyzed, respectively.

In vitro P. falciparum assay. To test drugs against P. falciparum liver stages, an in vitro inhibition assay for P. falciparum LS parasites is used, similar to the P. yoelii assay described above, using immunofluorescence or quantitative RT-PCR to assess inhibition. For immunofluorescence studies, transgenic P. falciparum sporozoites expressing luciferase encoded by a genomic copy of the luciferase gene with a promoter active in liver stages are used to infect human HC-04 cells (Sattabongkot et al. (2006) Am. J. Trop. Med. Hyg. 74(5):708-15). The other assay parameters are as described above and in EXAMPLE 6. In vitro P. vivax and P. ovale assays. To test drugs against P. vivax liver stages, an in vitro inhibition assay for P. vivax LS parasites is used. P. vivax sporozoites isolated from infected human or primate subjects are used to infect human HC-04 cells. The other assay parameters are as described above and in EXAMPLE 6. To test drugs against P. ovale liver stages, an in vitro inhibition assay for P. vivax LS parasites is used. P. vivax sporozoites isolated from infected human or primate subjects are used to infect human HC-04 cells. The other assay parameters are as described above and in EXAMPLE 6. In vivo P. yoelii assay. To test the efficacy of FAS II inhibitors on LS in vivo and to measure causal prophylactic activity, BALB/c mice will be treated with inhibitor compounds (separately or in combination) at -1, 0, +1 and +2 days after inoculation of 10,000 PyGFP sporozoites by intravenous injection. A control group is treated with PBS. Parasite burden in the infected livers of a subset of mice is assessed by real-time quantitative PCR analysis using specific primers against P. yoelii 18S rRNA. The remaining mice are assessed for blood stage patency.

Each of the references cited herein is hereby incorporated by reference. While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for inhibiting the growth of liver stage Plasmodium parasites in a vertebrate subject, comprising the step of administering an effective amount of an inhibitor of apicoplast fatty acid synthesis to a vertebrate subject in need thereof.

2. The method of Claim 1, wherein the inhibitor of apicoplast fatty acid synthesis is a type II fatty acid synthesis pathway inhibitor.

3. The method of Claim 1, wherein the inhibitor of apicoplast fatty acid synthesis is an inhibitor of at least one of ACP, FabB/F, FabD, FabG, FabH, Fabl, and FabZ.

4. The method of Claim 1, wherein the inhibitor of apicoplast fatty acid synthesis is an inhibitor of at least one of ACCase, PHD El -alpha, PDH El -beta, PDH E2, PDH E3, and a PEP/phosphate translocator.

5. The method of Claim 1, wherein the Plasmodium parasites are one of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale parasites.

6. The method of Claim 1, wherein the vertebrate subject is a human subject.

7. A method for preventing malaria in a vertebrate subject, comprising the step of administering to a vertebrate subject prior to the appearance of blood stage Plasmodium parasites an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit fatty acid synthesis by liver stage Plasmodium parasites in the subject.

8. The method of Claim 7, wherein the inhibitor of apicoplast fatty acid synthesis is an inhibitor of at least one of ACP, FabB/F, FabD, FabG, FabH, Fabl, and FabZ.

9. The method of Claim 7, wherein the inhibitor of apicoplast fatty acid synthesis is an inhibitor of at least one of ACCase, PHD El -alpha, PDH El -beta, PDH E2, PDH E3, and a PEP/phosphate translocator.

10. The method of Claim 7, wherein the Plasmodium parasites are one of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale parasites.

11. The method of Claim 7, wherein the vertebrate subject is a human subject.

12. A method for treating a Plasmodium infection in a vertebrate subject, comprising the step of administering to a vertebrate subject in need thereof an amount of an inhibitor of apicoplast fatty acid synthesis effective to inhibit fatty acid synthesis by the liver stage Plasmodium parasite in the subject.

13. The method of Claim 7, wherein the inhibitor of apicoplast fatty acid synthesis is an inhibitor of at least one of ACP, FabB/F, FabD, FabG, FabH, Fabl, and FabZ.

14. The method of Claim 7, wherein the inhibitor of apicoplast fatty acid synthesis is an inhibitor of at least one of ACCase, PHD El -alpha, PDH El -beta, PDH E2, PDH E3, and a PEP/phosphate translocator.

15. The method of Claim 7, wherein the Plasmodium parasites are one of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale parasites.

16. The method of Claim 7, wherein the vertebrate subject is a human subject.