WO2013108272A2 - Blood stage malaria vaccine - Google Patents

Blood stage malaria vaccine Download PDF

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WO2013108272A2
WO2013108272A2 PCT/IN2013/000041 IN2013000041W WO2013108272A2 WO 2013108272 A2 WO2013108272 A2 WO 2013108272A2 IN 2013000041 W IN2013000041 W IN 2013000041W WO 2013108272 A2 WO2013108272 A2 WO 2013108272A2
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pfrh5
pff2
pfrh2
pfrh4
msp
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PCT/IN2013/000041
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French (fr)
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WO2013108272A3 (en
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Deepak GAUR
Virander Singh CHAUHAN
Chetan Chitnis
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International Centre For Genetic Engineering And Biotechnology
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Priority to IN177DE2012 priority
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Publication of WO2013108272A3 publication Critical patent/WO2013108272A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/002Protozoa antigens
    • A61K39/015Hemosporidia antigens, e.g. Plasmodium antigens
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445Plasmodium
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/408Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa
    • Y02A50/411Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa of the genus Plasmodium, i.e. Malaria
    • Y02A50/412Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa of the genus Plasmodium, i.e. Malaria the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera

Abstract

The invention relates to a blood stage malaria vaccine. The vaccine comprises merozite antigens. The merozite antigens are selected from the group consisting of PfF2, PfRHl, PfRH2, PfRH4, PfRH5, AARP and PfMSP-fusion. The vaccine targets merozite surface proteins and the erythrocyte binding domains of multiple parasite adhesins blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. The inventions also provide a method of treatment wherein the antigens block the sialic acid independent as well as sialic acid dependent binding of the parasite P. falciparum to the erythrocyte.

Description

BLOOD STAGE MALARIA VACCINE

FIELD OF THE INVENTION

The invention relates to a blood stage malaria vaccine. In particular the invention provides a malaria parasite neutralizing vaccine based on a combination of novel P. falciparum blood stage merozoite antigens that play an important role in erythrocyte invasion. The vaccine targets merozoite surface proteins and the erythrocyte binding domains of multiple parasite adhesins blocking their interaction with their receptors and thus inhibiting erythrocyte invasion.

BACKGROUND OF INVENTION

Malaria is a leading public health threat with almost three billion people at risk of contracting the disease. Plasmodium falciparum, the causative pathogen of the most severe form of malaria, accounts for an estimated 250 million cases worldwide that lead to 800,000 deaths annually particularly among young children and pregnant women in the world's most impoverished continent of Africa (WHO. (2008 World Malaria Report). An effective vaccine against malaria would be a great asset in controlling and eliminating the disease. Malaria vaccine development has been hindered by the enormous complexity of the parasite and its life cycle, widespread antigenic polymorphisms and inadequate knowledge of host-parasite interactions.

The life cycle of P. falciparum involves two hosts, the Anopheles mosquito and the human host [Bray, R. S. et al, (1982) Br Med Bull 38, 117-22]. During a blood meal, the Plasmodium infected mosquito injects parasites (sporozoites) into the human host, which migrate to the liver where they invade hepatocytes (Pre-erythrocytic phase/liver stage). In the hepatocytes, the sporozoite differentiates and divides to form an exo-erythrocytic schizont containing thousands of invasive merozoites that are released in the bloodstream and further invade erythrocytes (blood stage). In the erythrocyte the merozoite develops through different stages and multiplies over a 48 hour cycle. Thereafter, the infected cell undergoes egress and releases 16-32 merozoites which further invade uninfected erythrocytes. During the blood stage, some parasites form gametocytes that are taken up by the mosquito during a blood meal. The sexual stage of the parasite life cycle is completed in the mosquito. The blood stage of the life cycle is responsible for all the clinical symptoms associated with malaria. The global efforts to develop malaria vaccines are based on few parasite antigens expressed at different stages of the parasite life cycle that have been tested individually in clinical trials [Crompton PD et al, (2010) J Clin Invest. Dec 1 ; 120(12):4168-78]. The leading P. falciparum malaria vaccine candidate, RTS,S, targets the pre-erythrocytic stage, is currently being tested in a Phase III trial and yields 30-50% efficacy against clinical malaria [Regules JA et. a/.,(2011) Expert Rev Vaccines, May;10(5):589-99].. Thus, there is a clear need to develop vaccines with higher efficacy. Vaccines that target the blood stage of the malaria parasite would help protect against clinical malaria and reduce risk of severe malaria and death. Unfortunately, the leading blood stage vaccine candidates based on merozoite surface protein-1 (MSP1) and apical merozoite antigen-1 (AMA1) have not shown any protective efficacy in field trials (Spring MD et ah, PLoS One 4(4): e5254 and Ogutu BR et al, PLoS One. 4(3): e4708). The lack of efficacy in field trials with MSP1 and AMA1 suggests that targeting individual parasite proteins involved in the complex process of erythrocyte invasion is unlikely to be effective as P. falciparum is known to exhibit redundancy and invades erythrocytes through multiple pathways. Moreover, the presence of extensive sequence polymorphisms in MSP1 and AMA1 is also thought to be responsible for the lack of efficacy against P. falciparum field isolates (Ferreira MU et al., Gene. Jan 30; 304:65-75 and Duan J et al, Proc Natl Acad Sci U S A 2008,105:7857-7862).

US Application 20090175895 provides compositions comprising antigens PfRH and EBA-175 of P. falciparum.

Indian Patent Application No. 1737/DEL/2008 discloses a fusion protein malaria vaccine comprising of P. falciparum merozoite surface protein-1 (PfMSP-l 19) and P. falciparum merozoite surface protein-3 (PfMSP-3n) and the process of preparation and expression of said protein.

Indian Patent Application No. 1844/DEL/2008 discloses the asparagine rich P. falciparum merozoite protein (PfAARP) as a malarial vaccine; a recombinant protein prepared from said protein and the process of preparation of said protein.

Apart from MSP-1 and AMA-1, there are not many essential parasite ligands involved in P. falciparum erythrocyte invasion. Two families of P. falciparum erythrocyte binding proteins - EBA (Erythrocyte binding antigens) and PfRH (P. falciparum reticulocyte binding-like homologous proteins) have been identified as major determinants of erythrocyte invasion (Gaur D et al., Curr. Opin. Microbiol. 14:422-428; Gaur D et al., Int. J. Parasitol. 34:1413-1429; Cowman AF et al, Cell 124:755-766). However, due to redundancy, EBA or PfRH proteins (with the exception of Pf H5) are not essential for the parasite and their antibodies individually do not block invasion in a strain- transcending manner (Gaur D et al, Curr. Opin. Microbiol. 14:422-428; Gaur D et al, Int. J. Parasitol. 34:1413-1429; Cowman AF et al, Cell 124:755-766; Hayton K et al, Cell Host Microbe 4:40-51 ; Baum J et al, Int. J. Parasitol. 39:371-380; Crosnier C et al, Nature 480:534-537; Douglas AD et al, Nat. Commun. 2:601; Lopaticki S et al, Infect. Immun. 79: 1 107-11 17)

P. falciparum has the ability to switch its invasion phenotype (Stubbs J et al, Science 309:1384-1387; Gaur D et al, Mol. Biochem. Parasitol. 145:205-215) and generate polymorphisms to enable immune escape. Thus, targeting single antigens is unlikely to be effective for blood stage malaria vaccines. Analogous to the anti-malarial combinatorial drugs administered to prevent onset of drug resistance (White NJ., Parasitologia. 41 :301- 308), a combination vaccine approach that targets multiple antigens may be more effective in limiting the parasite's ability to escape host immunity. Therefore, the inventors of present invention have taken an approach wherein a receptor blocking blood stage malaria vaccine is based on targeting the functional erythrocyte binding domains of key merozoite ligands involved in erythrocyte invasion, which would simultaneously block diverse invasion pathways and overcome the redundancy exhibited by P. falciparum producing a significant inhibition of erythrocyte invasion.-Another effective approach undertaken by the inventors is to target both merozoite surface proteins and key parasite ligands, which produce highly potent inhibition of erythrocyte invasion at much lower antibody concentrations.

Recently, few reports have demonstrated that targeting combinations of merozoite antigens (including EBA and PfRH) yielded potent invasion inhibition (Lopaticki S et al, Infect. Immun. 79: 1107-1117; Chen L et al, PLoS Pathog. 7:el002199; Arumugam TU et al, Infect. Immun. 79:4523^532). However, these studies did not demonstrate invasion inhibitory efficacy against heterologous P. falciparum clones, which exhibit different invasion phenotypes. Demonstration of potent strain-transcending invasion inhibition against multiple P. falciparum clones is a major prerequisite for taking any candidate antigen into vaccine development. Another important challenge is to identify potent antigen combinations from the large expanding repertoire of merozoite ligands that are involved in erythrocyte invasion. To identify potent antigen combinations, it is not feasible to co-immunize all possible antigen mixtures and assay their invasion inhibitory activity. Recently, potent parasite neutralizing antibodies have been raised using viral vector based DNA vaccines, which are effective both individually and in combinations (Douglas AD et al., Nat. Commun. 2:601). However, considering that the target product profile of malaria vaccines is aimed at young infants and children, it is important to test different delivery platforms so as to identify the safest, most efficacious vaccine that is also easy to administer under mass immunization. While, live viral vectored vaccines have their own advantages, their limitations are pre-existing immunity prevalent in human populations particularly against adenoviruses and most importantly the potential interference between immunizing a number of viral vector vaccines against different diseases. The subunit vaccine approach based on formulations of recombinant proteins and adjuvant poses as a safe and effective platform for administering a vaccine for large masses. Here the challenge lies in being able to produce the recombinant protein with a structural integrity that yields potent neutralizing antibodies against the respective pathogens.

Accordingly, there is still a need for the development of a vaccine that can provide an effective protection against malarial parasite.

OBJECTS OF THE INVENTION

It is an object of the invention to target multiple parasite proteins to simultaneously block multiple pathways and achieve synergistic inhibition of the invasion process.

It is another object of the invention to address the polymorphisms for development of a vaccine effective against diverse field isolates

It is another object of the invention to provide a blood stage vaccine for the treatment and prevention of malaria.

It is another object of the invention to provide full length recombinant protein PfRH5.

SUMMARY OF INVENTION

The present invention provides a blood stage malaria vaccine comprising merozoite antigens. In particular, the vaccine comprises a combination of merozoite antigens.

In one embodiment, the merozoite antigens are selected from the group consisting of PfF2, PfRHl, PfRH2, PfRH4, PfRH5, AARP and PfMSP-fusion.

In an embodiment, the PfMSP-fusion (MSPli9-MSP3) antigen encodes a protein having SEQ ID 19 comprises a conserved 19 kda C-terminal region of MSP-1 and a functional 24 kDa region of MSP-3. In another embodiment, the PfRHl antigen encodes a protein having SEQ ID 3 comprises an amino acid region 500-833 of the 350 kDa native parasite protein having 2971 amino acids.

In another embodiment, the PfRH2 antigen encodes a protein having SEQ ID 24 comprises the amino acid region 495-860 of the 375 kDa native parasite protein having 3310 amino acids.

In another embodiment, the PfRH4 antigen encodes a protein having SEQ ID 7 comprises the amino acid region 328-588 of the 250 kDa native parasite protein having 1716 amino acids.

In another embodiment, the PfRH5 antigen encodes a protein having SEQ ID 11 comprises the amino acid region 28-526 of the 66 kDa native parasite protein having 526 amino acids.

In another embodiment, the PfAARP antigen encodes a protein having SEQ ID 14 comprises the amino acid region 20-107 of the 24 kDa native parasite protein having 217 amino acids.

In another embodiment, the PfF2 antigen encodes a protein having SEQ ID 17 comprises the amino acid region 447-795 of the 175 kDa native parasite protein having 1502 amino acids.

In another embodiment, the vaccine comprises of two or more merozoite antigens. In the preferred embodiment, the vaccine comprises of three merozoite antigens.

In another embodiment, the merozoite antigens are selected from the group consisting of PfAARP + PfRH5 + PfF2, PfAARP + PfRH5 + PfRHl, PfAARP + PfRH5 + PfRH2, PfAARP + PfRH5 + PfRH4, PfAARP + PfRH2 + PfF2, PfAARP + PfRH2 + PfRH4, PfAARP + PfRH2 + PfRHl, PfAARP + PfRHl + PfRH4, PfAARP + Pff2 + PfRHl, PfAARP + PfF2 + PfRH4, PfRH5 + PfRH2 + PfF2, PfRH5 + PfRH2 + PfRHl, PfRH5 + PfRH2 + PfRH4, PfRH5 + PfF2 + PfRHl , PfRH5 + PfF2 + PfRH4, PfRH5 + PfF2 + PfRH2, PfRH5 + PfRHl + PfRH4, PfRHl + PfRH2 + PfRH4, PfF2 + PfRHl + PfRH4, PfF2 + PfRH2 + PfRH4.

In another embodiment, the merozoite antigens produce a potent invasion inhibition at an an IgG content of 9-14 mg/ml.

In another embodiment, the merozoite antigens are selected from the group consisting of MSP(Fusion) + PfAARP + PfRH5, MSP(Fusion) + PfAARP + PfRH2, MSP(Fusion) + PfAARP + PfRHl, MSP(Fusion) + PfAARP + PfRH4, MSP(Fusion) + PfAARP + PfF2, MSP(Fusion) + PfRH5 + PfRH2, MSP(Fusion) + PfRH5 + PfRHl, MSP(Fusion) + PfRH5 + PfRH4, MSP(Fusion) + PfRH5 + PfF2, MSP(Fusion) + PfRH2 + PfRHl, MSP(Fusion) + PfRH2 + PfRH4, MSP(Fusion) + PfRH2 + PfF2, MSP(Fusion) + PfRHl + PfRH4, MSP(Fusion) + PfRHl + PfF2, MSP(Fusion) + PfRH4 + PfF2.

In another embodiment, the merozoite antigens produce a potent invasion inhibition at an IgG content of 1.5-6 mg/ml.

In another embodiment, the merozoite antigens provided in the present invention block the sialic acid independent as well as sialic acid dependent binding of the parasite P. falciparum to the erythrocyte.

In yet another embodiment, the present invention provides a malaria vaccine composition comprising two or more erythrocyte binding merozoite antigens.

In one embodiment the malaria vaccine composition comprises pharmaceutically acceptable excipients and/or adjutants.

In another embodiment, the merozite antigens are selected from the group selected from PfF2, PfRHl , PfRH2, PfRH4, PfRH5, AARP and Pf SP-fusion.

In yet another embodiment, the invention provides a full length recombinant PfRH5 protein having SEQ ID No. 11

In another embodiment, the invention provides a method of preparation of recombinant PfRH5 protein in E. coli, wherein the method comprises inserting a full length DNA sequence of SEQ ID 10 in the E. coli expression vector pET-24b; transforming E. coli BL21 (DE3) cells with the vector; culturing the transformed cells in super broth at 37°C; inducing the cultured cells with ImM IPTG so that OD600 is around 0.8-0.9; harvesting the cells as pellets by centrifugation at 3000g; and obtaining the protein as inclusion bodies by cell lysis of the pellets .

In another embodiment, the method comprises inserting the DNA sequence downstream of the T7 promoter.

In another embodiment, the E. coli expression vector pET-24b has 6-histidine (6-His) tag at C-terminal end.

In another embodiment, the method also comprises purification of rPfRH5 protein by

Ni-NTA (nitrilotriacetic acid) affinity chromatography of the inclusion bodies. The purified rPfRH5 protein is refolded by rapid dilution method in MES based buffer. The refolded protein is purified by dialysis with SP- sepharose cation exchange column.

In another embodiment, the recombinant PfRH5 protein exhibits specific erythrocyte binding activity consistent with that of the native PfRH5 parasite protein.

In still another embodiment, the present invention provides a method of treatment or prevention of a pathological condition wherein the vaccine according to present invention is useful.

In another embodiment, the invention provides use of merozoite antigens for the blocking the sialic acid independent and sialic acid dependent receptors on the surface of P. falciparum wherein the merozoite antigens comprises a combination of two or more, preferably, three antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts receptor blocking mechanism and key target ligands.

Figure 2 depicts portfolio of Blood Stage Malaria Antigens.

Figure 3 depicts erythrocyte binding phenotypes of the native parasite proteins and their recombinant receptor binding regions. (A) Erythrocyte binding of the native PfRHl, PfRH2, PfRH4 and EBA-175 proteins from 3D7 culture supematants incubated with untreated (U) erythrocytes and different enzyme- treated erythrocytes (N: neuraminidase- treated; T: trypsin-treated; C: chymotrypsin-treated). The parasite proteins were detected in the eluate fractions by immunoblotting using the respective antibodies. (B) Erythrocyte binding of the respective recombinant proteins (rPfRHl40, rPf H240, rPfRH430, PfF2) raised against the erythrocyte binding domains of the four native parasite proteins with a similar set of erythrocytes.

Figure 4 depicts PfRH antibodies block erythrocyte binding of parasite and recombinant PfRH proteins. The erythrocyte binding activity of the (A) native PfRHl/PfRH2 parasite proteins and their (B) recombinant receptor binding domains were assayed in the presence of increasing amounts of the purified rabbit IgG against each protein (1 to 160 μg/ml). The native proteins from the culture supernatant of the 3D7 parasite clone or the recombinant proteins were eluted from normal erythrocytes in the presence of different amounts of the purified IgG and detected by immunoblotting. As a control, the purified IgG against PfRHl or PfRH2 had no effect on the binding of native EBA-175 from culture supernatant or the recombinant PfF2 binding domain of EB A- 17 . Figure 5 A is expression PfRH240 in E. coli BL21(DE3) both Uninduced and Induced with ImM IPTG.

Figure 5 B depicts the purification of rPfRH240 by Metal affinity Chromatography. Figure 5 C depicts (A): Anion Exchange purified protein and (B): Final purified protein in reducing and non-reducing conditions with corresponding western blots probed with anti-His antibody.

Figure 6 is a schematic representation of Recombinant PfRH5.

Figure 7 A Expression of rPfRH5 in BL21DE3 cells post 1, 2 and 4hr of induction Figure 7 B Purification of recombinant PfRH5 protein from Inclusion bodies

Figure 7 C Final product after refolding and concentration of rPfRH5

Figure 8 depicts Erythrocyte invasion inhibitory activity of PfRH antibodies in combination against P. falciparum 3D7 clone. Total IgG purified from rabbit sera raised against the receptor binding regions of PfRHl, PfRH2, PfRH4 were tested for its invasion inhibitory activity individually (2.5-10 mg/ml) and in combination/ Combinations of two PfRH IgG were assessed at two concentrations (2.5 + 2.5 mg/ml, 5.0 + 5.0 mg/ml) and combinations of three PfRH IgG were tested at 3.3 mg/ml each. AMA-1 IgG (5 mg/ml) was used as a positive control. Negative control is an average of inhibition of purified IgG from pre-immune rabbit sera and a control rabbit immunized with a non-related peptide. Three independent assays were performed in duplicate. The error bars show the standard error of the mean.

Figure 9 depicts Invasion inhibitory activity of antibody combinations against the P. falciparum clones 3D7 & Dd2.

Figure 10 depicts Invasion inhibitory activity of antibody combinations five diverse P. falciparum clones.

Figure 11 is graphical representation of Immunogenicity (antibody responses) of the antigens in mice when used immunized alone and in combination.

Figure 12 depicts invasion inhibitory activity of antibodies raised against antigen mixtures against the P. falciparum clones 3D7 & Dd2.

Figure 13 depicts Antibodies raised against the co-immunized antigen mixtures exhibit potent invasion inhibition.

Figure 14 depicts additive invasion inhibitory effect observed with combinations of two antibodies against P. falciparum 3D7 clone. Figure 15 A depicts Erythrocyte binding activity of PfRH5.

Figure 15 B depicts the rPfRH5 protein bound to erythrocytes conforming its functionally folded conformation

Figure 16 depicts rPfRH5 antibodies specifically block erythrocyte binding of the native PfRH5 protein.

Figure 17 depicts anti-BSG monoclonal antibodies block erythrocyte binding activity of PfRH5.

Figure 18 depicts rPfRH5 antibodies exhibit strain transcending activity.

Figure 19 depicts Invasion inhibitory activity with triple antibody combinations Figure 20 depicts Invasion inhibitory activity with triple antibody combinations involving MSP-fu antibodies and two other Merozoite antibodies.

Figure 21 depicts Invasion inhibitory activity of triple antibody combinations.

Figure 22 depicts Invasion inhibition results with 15 MSP-Fusion based antibody combinations against Pf clone 3D7 Rabbit K3AR3 (Abexome).

Figure 23 depicts Invasion inhibition results with PfRH5 based triple antibody combinations against Pf clone 3D7.

Figure 24 depicts Invasion inhibition results with 15 MSP-Fusion based antibody combinations against Pf clone 3D7 Rabbit 372 (Abexome).

Figure 25 depicts Invasion inhibition results with 15 MSP-Fusion based antibody combinations against Pf clone Dd2 [Rabbit 372 (Abexome).

Figure 26 depicts Invasion inhibition results with 10 PfRH5 based antibody combinations against Pf clone Dd2.

DETAIL DESCRIPTION OF THE INVENTION

The invention provides a blood stage malaria vaccine for P. falciparum that is based on a combination of novel merozoite antigens that belong to the P. falciparum reticulocyte binding-like homologous (PfRH) protein family (PfRHl, PfRH2, PfRH4, PfRH5), P. falciparum Apical Asparagine Rich Protein (AARP), P. falciparum Erythrocyte Binding Antigen 175 (EBA-175) and P. falciparum MSP-fusion (MSP119-MSP3U).

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood- stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain- transcending invasion inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently.

The present invention provides combination blood-stage vaccines against P. falciparum that targets merozoite surface proteins and the erythrocyte binding domains of multiple parasite adhesions, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition.

The PfRH (PfRHl, PfRH2, PfRH4, PfRH5), PfAARP, PfF2 proteins play important receptor binding roles in erythrocyte invasion and define the invasion phenotypes of different parasite strains. These characteristics along with the fact that they are highly conserved (less polymorphic) as compared to other parasite antigens such as AMAl makes them promising candidates for inclusion in a blood stage malaria vaccine. MSP-119 is a critical conserved C- terminal region of full length MSP-1 that elicits invasion inhibitory antibodies that act by blocking the processing of MSP-1 during invasion. MSP-3 has been shown to induce cytophilic antibodies that neutralize parasites by the mechanism of antibody dependent cellular inhibition (ADCI).

In the present invention the inventors have demonstrated two step screening approach that allowed identification of an antigen combination from a pool of six merozoite proteins that elicit potent strain-transcending neutralizing antibodies.

The inventors of present invention have first evaluated the invasion inhibitory activity of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands including the novel PfAARP, EBA-175(PfF2), PfRHl, PfRH2, PfRH4,( Thompson J et al, Mol. Biochem. Parasitol. 134:225-232; Green JL et ah, Mol. Biochem. Parasitol. 150:114-1 17), which are localized in different apical organelles and get translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination EBA-175(PfF2)+PfRH2+PfAARP produced the most efficacious strain-transcending inhibition (80% at an IgG concentration of 10 mg/ml) of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for co-immunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. The present invention includes antigens of the PfRH family of proteins (PfRHl, PfRH2 PfRH4), EBA-175, P. falciparum Apical Asparagine Rich Protein, PfAARP (Wickramarachchi T et al., PLoS One 3:el732; Crompton PD et al., J. Clin. Invest. 120:4168^1178) and Plasmodium Thrombospondin Apical Merozoite Protein, PTRAMP (Thompson J et al., Mol. Biochem. Parasitol. 134:225-232; Green JL et al., Mol. Biochem. Parasitol. 150:114-117) that bind erythrocytes with different specificities and have been shown to be major determinants of different invasion pathways (Fig. 1) (Rayner JC et al., J. Exp. Med. 194:1571-1581; Triglia T et al, Mol. Microbiol. 55:162-174; Duraisingh MT et al, EMBO J. 22:1047-1057; Sahar T et al, PLOS One 6:el7102; Gunalan K et al, Infect. Immun. 79:3421-3430; Kaneko O et al, Mol. Biochem. Parasitol. 121:275-278; Gaur D et al, Proc. Natl. Acad. Sci. USA. 104:17789-17794; Tham WH et al, Proc. Natl. Acad. Sci. USA. 107:17327-17332; Gao X et al, PLoS Pathog. 4:el000104). The erythrocyte binding domains of these adhesins have been elucidated (Sahar T et al, PLOS One 6:el7102; Gaur D et al, Proc. Natl. Acad. Sci. USA. 104:17789-17794; Gao X et al, PLoS Pathog: 4:el000104; Wickramarachchi T et al, PLoS One 3:el732) PTRAMP contains the adhesive thrombospondin repeat (TSR) domain that has been implicated to play a conserved role in erythrocyte invasion (Thompson J et al, Mol. Biochem. Parasitol. 134:225-232; Green JL et al, Mol. Biochem. Parasitol. 150:114-117) The antigens represent merozoite adhesins that are localized in different apical organelles such as the rhoptries and micronemes (Fig. 1), which release their contents in a sequential manner at different time points during invasion (Singh S et al, PLoS Pathog. 6: el 000746) PfRHl, PfRH2 and PfAARP are localized in the neck of the rhoptries (Duraisingh MT et al, EMBO J. 22:1047-1057; Gao X et al, PLoS Pathog. 4:e 1000104; Wickramarachchi T et al, PLoS One 3:el732) whereas PTRAMP is located in the bulb of the rhoptries (Siddiqui FA and Chitnis CE, Unpublished data). PfEBA- 175 and PfRH4 are localized in the micronemes (Kaneko O et al, Mol. Biochem. Parasitol. 121:275-278; Gaur D et al, Proc. Natl. Acad. Sci. USA. 104:17789-17794; Sim B et al, : Mol. Biochem. Parasitol. 51:157-159; Healer J et al, Infect. Immun. 70:5751-5758) Further, these antigens bind different erythrocyte receptors and mediate different invasion pathways which are defined by their dependence on sialic acids and sensitivity to enzymes such as trypsin (Fig. 1). Therefore, the antigens selected for present invention represents a combinatorial diversity involving different parasite ligands mediating distinct invasion pathways and probably even different steps of erythrocyte invasion. The PfMSP-fusion protein includes a conserved 19 kDa C-terminal region of MSP- 1 and a functional 24 kDa region of MSP-3 that elicits Antibody Dependent Cellular Inhibition (ADCI) activity. The PfRHl protein includes the amino acid region 500-833 of the 350 kDa native parasite protein (comprising of 2971 amino acids) and has been demonstrated to be the erythrocyte binding domain of PfRHl, which binds with a sialic acid containing receptor on the erythrocyte receptor. The PfRH2 protein includes the amino acid region 495-860, which is identical between both P. falciparum PfRH2 paralogues (PfRH2a and PfRH2b)and has been demonstrated to constitute the erythrocyte binding domain of PfRH2, which binds with a sialic acid independent receptor on the erythrocyte receptor. The PfRH4 protein includes the amino acid region 328-588 of the 250 kDa native parasite protein (comprising of 1716 amino acids) that has been demonstrated to constitute the erythrocyte binding domain of PfRH4, which binds with its' sialic acid independent receptor on the erythrocyte surface, Complement Receptor 1. The PfRH5 protein includes the amino acid region 28-526 of the 66 kDa native parasite protein (comprising of 526 amino acids) that has been demonstrated to constitute the erythrocyte binding domain of PfRH5, which binds with its' receptor on the erythrocyte surface, Basigin. The PfAARP protein includes the amino acid region 20-107 of the 24 kDa native parasite protein (comprising of 217 amino acids) that has been demonstrated to constitute the erythrocyte binding domain of PfAARP, which binds with its' sialic acid dependent receptor on the erythrocyte receptor. The PfF2 protein includes an amino acid region 447-795 of the 175 kDa Erythrocyte Binding Antigen (EBA-175) native parasite protein (comprising of 1502 amino acids) that has been demonstrated to constitute the erythrocyte binding domain of EBA-175, which binds with its' sialic acid dependent receptor on the erythrocyte receptor, Glycophorin A.

In an embodiment, the functional receptor binding domains of PfRH, PfAARP, PfF2 proteins as recombinant proteins in their correct conformations are generated. Also specific " anti-PfRH (PfRHl, PfRH2, PfRH4, PfRH5), anti-PfAARP, anti-PfF2 antibodies are generated that block the erythrocyte binding activity of the native parasite proteins. It is also shown that combinations of antibodies, against key parasite ligands that mediate invasion, including the PfRH family (PfRHl, PfRH2, PfRH4, PfRH5), PfAARP, PfF2 proteins efficiently blocks erythrocyte invasion in synergistic manner.

In another embodiment of the present invention the merozoite antigens combination are selected from the group consisting of PfAARP + PfRH5 + PfF2, PfAARP + PfRH5 + PfRHl, PfAARP + PfRH5 + PfRH2, PfAARP + PfRH5 + PfRH4, PfAARP + PfRH2 + PfF2, PfAARP + PfRH2 + PfRH4, PfAARP + PfRH2 + PfRHl, PfAARP + PfRHl + PfRH4, PfAARP + PfF2 + PfRHl, PfAARP + PfF2 + PfRH4, PfRH5 + PfRH2 + PfF2, PfRH5 + PfRH2 + PfRHl, PfRH5 + PfRH2 + PfRH4, PfRH5 + PfF2 + PfRHl, PfRH5 + PfF2 + PfRH4, PfRH5 + PfF2 + PfRH2, PfRH5 + PfRHl + PfRH4, PfRHl + PfRH2 + PfRH4, PfF2 + PfRHl + PfRH4, PfF2 + PfRH2 + PfRH4. These merozoite antigens combinations produce potent invasion inhibition at an IgG content of 9- 14 mg/ml.

In another embodiment the merozoite antigens are selected from the group consisting of MSP(Fusion) + PfAARP + PfRH5, MSP(Fusion) + PfAARP + PfRH2, MSP(Fusion) + PfAARP + PfRHl, MSP(Fusion) + PfAARP + PfRH4, MSP(Fusion) + PfAARP + PfF2, MSP(Fusion) + PfRH5 + PfRH2, MSP(Fusion) + PfRH5 + PfRHl, MSP(Fusion) + PfRH5 + PfRH4, MSP(Fusion) + PfRH5 + PfF2, MSP(Fusion) + PfRH2 + PfRHl, MSP(Fusion) + PfRH2 + PfRH4, MSP(Fusion) + PfRH2 + PfF2, MSP(Fusion) + PfRHl + PfRH4, MSP(Fusion) + PfRHl + PfF2, MSP(Fusion) + PfRH4 + PfF2. These merozoite antigens combinations produce potent invasion inhibition at an IgG content of 1.5-6 mg/ml.

In another embodiment, a full length PfRH5 parasite protein as a recombinant protein in E. coli is provided that exhibits a functional activity and structural integrity similar to that of the native parasite protein. The PfRH5 according to present invention is capable of eliciting antibodies capable of preventing invasion of P. falciparum parasites into human erythrocytes. It is the only unique parasite ligand among the EBA/PfRH proteins that is known to be essential for erythrocyte invasion. PfRH5 has been demonstrated to bind with the basigin receptor on the erythrocyte surface. Antibodies against both PfRH5 and the basigin (BSG) receptor that impede this molecular interaction potently blocked erythrocyte invasion of wide number of P. falciparum clones, implying that the PfRH5-BSG interaction has a conserved critical role during erythrocyte invasion.

PfRH5 is a key determinant of species specific erythrocyte invasion. Genetic analysis of a P. falciparum cross between two parental clones 7G8 x GB4 and their progeny mapped the PfRH5 gene on chromosome 4 as the loci responsible for mediating invasion of Aotus nancymiae erythrocytes and infectivity of Aotus monkeys by P. falciparum. It was also demonstrated that PfRH5 is an erythrocyte binding ligand in which single point mutations critically affected the specificity of binding to both human and Aotus erythrocytes. Further, PfRH5 is unique in being the only parasite erythrocyte binding ligand that is essential for the parasite as it cannot be genetically knocked out, suggesting a crucial role in erythrocyte invasion.

PfRH5 is an exceptional member of the PfRH family of proteins as unlike other PfRH homologues (RH1, RH2a, RH2b, RH4) it is much smaller in size (66 kDa) and lacks a transmembrane domain. Recently, PfRH5 has been shown to be localized on the parasite surface in association with another parasite molecule, PfRipr (PFC1045c). While, PfRH proteins are differentially expressed among different P. falciparum clones that exhibit phenotypic variation in their invasion properties, the expression of PfRH5 was found to be consistent among these parasite clones.

PfRH5 is reported to bind with the CD 177 IgG super family member, Basigin on the erythrocyte surface. The significance of this interaction is highlighted by the demonstration that anti-BSG antibodies blocked erythrocyte invasion by a large number of P. falciparum clones that are known to exhibit different invasion phenotypes. While, this substantiated the importance of the PfRH5-BSG interaction during erythrocyte invasion, the challenge from a vaccine stand point has been to produce recombinant PfRH5 that would elicit similar potent invasion inhibitory antibodies. A heterologous prime-boost strategy based on the adenoviral/MVA viral vector delivery platform was used to generate anti-PfRH5 antibodies that efficiently inhibited erythrocyte invasion by multiple heterologous P. falciparum clones. Recently, a recombinant PfRH5 protein was produced from mammalian HEK293 cells that had to be mutated so as to produce a non- glycosylated protein. Further, this recombinant protein was expressed as a fusion with a C4d domain and a biotin or pentameric B-lactamse tag (Crosnier C et al., Nature 480:534-537; Bustamante LY, Vaccine. Jan 2;31(2):373-9). While, antibodies against this modified fusion PfRH5 protein did exhibit invasion inhibitory activity (Bustamante LY, Vaccine. Jan 2;31(2):373-9), production of a non-mutated, wild type PfRH5 protein on its own without any fusion partner in a production friendly system such as E. coli would be highly beneficial.

Previous reports of production of PfRH5 recombinant proteins in E.coli did not produce the full length protein and instead were focused on expressing smaller fragments of 143-168 amino acids. Both these recombinant fragments failed to elicit invasion inhibitory antibodies. This is consistent with the recent report using the adeno-MVA prime boost approach that demonstrated potent invasion inhibitory antibodies only against full length PfRH5 and not against the 168 amino acid fragment of PfRH5. In present invention it has also been validated that the full length recombinant PfRH5 protein would be equally efficacious in eliciting potent invasion inhibition as observed with the viral vector delivery platform. The inventors of present invention have successfully produced full length PfRH5 as a recombinant protein in E. coli that exhibits specific erythrocyte binding activity consistent with that of the native PfRH5 parasite protein. This E. coli produced full length PfRH5 recombinant protein elicited potent invasion inhibitory antibodies that blocked erythrocyte invasion by a number of heterologous parasite clones.

The present invention shows that anti-basigin antibodies block the erythrocyte binding activity of native PfRH5 and a recombinant PfRH5 protein produced with the wild- type parasite sequence. The data given confirms that basigin acts as the erythrocyte receptor for the PfRH5 parasite protein and strongly supports the development of PfRH5 as a blood- stage malaria vaccine candidate.

The combination of antigens selected from PfRH, PfAARP and PfF2 exhibit 80% inhibition of P. falciparum at low IgG concentration in the range of 9 to 14 mg/ml.

In order to further augment the activity of the parasite neutralizing antibodies, key parasite ligands (PfRH, PfAARP and PfF2) are targeted in combination with Merozoite surface protein 1 (MSP-1 through the MSP-Fusion antigen), which exhibit 80% inhibition of P. falciparum at lower IgG,concentration in the range of 1.5 to 6 mg/ml.

MSP-1 has been well described in the publication (Moss DK, Remarque EJ, Faber BW, Cavanagh DR, Arnot DE, Thomas AW, Holder AA. 2012. Infect Immun. Mar; 80(3):1280-7). MSP-1 is a well characterized merozoite surface protein (Holder AA. 2009. Parasitology 136: 1445-1456 ) and is essential during the invasive blood stage (Combe A, et al. 2009. Cell Host Microbe 5:386 -396.). It is synthesized in schizonts as a 190-kDa protein, which is cleaved by P. falciparum subtilisin 1 (PfSUBl) at the end of schizogony into four polypeptides of characteristic length: p83, p42, p38, and p30 (Child MA, et al. 2010. Mol. Microbiol. 78:187-202 ). These fragments remain associated together on the parasite's surface via non-covalent bonds, along with several other surface proteins (Pachebat JA, et al. 2007. Mol. Biochem. Parasitol. 151:59-69 and anchored to the plasma membrane via the C- terminal glycosyl phosphatidyl inositol (GPI) moiety located on the 42-kDa fragment (MSP- 142) (Gerold P, et al. 1996. Mol. Biochem. Parasitol. 75:131-143.). MSPl may play a role in the initial binding of the merozoite to an erythrocyte (Perkins ME. 1988. J. Immunol. 141:3190 -3196.). During the final stages of erythrocyte invasion, MSP- 142 undergoes a second cleavage event called secondary processing and mediated by another parasite subtilisin (PfSUB2) [Harris PK, et al. 2005. PLoS Pathog. 1:241-251.], generating MSP-133 and MSP-119 (19-kDa carboxy-terminal region). The result of this cleavage is the shedding of the majority of the MSP-1 and its associated protein complex, a process that has been linked with loss of the merozoite coat during erythrocyte invasion (Blackman MJ, Holder AA. 1992. Mai. Biochem. Parasitol. 50:307-315. ) However, MSP-119 remains attached to the merozoite due to the GPI anchor and is taken into the erythrocyte (Dluzewski AR, et al. 2008. PLoS One 3:e3085.). The role MSP-119 plays in subsequent intracellular parasite development is unclear, although it is the first known marker for the developing food vacuole where it persists until the end of the intracellular cycle and is discarded in the residual body together with products of digestion such as hemozoin (Dluzewski AR, et al. 2008. PLoS One 3:e3085.) There is abundant evidence that antibodies to MSPl 19 can interfere with parasite growth, and a range of mechanisms have been proposed, ranging from steric inhibition of parasite binding to erythrocytes and inhibition of SUB2-mediated secondary processing to the recruitment of cellular functions through Fc-mediated mechanisms (Bergmann-Leitner ES, et al. 2009. Malar. J. 8:183; Blackman MJ et al. 1994 J. Exp. Med. 180:389 -393. Gilson PR, et al 2008. Mol. Microbiol. 68:124 -138.; Guevara Patino JA et al, 1997. J. Exp. Med. 186:1689 -1699. Mcintosh RS, et al. 2007. PLoS Pathog. 3:e72. Murhandarwati EE, et al. 2010. Infect. Immun. 78:680-687.

The structure of P. falciparum MSP-119 has been elucidated and used to define the epitopes recognized by monoclonal antibodies (MAbs) that when bound inhibit secondary processing (Tham WH et al, Proc. Natl. Acad. Sci. USA. 107:17327-17332; Gao X et al, PLoS Pathog. 4:el000104; Pandey KC et al, Mol. Biochem. Parasitol. 123:23-33). MSP-119 antibodies inhibit erythrocyte invasion and parasite growth, with some MSPl -specific antibodies shown to inhibit the proteolytic processing of MSPl that occurs at invasion. In addition to inhibition of processing MSP-119 antibodies have been reported to neutralize parasite growth by delaying intracellular parasite development. MSP-119

MSP-119 -specific antibodies are taken up into invaded erythrocytes, where they persist for significant periods and result in delayed intracellular growth of the parasite. This delay may result from antibody interference with coalescence of MSPl 19 containing vesicles with the food vacuole. Thus, antibodies specific for MSPl 19 mediate inhibition of parasite growth by at least three mechanisms: inhibition of MSPl processing, direct inhibition of invasion, and inhibition of parasite development following invasion. The balance between mechanisms may be modulated by modifying the immunogen used to induce the antibodies.

MSP-119 antibodies block erythrocyte invasion very potently in a strain-transcending manner. However, the problem has been that MSP-119 is a very small molecule and poorly immunogenic. This problem of its poor immunogenicity has been resolved by the MSP- : Fusion protein that induces strong antibody responses against MSP-119. MSP-Fusion antibodies in combination with atleast antibodies against two other key ligands such as i PfAARP, PfRH5 or PfRH2, PfRHl, PfRH4, or PfF2 exhibit a potent additive effect in erythrocyte invasion.

The combination of MSP(Fusion) with other two antigens provide high inhibition of

80% at a lower total IgG concentration of 1.5 to 3 mg/ml Thus, the present invention provides a novel combination of merozite antigens which demonstrate the maximum inhibition at the lowest possible IgG concentration. It is for the first time novel antibody combinations that yield very high parasite growth inhibition against heterologous parasite clones at highly achievable human IgG concentrations have been provided.

Individually, the antibodies produces 10-33% inhibition, with the PfAARP antibodies being most potent (33% inhibition) followed by the PfRH2 antibodies. The combinations involving antibodies against up to 3 antigens show synergistic effect and the following combinations show the highest invasion inhibitory rates against P. falciparum 3D7: PfF2+PfRH2+AARP, PfRH2+PfRH4+AARP, PfF2+PfRH4+PfAARP, PfRHl +PfRH2+PfAARP, PfF2+PfRH2+PfRH4 and PfRHH PfRH4 +PfAARP. The highest inhibition (78%) is seen with the combination PfF2+PfRH2+PfAARP, which is higher than that observed with the combination of the three PfRH antibodies (65%) (Fig. 10).

In addition, the invasion inhibition experiments are performed with the P. falciparum sialic acid dependent clone Dd2 (Fig. 12). Antibodies against PfAARP have similar invasion inhibitory activity against Dd2 as observed with 3D7. Interestingly antibodies against PfF2 and PfRHl, which are sialic acid binding proteins, have higher invasion inhibitory activity against Dd2 compared to 3D7. In case of antibody combinations, the highest invasion inhibitory rates against P. falciparum Dd2 are observed with the following: PfF2+PfRH 1 +Pf AARP, PfF2+PfRH2+PfAARP and PfF2+PfRH4+ PfAARP (Fig. 12). In case of Dd2 the combination PfF2+ PfRH 1+Pf AARP yield the highest invasion inhibitory activity (74%), with the PfF2+PfRH2+PfAARP combination yielding a 64% invasion inhibition.

It is quite evident that combinations of antibodies against PfAARP, PfRH2, PfRHl, PfRH4 and PfF2 exhibited high invasion inhibitory rates against the P. falciparum clones 3D7 and Dd2. While, the combinations PfAARP+PfRH2+PfF2 and Pf AARP+PfRH 1 +PfF2 exhibit the maximum invasion inhibition against 3D7 (78%) and Dd2 (74%), respectively. The combination PfAARP+PfRH2+PfF2 observed to be most effective against both parasite clones as it inhibit invasion of Dd2 by 64%. The antibody combinations involving PTRAMP observed not to yield a synergistic inhibition of invasion against either P. falciparum clone 3D7 or Dd2.

This confirms that the high synergy obtained with the combinations of three antibodies against portfolio of antigens is not a non-specific effect that would be observed with any combination of antibodies.

Antibodies generated against the 3D7 sequence of all candidate antigens exhibited efficacious inhibition with other heterologous parasite clones. The invasion inhibition activity of three combinations (PfAARP+PfRH2+PfF2, PfRHl +PfRH2+PfRH4, PfRHl+PfRH2+PfF2) with five parasite clones - 3D7, 7G8, HB3, Dd2 and MCamp is analysed. 3D7, 7G8, HB3 are sialic acid independent clones whereas Dd2, MCamp are sialic acid dependent clones. The antibody combinations according to present invention exhibit synergistic effect and are more potent then the individual antibody. The antibody combination PfAARP+PfRH2+PfF2 that is found to be most potent in the previous assays with which is most potent with 3D7, Dd2 is consistently observed to efficiently block erythrocyte invasion against 7G8, HB3 and MCamp. While the other two antibody combinations (PfRHl +PfRH2+PfRH4, PfRHl +PfRH2+PfF2) inhibit invasion of the other parasite clones, its efficiency is comparatively lower than that of the PfAARP+PfRH2+PfF2 combination. In addition, the inhibition by these two combinations against all the clones is not balanced as observed for the PfAARP+PfRH2+PfF2 combination. This data strongly suggests that polymorphisms in parasite strains do not affect the effectiveness of the antigen based vaccine provided by the present invention.

It is therefore evident that combination of antibodies against different antigens provides synergy yielding highly efficient inhibition of invasion. The inventors have therefore developed receptor blocking blood stage vaccine based on novel combination of parasite proteins that play a crucial role in attachment and invasion of the host erythrocyte. Achieving such high invasion inhibitory activity against conserved functional regions of parasite proteins involved in invasion may be a key to the development of an effective blood stage vaccine against malaria.

It is shown that antibodies targeting multiple erythrocyte binding parasite proteins involved in different invasion pathways block erythrocyte invasion highly efficiently. The combination of antibodies against different antigens provides synergy yielding highly efficient inhibition of invasion.

PfRH antibody combinations produce an synergistic inhibition of erythrocyte invasion by P. falciparum. The invasion inhibitory activity of different combinations of purified total IgG against each of the three PfRH proteins is assessed over one cycle against the P. falciparum clone, 3D7 (Sahar T et al, PLOS One 6:el7102; Gaur D et al, Proc. Natl Acad. Sci. USA. 104: 17789-17794) that invades erythrocytes using both sialic acid dependent and independent pathways (Figures 11). The purified total IgGs were tested individually (2.5 mg/ml, 3.3 mg/ml. 5.0 mg/ml, 10.0 mg/ml) as well as in double (2.5 mg/ml and 5 mg/ml each) and triple antibody combinations (3.3mg/ml each) (Figure 11).

The maximum total IgG concentration tested was 10 mg/ml as this is close to the physiological concentration of IgG in human sera (Walliker D et al., Science 236: 1661- 1666). Individual PfRH antibodies exhibited a dose dependent invasion inhibition confirming a specific effect (Figure 1 1). Anti-PfRH240 IgG exhibited maximum inhibition of 54% (10 mg/ml) and 29% (5 mg/ml) (Figure 11). Anti-PfRH140 and anti-PfRH430IgG are less potent with an inhibition of 22-30% at 10 mg/ml (Figure 11). PfRHIgG combinations at 2.5 mg/ml each do not produce any significant increase in invasion inhibition, however, the combinations at 5.0 mg/ml each, produced an additive effect (Figure 1 1). Individually, the three PfRH IgGs at 5 mg/ml blocked invasion by 17-29% (Figure 1 1). Among the double combinations, PfRH2+PfRH4 IgG inhibited invasion by 54%, while the inhibition of PfRHl+PfRH2 and PfRHl+PfRH4 is 30-37% (Figure l l).The most potent additive inhibition was observed with the combination of three antibodies (PfRHl+PfRH2+PfRH4; 3.3 mg/ml each) that produced 66% inhibition compared to the low inhibition (< 12%) exhibited by each individual IgG at 3.3 mg/ml (Figure 11).

PfRH5 antibodies are highly potent and strain-transcending in nature (Figure 20). They exhibit maximum invasion inhibition among all antibodies against parasite ligands alone and are secomd only to MSP-Fusion antibodies. Antibody combinations with PfRH5 antibodies also produced strong additive inhibition of erythrocyte invasion as observed for the previous set of antibody combinations.

Invasion inhibitory activity of combinations of antibodies against PfRH proteins, PfF2, PfAARP and PTRAMP.

To further assess if the inhibition obtained by targeting PfRH proteins could be augmented by inclusion of other key merozoite target antigens, the invasion inhibitory activity of all 20 possible triple antibody combinations of purified total IgG (3.3 mg/ml each) against a pool of 6 antigens (RHl, RH2, RH4, PfF2, AARP, PTRAMP) are tested. Invasion inhibition is assayed against the P. falciparum clones, 3D7 and Dd2 (Figure 12). Against 3D7, individual IgG against each of the 6 antigens produced 8-25% inhibition, with AARP IgG being most potent (25%) at 3.3 mg/ml (Figure 12). Different combinations of three IgGs displayed potent inhibition (62-79%) of erythrocyte invasion such as PfF2+RH2+AARP, RH2+RH4+AARP, PfF2+RH4+AARP, RH 1 +RH2+ AARP, PfF2+RH2+RH4 and RH1+RH4+AARP (Figure 12). Maximum inhibition (79%) is elicited by PfF2+RH2+AARP IgG, which is higher than the 66% observed with RH1+RH2+RH4 IgG (Figure 12). Against the sialic acid dependent clone Dd2, AARP IgG inhibited erythrocyte invasion with same efficiency as observed with 3D7 (Fig. 4B). Total IgG against PfF2 and PfRHl, which are sialic acid binding proteins, exhibited higher inhibition against Dd2 compared to 3D7 (Figure 12). This is consistent with the fact that Dd2 is known to express greater levels of PfRHl and utilize it for invasion (Triglia T et al., Mol. Microbiol. 55:162-174). Conversely, PfRH2 and PfRH4 IgG exhibited poor inhibition against Dd2, again consistent with Dd2 expressing low levels of these two proteins (Stubbs J et al, Science 309:1384-1387; Gaur D et al, Mol. Biochem. Parasitol. 145:205-215; Duraisingh MT et al, EMBO J. 22: 1047-1057). Similar to 3D7, some triple IgG combinations such as PfF2+RH 1 + AARP, PfF2+RH2+AARP and PfF2+RH4+AARP displayed a potent invasion inhibition (62-75%) against Dd2, (Figure 12). PfF2+RH 1 +AARP yielded the maximum invasion inhibition (75%) against Dd2 (Figure 9), which is consistent with the three antigens being involved in sialic acid dependent invasion. However, this combination only yielded 48% inhibition against the 3D7 clone (Figure 12). The most effective combination against 3D7, PfF2+RH2+AARP, exhibited the second highest inhibition (68%) against Dd2 (Figure 12) and is therefore, considered to be most efficacious against both parasite clones. The efficacy of PfF2+RH2+AARP is further analyzed against three other diverse P. falciparum clones. In addition to 3D7 and Dd2, inhibition of invasion by the sialic acid independent clones (7G8, HB3) and the sialic acid dependent clone (MCamp) is tested. PfF2+RH2+AARP IgG inhibited the invasion of all five clones with invasion inhibition efficiency ranging between 67-79% (Figure 13). Two other IgG combinations (RH 1 +RH2+RH4, RHl+RH2+PfF2) were also tested for inhibition of invasion by multiple clones. The invasion inhibitory efficiency of these combinations is not similar for all clones (Figure 13) with broad inhibition between 36-67% (Figure 13). Thus, the PfF2+RH2+AARP antibody combination is identified from first step of screening to exhibit the most potent, strain-transcending, invasion inhibitory activity.

MSP-Fusion antibody combinations produce synergistic inhibition of erythrocyte invasion by P. falciparum. MSP-Fusion antibodies exhibit a strong dose dependent activity with 85-95% inhibition at an IgG concentration of 10 mg/ml; 60-70% inhibition at an IgG concentration of 5 mg/ml; 50-65% inhibition at an IgG concentration of 2.5 mg/ml; 40-50% inhibition at an IgG concentration of 1.0 mg/ml.The MSP-Fusion antibodies have been tested from 4 different rabbits (Figures 22 and 24). Various combination of 15 triple antibody comprising of antibodies against MSP-Fusion along with those against two other major ligands (from a pool of 6 - RH1, RH2, RH4, RH5, AARP, PfF2) have been tested. These antibody combinations are tested at an individual IgG concentration of 0.5 mg/ml, 1.0 mg/ml and 2.0 mg/ml for each of the three antibodies comprising the triple combination (Fig. 20-22 and 24-25).At these three IgG concentrations, the individual purified antibodies exhibited a dose dependent invasion inhibitory activity with the maximum inhibition displayed by MSP- Fusion IgG followed by PfRH5 IgG> AARP IgG / PfRH2 IgG.The MSP-Fusion IgG based combinations are highly potent and blocked invasion by 50-70% at a total concentration of 1.5 mg/ml (0.5 mg/ml individual IgG); 60-80% at a total concentration of 3.0 mg/ml(1.0 mg/ml individual IgG); 68-85% at a total concentration of 6.0 mg/ml (2.0 mg/ml individual IgG).

Thus, by targeting the merozoite surface proteins along with two key parasite ligands, the same level of invasion inhibition is achieved at a three-fold lower IgG concentration than that observed for an antibody combination solely targeting only ligand-receptor interactions. Antibodies against MSP(Fusion)+AARP+RH5 blocked invasion by 70% and 80% at total concentrations of 1.5 mg/ml (0.5 mg/ml individual IgG) and 3.0 mg/ml (1.0 mg/ml individual IgG). The antibody combination AARP+PfF2+RH2 blocked invasion by 80% at a total IgG concentration of 10 mg/ml (3.3 mg/ml individual IgG).

The most potent MSP(fusion) based antibody combinations against the P. falciparum clone 3D7 are:

MSP(Fusion)+AARP+RH5 (80% at 3 mg.ml)

MSP(Fusion)+AARP+RH2 (75% at 3 mg.ml)

MSP(Fusion)+RH5+RH2 (77% at 3 mg.ml)

MSP(Fusion)+RH5+RH4 (72% at 3 mg.ml)

MSP(Fusion)+RH5+PfF2 (69% at 3 mg.ml)

The most potent MSP(fusion) based antibody combinations against the P. falciparum clone Dd2 are:

MSP(Fusion)+AARP+RH5 (74% at 3 mg.ml)

MSP(Fusion)+AARP+RHl (77% at 3 mg.ml)

MSP(Fusion)+AARP+ PfF2 (70% at 3 mg.ml)

MSP(Fusion)+RH5+PfF2 (65% at 3 mg.ml)

MSP(Fusion)+RH5+RHl (66% at 3 mg.ml)

MSP(Fusion) + RH1 + PfF2 (69% at 3 mg/ml)

PfRH5 IgG based combinations:

PfRH5 antibodies are highly potent and strain-transcending in nature (Figure 23 and 26). Antibody combinations with PfRH5 antibodies produce strong synergistic inhibition of erythrocyte invasion with the P. falciparum clone 3D7 as observed for the previous set of antibody combinations. Since, PfRH5 antibodies are highly potent, most of their antibody combinations also exhibit a high invasion inhibition.

A similar trend is observed with the Dd2 clone that exhibits a different invasion phenotype compared to 3D7.

The embodiments of present invention have now been illustrated herein below with the help of figures and should not be construed to limit the scope of invention:

Figure 1 depicts receptor blocking mechanism and key target ligands. The erythrocyte binding characteristics of the six antigens are tabulated with respect to the identity of their erythrocyte receptor, the type of the receptor with respect to its sialic acid content, and sensitivity to enzymes such as trypsin. These antigens are localized in different apical compartments and are believed to be released at different time points during invasion and in addition bind erythrocytes with different specificities, implying an involvement in different invasion pathways.

PfRHl, PfRH2, and PfAARP are localized in the neck of the rhoptries whereas PTRAMP is located in the bulb of the rhoptries. PfEBA-175 and PfRH4 are localized in micronemes.

Figure 2 is a schematic diagram of the six P. falciparum merozoite proteins expressed as recombinant proteins. These merozoite proteins involved in erythrocyte invasion. The erythrocyte binding domains against which the recombinant proteins have been produced are highlighted. Purified recombinant proteins against each of the six parasite proteins are analyzed on SDS-P AGE under reducing conditions.

The respective gene sequences encoding the receptor binding domains of the different merozoite antigens are cloned in the following T7 promoter-based pET expression vectors, obtained from Novagen (EMDMillipore):

rPfRH240 (pET-24b), rPfRH430 (pET-1 la), rPfAARP20-107 (pET-28a), and EBA- 175 (rPfF2) (pET-28a).

The identification of the erythrocyte binding domain of each parasite adhesion and expression as a recombinant protein are described as follows:

P. falciparum exhibits redundancy in its repertoire of invasion molecules that enables it to invade human erythrocytes through multiple pathways. P. falciparum reticulocyte homology (PfRH) proteins, which belong to a multi-gene family of parasite ligands (PfRHl, PfRH2a, PfRH2h, PfRH3, PfRH4 and PfRH5) bind erythrocytes and are homologous to the P. vivax reticulocyte binding proteins (PvRBPs). PfRH proteins bind human erythrocytes with different specificities. (Gaur D et al, Int J Parasitol (2004) 34:1413-29 and Cowman AF et al, Cell (2006) 124: 755-66). Most importantly the differential expression of PfRH proteins determines the invasion phenotypes of P. falciparum strains highlighting their critical role in invasion [Cowman AF et al, Cell (2006) 124: 755-66; Taylor HMet a/.,Infect Immun.(2002) Oct;70(10):5779-89 and Triglia Tet /.,Mol Microbiol(2005) 55(1): 162-74].

PfRHl binds a sialic acid containing erythrocyte receptor and its genetic disruption results in reduced invasion through sialic acid dependent pathway[Taylor HM et a/.,Infect Immun. (2002) Oct;70(10):5779-89 and Triglia Tet a/.,Mol Microbiol (2005) 55(1): 162-74]. PfRH2a and PfRH2b (PfRH2a/b), which are identical except for a unique region near the C- terminus, are predicted to bind erythrocytes in a sialic acid independent manner [Duraisingh MT et al, EMBO J 9(2003) 22: 1047-1057 and Dvorin JDet a/.,Mol Microbiol(2010) 75(4): 990-1006].

Sialic acid dependent P. falciparum strains express high levels of PfRHl and low levels of PfRH2a/b, whereas sialic acid independent strains express high levels of PfRH2a/b and low levels of PfRHl [Triglia let /.,Mol Microbiol (2005) 55(l):162-74]. PfRH3 is a pseudogene [Taylor HM et al, Infect Immun (2001) 69: 3635-3645]and is therefore not translated.

PfRH4 and PfRH5 bind erythrocytes in a sialic acid independent manner[Gaur Det al., 2007, Proc. Natl. Acad. Sci. USA 104: 17789-17794; Hayton K et a/.,2008, Cell Host Microbe 4(1):40-51 and Baum J et al, 2009,Int J Parasitol 39(3): 371-80. PfRH4 up regulation is shown to lead to a switch from sialic acid dependent to independent invasion [Stubbs J et al, (2005) Science 309: 1384-7 and Gaur D, et al, (2006) Mol Biochem Parasitol 145: 205-15]. It is not possible to delete the gene encoding PfRH5 suggesting that it plays an essential role in invasion [Baum Jet al, (2009) Int J Parasitol 39(3): 371-80].

The functional erythrocyte binding domains of PfRH4 (PfRH430) and PfRHl

(PfRHl 4o) have been recently elucidated[Gaur D et al, (2007) Proc. Natl. Acad. Sci. USA 104: 17789-17794 and Gao Xet a/.,(2008) PLoS Pathog, 4(7):e 1000104]. It is demonstrated that native PfRH2a/b from the parasite binds sialic acid independent receptors on human erythrocytes. Its receptor-binding domain comprising conserved 40 kDa region (PfRH24o) [Sahar T et /.,(2011) PLOS OneFeb;6 (2): el7102] is also mapped. Antibodies raised against PfRH240 demonstrate that PfRH2 is expressed only in the sialic acid independent clone (3D7) and not in the sialic acid dependent clone (MCamp)[Sahar T et a/.,(2011) PLOS OneFeb;6 (2): el 7102]. Further, the antibodies localized PfRH2 in the rhoptries consistent with previous immunoelectron microscopy studies) [Sahar T et a/.,(2011) PLOS OneFeb;6 (2): el7102]. The recombinant 40 kDa receptor-binding region (PfRH24o, amino acids 495- 860) binds erythrocytes with the same specificity as the native PfRH2 protein (Fig. 2). These specificities are defined by binding with different enzyme treated erythrocytes that reflect the nature of the erythrocyte receptor, such as neuraminidase that cleaves sialic acids. Proteins that bind neuraminidase treated erythrocytes would have a sialic acid independent binding phenotype. Both native PfRH2 and PfRH240 bound neuraminidase-treated and trypsin-treated erythrocytes but not chymotrypsin-treated erythrocytes (Fig. 2). Recombinant PfRH24o is thus correctly folded. In addition to recombinant PfRH240, the receptor-binding domains of PfRHl and PfRH4 as recombinant proteins is also expressed [Gaur D et al., (2007) Proc. Natl. Acad. Sci. USA 104: 17789-17794 and Gao Xet a/..(2008) PLoS Pathog. 4(7):el000104] (Fig. 1).

The erythrocyte binding domain of any PfRH protein was first reported for the PfRH4 protein by one of the inventors and is published in Proceedings of the National Academy of Sciences, USA (Gaur D et al, 2007). On the basis of clustal alignment of different members of the reticulocyte binding-like family such PfRHl and PvRBPl, a 260 residue region near the N-terminus of PfRH4 comprising of amino acids 328-588 was identified as the receptor binding domain of PfRH4. This recombinant protein (rPfRH430) is expressed against this region exhibited erythrocyte binding properties identical to that of the native parasite protein. Expression and purification PfRH (.PfRHl. PfRH2, PfRH4, PfRHl V PfF2. PfAARP and MSP-fusion is provided herein below:

PfRHl:

PfRH4 and PfRHl are closely related phylogenetically and have a strong homology in their N-terminal regions.

Expression & Purification of the recombinant PfRHl protein (rPfRHl4o):

For rPfRHl40, an Escherichia coli codon-optimized synthetic gene encoding amino acids 500 to 833 of the receptor binding domain of PfRHl is obtained from GeneArt (Life Technologies) and cloned into pET-24b using the Ndel and Xhol restriction enzymes.

For rPfRHUo, anEscherichia coli codon-optimized synthetic gene encoding amino acids 500 to 833 of the receptor binding domain of PfRHl is obtained: from GeneArt (Life Technologies) and cloned into pET-24b using the Ndel and Xhol restriction enzymes.

20 ul aliquot of BL21 (DE3) competent cells (Novagen) is transformed with 1 ng RH1 plasmid (RH4-l/pETl la). Few colonies around 10 are obtained, 2 single colonies are picked and 5 ml LB-amp is inoculated for 5 hours. After 5 hours, 25 ul of each culture is inoculated into 50 ml LB-amp overnight for 13-14 hours.10 ml of the 50 ml culture is seeded into 1 L each LB amp.Total 40 ml is seeded into 4 L LB amp (4 large spinner flasks). OD 600 nm = 0.05 is started at time O (11:45 am). Cultures are monitored for absorbance. After 3 hours (2:45 pm) OD 600nm = 1.2 cultures are induced with 1 raM IPTG (954 mg dissolved in 20 ml LB amp; 5 ml added in each flask). After 3.5 hours, culture spun in bags using Beckman rotor and bottles (MVDB). Weight of E. coli pellet: 2 pellets is 7 g and 2 pellets is 6.6 g. Pellets are frozen at -80°C. Culture is resuspended in Lysis buffer and mixed at°4 C for 1 hour.[lysis buffer composition is: lOmM Tris, lOmM EDTA, lOOmM NaCl, pH 8.0] Suspension is passed through microfluidizer. Spinned 25 000 x g X 30 min. at 4 C. pellet are solubilized in 25 ml of solubilization buffer(6M Guanidine HC1, 10 mMTris pH 8.0, 100 mM sodium phosphate, lOmM imidazole, 1.0 mM Beta-mercaptoethanol). Protein is then purified on a Ni-NTA column (NiNTA column elution buffers' composition: 20 mM sodium phosphate, 0.25 NaCl, 6 M GmHCl, 500 Imidazole, 10 Beta-Mercaptoethanol). Run gel and collected fractions containing Rhls protein. Refolded the protein in Buffer 4 using a refold column.

Buffer 4 composition (for 1 L):

55 mMTris pH 8.2

264 mMNaCl

l l mMKCl

2.2 mM Mg C12

2.2 mMCa C12

440 mM Sucrose

550 mM L-Arg

Run gel and collect fractions containing Rhls protein, Protein run on a sizing column with Ix PBS pH7.4.

DNA sequence of the PfRHl gene The region highlighted is the DNA gene sequence that encodes the receptor binding region of the PfRHl protein. (SEQ. ID NO.l):

ATGCAAAGGTGGATTTTCTGCAACATTGTTTTGCATATATTAATTTACTTAGCAG AATTTAGCCATGAACAGGAAAGTTATTCTTCCAATGAAAAAATAAGAAAGGACT ATTCAGATGATAATAATTATGAACCTACCCCTTCATATGAAAAAAGAAAAAAAG AATATGGAAAAGATGAAAGTTATATAAAAAATTACAGAGGTAATAATTTTTCCT ATGATTTGTCTAAAAATTCTAGTATATTTCTTCACATGGGTAACGGTAGTAACTC GAAAACACTAAAAAGATGTAACAAGAAAAAAAATATAAAGACCAATTTTTTAAG ACCTATCGAGGAAGAGAAAACGGTATTAAATAATTATGTATATAAAGGTGTAAA TTTTTTAGATACAATAAAAAGAAATGATTCCTCTTATAAATTTGATGTTTATAAA GATACTTCCTTTTTAAAAAATAGAGAATATAAAGAATTAATTACTATGCAGTATG ATTATGCTTATTTAGAAGCAACAAAAGAGGTTCTTTATTTAATTCCGAAGGATAA AGATTATCACAAATTTTATAAAAATGAACTTGAGAAAATTCTTTTCAATTTAAAA GATTCACTTAAATTATTAAGAGAAGGATATATACAAAGCAAACTGGAAATGATT AGAATCCATTCGGATATAGATATATTAAATGAGTTTCATCAAGGAAATATTATAA ACGATAATTATTTTAATAATGAAATAAAAAAAAAAAAGGAAGACATGGAAAAA TATATAAGAGAATATAATTTATACATATATAAATATGAAAATCAGCTTAAAATA AAAATACAGAAATTAACAAATGAAGTTTCTATAAATTTAAATAAATCTACATGT GAAAAGAATTGTTATAATTATATTTTAAAATTAGAAAAATATAAAAATATAATA AAAGATAAGATAAATAAATGGAAAGATTTACCAGAAATATATATTGATGATAAA AGTTTCTCATATACATTTTTAAAAGATGTAATAAATAATAAGATAGATATATATA AAACAATAAGTTCTTTTATATCTACTCAGAAACAATTATATTATTTTGAATATAT ATATATAATGAATA AAAAT AC ATTAAACCTACTTTC ATATAATATAC AAAAAAC AGATATAAATTCTAGTAGTAAATACACATATACAAAATCTCATTTTTTAAAAGAT AATCATATATTGTTATCTAAATATTATACTGCCAAATTTATTGATATCCTAAATAA AACATATTATTATAATTTATATAAAAATAAAATTCTTTTATTCAATAAATATATTA

Figure imgf000028_0001
CGATATAAAAATACAAGAAACATTAAAACAAATAACTCATATTGTTAACAATAT AAAAACCATCAAAAAGGATTTGCTCAAAGAATTTATTCAACATTTAATAAAATA TATGAACGAAAGATATCAGAATATGCAACAGGGTTATAATAATTTAACAAATTA TATTAATCAATATGAAGAAGAAAATAATAATATGAAACAATATATTACTACCAT ACGAAATATCCAAAAAATATATTATGATAATATATATGCTAAGGAAAAGGAAAT TCGCTCGGGACAATATTATAAGGATTTTATCACATCAAGGAAAAATATTTATAAT ATAAGGGAAAATATATCCAAAAATGTAGATATGATAAAAAATGAAGAAAAGAA GAAAATACAGAATTGTGTAGATAAATATAATTCTATAAAACAATATGTAAAAAT GCTTAAAAATGGAGACACACAAGATGAAAATAATAATAATAATAATGATATATA CGACAAGTTAATTGTCCCCCTTGATTC AATAAAACAAAATATCGATAAATACAAC ACAGAACATAATTTTATAACATTTACAAATAAAATAAATACACATAATAAGAAG AACCAAGAAATGATGGAAGAATTCATATATGCATATAAAAGGTTAAAAATTTTA AAAATATTAAATATATCCTTAAAAGCTTGTGAAAAAAATAATAAATCTATCAAT ACATTAAATGACAAAACACAAGAATTAAAAAAAATTGTAACACACGAAATAGAT CTTCTAC AAAAAGATATTTTAACAAGTCAAATATCAAATAAAAATGTTTTATTAT TAAACGATTTATTAAAAGAAATTGAACAATATATTATAGATGTACACAAATTAA AAAAAAAATCAAACGATCTATTTACATATTATGAACAATCCAAAAATTATTTCTA TTTTAAAAACAAAAAAGATAATTTTGATATACAAAAAACAATCAATAAAATGAA TGAATGGCTAGCTATCAAAAATTATATAAATGAAATTAATAAAAATTATCAAAC ATTATATGAAAAAAAAATAAATGTACTCCTACATAATTCAAAAAGTTATGTACA ATACTTTTATGATCATATAATAAATCTAATTCTTCAAAAAAAAAATTATTTGGAA AATACTTTAAAGACAAAAATACAAGATAACGAACATTCACTATATGCTTTACAA CAAAATGAAGAATACCAAAAGGTAAAGAACGAAAAGGATCAAAACGAAATTAA GAAAATTAAACAATTAATCGAAAAAAATAAAAATGATATACTTACATATGAAAA CAACATTGAACAAATTGAACAAAAAAATATTGAGTTAAAAACAAATGCTCAAAA TAAGGATGATCAAATAGTAAATACCTTAAATGAGGTTAAGAAAAAAATAATATA TACATATGAAAAGGTAGATAATCAAATATCGAACGTTTTAAAAAATTATGAAGA AGGAAAAGTAGAATATGATAAAAATGTTGTACAAAATGTTAACGATGCGGATGA TACAAACGATATTGATGAAATAAACGATATTGATGAAATAAACGATATTGATGA AATAAACGATATTGATGAAATAAACGATATTGATGAAATAAAAGACATTGACCA TATAAAACATTTTGACGATACAAAACATTTTGACGATATATACCATGCTGATGAT ACACGTGATGAATACCATATAGCCCTTTCAAATTATATAAAGACAGAACTAAGA AATATAAACCTGCAAGAAATAAAAAACAATATAATAAAAATATTTAAAGAATTC AAATCTGCACACAAAGAAATTAAAAAAGAATCAGAACAAATTAATAAAGAATTT ACCAAAATGGATGTCGTCATAAATCAATTAAGAGATATAGACAGACAAATGCTT GATCTTTATAAAGAATTAGATGAAAAATATTCTGAATTTAATAAAACAAAAATT GAAGAAATAAATAATATAAGGGAAAATATTAATAATGTGGAAATATGGTATGAA AAAAATATAATTGAATATTTCTTACGTCATATGAATGATCAAAAAGATAAAGCT GCAAAATATATGGAAAACATTGATACATATAAAAATAATATTGAAATTATTAGT AAACAAATAAATCCAGAAAATTATGTTGAAACATTAAACAAATCAAATATGTAT TCTTATGTAGAAAAGGCTAATGATCTATTTTATAAACAAATAAATAATATAATCA TAAATT C AAATC AACTAAAAAACGAAGCTTTTAC AATAGATGAATTAC AAAATA TTCAAAAAAACAGAAAAAATCTTCTTACAAAGAAACAACAAATTATTCAGTATA CAAATGAAATAGAAAATATATTTAATGAAATTAAAAATATTAATAACATATTAG TCTTAACAAATTATAAATCTATCCTTCAAGATATATCACAAAATATAAATCATGT TAGTATATATACGGAACAATTACATAATTTATATATAAAATTAGAAGAAGAAAA AGAAC AAATGAAAAC ACTCTATC ATA AATC AAATGTGTTAC ATAACC AAATTAA TTTTAATGAAGATGCTTTTATTAATAATTTATTAATTAATATAGAAAAAATTAAA AATGATATTACACATATAAAGGAAAAAACAAATATATATATGATAGATGTAAAC AAATCTAAAAATAATGCTCAACTATATTTTCATAATACACTAAGAGGTAATGAA AAAATAGAATATTTAAAAAATCTTAAGAATTCAACAAACCAACAAATAACTTTA CAAGAATTAAAACAAGTACAAGAAAATGTTGAGAAGGTAAAAGATATATACAA TCAAAGTATAAAATATGAAGAAGAAATTAAAAAAAATTATCATATTATAACAGA TTATGAGAATAAAATAAATGATATTTTACATAATTCATTTATTAAACAAATAAAT ATGGAATCTAGCAATAATAAAAAACAAACAAAACAAATTATAGACATAATAAAC GATAAAACATTTGAAGAACATATAAAAACATCCAAAACCAAAATAAACATGCTA AAAGAACAATCACAAATGAAACATATAGACAAAACTTTATTAAATGAACAAGCA CTCAAATTATTTGTAGATATTAATTCTACTAATAATAATTTAGATAATATGTTATC TGAAATAAATTCTATACAAAATAATATACATACATATATCCAAGAAGCAAACAA ATCATTTGACAAATTTAAAATTATATGTGATCAAAATGTAAACGATTTATTAAAC AAATTAAGTTTAGGAGATCTAAATTATATGAATCATTTAAAAAATCTGCAAAAC GAAATAAGAAACATGAATCTAGAAAAAAATTTCATGTTAGATAAAAGTAAAAAA ATAGATGAGGAAGAAAAAAAATTAGATATATTAAAAGTTAACATATCAAATATA AATAATTCTTTAGATAAATTAAAAAAATATTACGAAGAAGCGCTCTTTCAAAAG GTTAAAGAAAAAGCAGAAATTCAAAAGGAAAATATAGAAAAAATAAAACAAGA AATAAATACACTGAGCGATGTTTTTAAGAAACCATTTTTTTTTATACAACTTAAT ACAGATTCATCACAACATGAAAAAGATATAAACAATAATGTAGAAACATATAAA AATAATATAGATGAAATATATAATGTTTTTATACAATCATATAATTTAATACAAA AATATTCTTCAGAAATTTTTTCATCCACCTTGAATTATATACAAACAAAAGAAAT AAAAGAAAAATCCATAAAGGAACAAAACCAATTAAATCAAAATGAAAAGGAAG CATCTGTTTTATTAAAAAATATAAAAATAAATGAAACCATAAAATTATTTAAACA AATAAAAAATGAAAGACAAAACGATGTACACAATATAAAAGAGGACTATAACT TGTTACAACAATATTTAAATTATATGAAAAATGAAATGGAACAATTAAAAAAAT ATAAAAATGATGTTC ATATGGATAAAAATTATGTTGAAAATAATAATGGTG AAA AAGAAAAATTACTTAAAGAAACCATTTCTTCATATTATGATAAAATAAATAATAT AAATAATAAGCTATATATATATAAAAACAAAGAAGACACTTATTTTAATAATAT GATCAAAGTATCAGAAATTTTAAACATAATTATAAAAAAAAAACAACAAAATGA ACAAAGAATTGTTATAAATGCAGAATATGACTCTTCATTAATTAATAAGGATGA AGAAATTAAAAAAGAAATTAATAATCAAATAATTGAATTAAATAAAC ATAATGA AAATATTTCCAATATTTTTAAGGATATACAAAATATAAAAAAACAAAGTCAAGA TATTATCACAAATATGAACGACATGTATAAAAGTACAATCCTTTTAGTAGACATC ATACAGAAAAAAGAAGAAGCTCTAAATAAACAAAAAAATATTTTAAGAAATAT AGACAATATATTAAATAAAAAAGAAAATATTATAGATAAAGTTATAAAATGTAA TTGTGATGATTATAAAGATATCTTAATACAAAACGAAACGGAATATCAAAAATT ACAAAATATAAATCATACATATGAAGAAAAAAAAAAATCAATAGATATATTAAA AATTAAAAATATAAAACAAAAAAATATTCAAGAATATAAAAACAAATTAGAAC AAATGAATACAATAATTAATCAAAGTATAGAACAACATGTATTCATAAACGCTG ATATTTTACAAAATGAAAAAATAAAATTAGAAGAAATCATAAAAAATCTAGATA TACTAGATGAACAAATTATGACATATCATAATTCAATAGATGAATTATATAAACT AGGAATACAATGTGACAATCATCTAATTACAACTATTAGTGTTGTTGTTAATAAA AATACAACAAAAATTATGATACATATAAAAAAACAAAAAGAGGATATACAAAA AATTAATAACTATATTCAAACAAATTATAATATAATAAATGAAGAAGCTCTACA ATTTCACAGGCTCTATGGACACAATCTTATAAGTGAAGATGACAAAAATAATTTG GTACATATTATAAAAGAACAAAAGAATATATATACACAAAAGGAAATAGATATT TCTAAAATAATTAAACATGTTAAAAAAGGATTATATTCATTGAATGAACATGATA TGAATCATGATACACATATGAATATAATAAATGAACATATAAATAATAATATTTT ACAACCATACACACAATTAATAAACATGATAAAAGATATTGATAATGTTTTTATA AAAATACAAAATAATAAATTCGAACAAATACAAAAATATATAGAAATTATTAAA TCTTTAGAACAATTAAATAAAAATATAAACACAGATAATTTAAATAAATTAAAA GATACACAAAACAAATTAATAAATATAGAAACAGAAATGAAACATAAACAAAA ACAATTAATAAACAAAATGAATGATATAGAAAAGGATAATATTACAGATCAATA TATGCATGATGTTCAGCAAAATATATTTGAACCTATAACATTAAAAATGAATGAA TATAATACATTATTAAATGATAATCATAATAATAATATAAATAATGAACATCAAT TTAATCATTTAAATAGTCTTCATACAAAAATATTTAGTCATAATTATAATAAAGA ACAACAACAAGAATATATAACCAACATCATGCAAAGAATTGATGTATTCATAAA TGATTTAGATACTTACCAATATGAATATTATTTTTATGAATGGAATCAAGAATAT AAACAAATAGACAAAAATAAAATAAATCAACATATAAACAATATTAAAAATAAT CTAATTCATGTTAAGAAACAATTTGAACACACCTTAGAAAATATAAAAAATAAT GAAAATATTTTCGACAACATACAATTGAAAAAAAAAGATATTGACGATATTATT ATAAACATTAATAATACAAAAGAAACATATCTAAAAGAATTGAACAAAAAAAA AATGTTACA AAATAAAAAAAA AGTTGATGAAAA ATC AGAAATAAATAATC ATC A CACATTACAACATGATAATCAAAATGTTGAACAAAAAAATAAAATTAAAGATCA TAATTTAATAACCAAGCCAAATAACAATTCATCAGAAGAATCTCATCAAAATGA ACAAATGAAAGAACAAAACAAAAATATACTTGAAAAACAAACAAGAAATATCA AACCACATCATGTTCATAATCATAATCATAATCATAATCAAAATCAAAAAGATTC AACAAAATTACAGGAACAAGATATATCTACACACAAATTACATAATACTATACA TGAGCAACAAAGTAAAGATAATCATCAAGGTAATAGAGAAAAAAAACAAAAAA ATGGAAACCATGAAAGAATGTATTTTGCCAGTGGAATAGTTGTATCCATTTTATT TTTATCTAGTCTTGGATTTGTTATAAATAGTAAAAATAATAAACAAGAATATGAT AAAGAGCAAGAAAAACAACAACAAAATGATTTTGTATGTGATAATAACAAAATG GATGATAAAAGCACACAAAAATATGGTAGAAATCAAGAAGAGGTAATGGAGAT ATCTTTTGATAATGATTATATTTAA

Codon optimized DNA sequence for expression of rPf Hl4o/SEO. ID N0.2):

CATATGCTGCAAATCGTGCAACAGAAGCTGCTGGAGATCAAACAGAAAAAGAAC GATATTACACATAAGGTGCAACTGATTAATCACATTTACAAAAACATTCACGAC GAAATTCTGAACAAAAAGAACAACGAAATCACTAAGATTATCATTAACAATATC AAAGATCACAAAAAGGATCTGCAAGACTTATTGCTGTTCATTCAGCAGATCAAA CAATACAATATCTTAACGGACCACAAAATCAC^CAGTGCAATAACTATTACAAG GAAATTATCAAAATGAAAGAGGATATCAACCACATTCATATCTACATTCAGCCG ATCTTAAACAATCTGCATACTCTGAAACAGGTGCAGAACAACAAAATCAAATAC GAGGAACATATCAAACAGATTTTACAGAAGATTTACGATAAGAAAGAATCTCTG AAAAAGATCATTCTGCTGAAGGACGAGGCGCAGCTGGACATCACCCTGTTAGAC GATCTGATCCAGAAACAGACTCAGACCCAGACCCAGACGCAGAAACAGACCTTA ATCCAGAACAACGAAACCATTCAGCTGATTAGTGGTCAGGAAGATAAACACGAA AGCAACCCGTTCAATCATATCCAGACGTACATCCAGCAGAAAGATACTCAGAAC AAAAACATCCAGAACTTACTGAAATCACTGTACAATGGTAACATTAACACGTTT ATCGATACAATCAGCAAATACATCCTGAAACAGAAAGATATCGAACTGACTCAG CACGTGTATACCGACGAAAAGATCAACGATTACCTGGAAGAAATCAAGAACGAA CAGAACAAAATCGACAAAACCATTGACGATATCAAGATCCAGGAAACACTGAA ACAGATCACCCACATCGTTAACAACATCAAAACCATCAAGAAAGACCTGCTGAA AGAATTCATTCAGCATCTTATCAAATACATGAATGAACGTTATCAAAACATGCAG CAAGGTTACAATAACCTGACGAACTACATTAACCAGTACGAGGAGGAAAATCTC GAG

Protein sequence of PfRHl The region highlighted is the erythrocyte binding domain of PfRHl (Receptor binding domain marked) (SEQ. ID NO.3):

MQRWIFCNIVLHILIYLAEFSHEQESYSSNEKIRKDYSDDNNYEPTPSYEKRKKEYG DESYIKNYRGNNFSYDLSKNSSIFLHMGNGSNSKTLKRCNKKKNI TNFLRPIEE EKTVLNNYVYKGVNFLDTIKRNDSSYKFDVY DTSFL NREYKELITMQYDYAY LEATKEVLYLIPKDKDYHKFYKNELEKILFNLKDSL LLREGYIQS LEMIRIHSDI DILNEFHQGNIINDNYF^EIKKKKEDMEKYIREYNLYIYKYENQLKIKIQKLTNEV SINLNKSTCEKNCYNYILKLE YKNII DKINKWKDLPEIYIDDKSFSYTFL DVIN NKIDIYKTISSFISTQKQLYYFEYIYIMNK TLNLLSYNIQKTDINSSSKYTYTKSHFL KDNHILLS YYTA FIDILN TYYYNLY NKILLFNKYII LRNDLKEYAF SIQFIQ DKIKKHKDELSIENILQEVNNIYIKYDTSI EISKYNNLIINTDLQIVQQKLLEIKQK NDmiKVQLINHIY NIHDEn.N KK^^^

LTpHKrrQCNNYY EilKM EDrNHiHfYlQPILlWLH

QKIYDK ESLKKI^^

Kl3lELTQHVYTDE¾

LKEFIQHLIKYMNERYQ^ DNIYAKEKEIRSGQYYKDFITSRKNIYNIRENISKNVDMI NEEKKKIQNCVDKYNS IKQYVKMLKNGDTQDENNNNNNDIYDKLIVPLDSIKQNIDKYNTEHNFITFTN IN THNKXNQEMMEEFIYAYKRLKILKILNISLKACEK NKSINTLNDKTQELKKIVTH EIDLLQKDILTSQISNKNVLLLNDLLKEIEQYIIDVHKL KKSNDLFTYYEQSKNYF YFK KKDNFDIQKTINKMNEWLAIKNYINEINKNYQTLYEKKINVLLHNSKSYVQ YFYDHIINLILQK NYLENTL TKIQDNEHSLYALQQNEEYQKVKNEKDQNEIKKI KQLIEKNKNDILTYENNIEQIEQKNIEL TNAQNKDDQIVNTLNEVK KIIYTYE V DNQISNVLKNYEEGKVEYDKNVVQNVNDADDTNDIDEINDIDEINDIDEINDIDEIN DIDEIKDIDHI HFDDT HFDDIYHADDTRDEYHIALSNYIKTELRNINLQEIKN II KIFKEFKS AHKEIKXESEQINKEFT MDVVINQLRDIDRQMLDLYKELDE YSEFN KTKIEEI>WIRENINNVEIWYEKNIIEYFLRH

KQINPENYVETLNKSNMYSYVEKANDLFY QINNIIINSNQLKNEAFTIDELQNIQK

NRKNLLTKKQQIIQYTNEIENIFNEIKNINNILVLTNYKSILQDISQNINHVSIYTEQL

HNLYI LEEEKEQMKTLYHKSNVLHNQINFNEDAFINNLLINIEKIKNDITHIKEKT NIYMIDVNKSKNNAQLYFHNTLRGNEKIE YL NLKNSTNQQITLQEL QVQENVE KVKDIYNQTIKYEEEIKKNYHIITDYENKINDILHNSFIKQINMESSNNKKQTKQIIDI INDKTFEEHI TSKTKINMLKEQSQMKHIDKTLLNEQALKLFVDINSTNNNLDNML SEINSIQN IHTYIQEANKSFDKFKIICDQNVNDLLNKLSLGDLNYMNHLKNLQNEI RNMNLEKNFMLDKSKKIDEEE KLDILKVNISNINNSLDKLKKYYEEALFQKVKE KAEIQKENIEKI QEINTLSDVFKKPFFFIQLNTDSSQHEKDINNNVETYKNNIDEIY NVFIQSYNLIQKYSSEIFSSTLNYIQTKEIKEKSI EQNQLNQNE EASVLLKNIKINE TI LFKQIKNERQNDVHNIKEDYNLLQQYLNYMKNEMEQLKKYKNDVHMDKNY VENNNGEKEKLLKETISSYYDKINNINNKLYIYKNKEDTYFNNMIKVSEILNIHKKK QQNEQWVINAEYDSSLINKDEEIKKEINNQIIELNimNENISNIFKDIQNIi KQSQDII TNMNDMYKSTILLVDIIQKKEEALNKQK ILRNIDNILNKKENIID VIKCNCDDYK DILIQNETEYQ LQNINHTYEEi KSIDILKIKNIKQ NIQEYKNKLEQMNTIINQSI EQHVFINADILQNEKIKLEEIIKNLDILDEQIMTYHNSIDELYKLGIQCDNHLITTISV VVlSlKNTTKJMIHIKKQKEDIQKINNYIQTNYNIINEEALQFHRLYGHNLISEDDKmr LVHIIKEQ NIYTQKEIDISKJIKHVKKGLYSLNEHDMNHDTHMNIINEHIN NILQP YTQLINMIKDIDNVFIKIQNNKFEQIQKYIEIIKSLEQLNKNINTDNLNKL DTQNKL INIETEMKHKQKQLINKMNDIEKDNITDQYMHDVQQNIFEPITLKMNEYNTLLND NHNNNIN EHQFNHLNSLHTKIFSHNYNKEQQQEYITNIMQRIDVFI DLDTYQYE YYFYETOQEYKQIDKNKINQHINNIKNNLIHV KQFEHTLENIKNNENIFDNIQLK KKDIDDniNINNTKETYLKELNK KMLQNKKKVDEKSEFN HHTLQHDNQNVEQ KNKI OHNLITKP NNSSEESHQNEQMKEQNKNILEKQTRNIKPHHVHNHNH HN QNQKDSTKLQEQDISTHKLHNTIHEQQSKDNHQGNREKKQKNGNHERMYFASGI VVSILFLSSLGFVINSKNNKQEYDKEQEKQQQNDFVCDNNKMDD STQKYGRNQ EEVMEISFDNDYI(2971 amino acids)

PfRH2:

Expression & Purification of the rPfRH24n_(Figures 5 A, B and Q:

An E.coli codon-optimized synthetic gene encoding amino acids 495 to 860 of the receptor binding domain of P RFOw is obtained from GeneArt (Life Technologies) and cloned into pET-24b expression vector using the Ndeland Xhol restriction enzymes.

E.coli BL21(DE3) cells (Novagen, San Diego, CA) are transformed with the expression plasmid and used to produce the recombinant rPfRH4o protein. Transformed E. coli BL21(DE3) are cultured in Luria broth at 37°C. Expression of rPfRH240 protein is induced with 1 mM IPTG when OD600 of the culture was in the range of 0.6 to 0.8. Cells are grown for 4 hours after induction and were harvested by centrifugation at 4500 g (Figure

5A).

Expression in BL21(DE3): U= Uninduced, In= Induced with ImM IPTG

Harvested cell pellet is lyzed by sonication in Lysis Buffer (lOmM Tris, 10 mM EDTA, 1 lOmM NaCl, pH-8) and the over expressed protein is found in inclusion bodies. The inclusion bodies are collected by centrifugation at 15000 g, washed twice with lOmM Tris, 10 mM EDTA, l lOmM NaCl, pH-8.0 and solubilized in 6M Guanidine-HCL containing buffer. The protein is purified from solubilized inclusion bodies by Ni-NTA (Nitrilotriacetic acid) affinity chromatography.

Purification of rPfRH240 by Metal affinity Chromatography

Ni-NTA purified rPfRH240 is refolded in a Tris based refolding buffer (55mM Tris pH 8.2, 264 mM NaCl, 11 mM KC1, 2.2 mM MgC12, 2.2 mM CaC12, 440 mM Sucrose, 550 mM L-Arginine). The refolding solution is incubated at 4oC for 24 hours with constant stirring. The refolded protein is then dialysed overnight against Phosphate buffered saline (PBS pH 7.4). Following dialysis the protein is loaded on Q-sepharose column (GE Healthcare) for further purification by Anion exchange chromatography (Figure 5B). DNA sequence of the PfRH2b gene (The highlighted region encodes the receptor binding region of the PfRH2 protein) (SEQ ID NO. 4):

ATGAAGACCACACTATTTTGTAGCATATCTTTTTGTAATATTATATTTTTCTTCTT AGAATTAAGTCATGAGCATTTTGTTGGACAATCAAGTAATACCCATGGAGCATCT TCAGTTACTGATTTTAATTTTAGTGAGGAGAAAAATTTAAAAAGTTTTGAAGGGA AGAATAATAATAATGATAATTATGCTTCAATTAATCGTTTATATAGGAAGAAACC ATATATGAAGAGATCGCTTATAAATTTAGAAAATGATCTTTTTAGATTAGAACCT ATATCTTATATTCAAAGATATTATAAGAAGAATATAAACAGATCTGATATTTTTC ATAATAAAAAAGAAAGAGGTTCCAAAGTATATTCAAATGTGTCTTCATTCCATTC TTTTATTC AAGAGGGT AAAG AAGA AGTTG AGGTTTTTTCT ATATGGGGTAGTAAT AGCGTTTTAGATCATATAGATGTTCTTAGGGATAATGGAACTGTCGTTTTTTCTGT TCAACCATATTACCTTGATATATATACGTGTAAAGAAGCCATATTATTTACTACA TCATTTTACAAGGATCTTGATAAAAGTTCAATTACAAAAATTAATGAAGATATTG AAAAATTTAACGAAGAAATAATCAAGAATGAAGAACAATGTTTAGTTGGTGGGA AAACAG ATTTTGATA ATTTACTTATAGTTTTAGAAAATGCGGAA A AAGCA AATGT TAGAAAAACATTATTTGATAATACATTTAATGATTATAAAAATAAGAAATCTAGT TTTTACAATTGTTTGAAAAATAAAAAAAATGATTATGATAAGAAAATAAAGAAT ATAAAGAATGAGATTACAAAATTGTTAAAAAATATTGAAAGTACAGGAAATATG TGTAAAACGGAATCATATGTTATGAATAATAATTTATATCTATTAAGAGTGAATG AAGTTAAAAGTACACCTATTGATTTATACTTAAATCGAGCAAAAGAGCTATTAG AATCAAGTAGCAAATTAGTTAATCCTATAAAAATGAAATTAGGTGATAATAAGA ACATGTACTCTATTGGATATATACATGACGAAATTAAAGATATTATAAAAAGAT ATAATTTTCATTTGAAACATATAGAAAAAGGAAAAGAATATATAAAAAGGATAA CACAAGCAAATAATATTGCAGACAAAATGAAGAAAGATGAACTTATAAAAAAA ATTTTTGAATCCTCAAAACATTTTGCTAGTTTTAAATATAGCAATGAAATGATAA GCAAATTAGATTCGTTATTTATAAAAAATGAAGAAATACTTAATAATTTATTCAA TAATATATTTAATATATTCAAGAAAAAATATGAAACATATGTAGATATGAAAAC AATTGAATCTAAATATACAACAGTAATGACTCTATCAGAACATTTATTAGAATAT GCAATGGATGTTTTAAAAGCTAACCCTCAAAAACCTATTGATCCAAAAGCAAAT

TTAGATAATGCTATAAG^

ATGATATTATTATATCTGAAA^G

Figure imgf000037_0001

Figure imgf000037_0002

AATTATTATGAAATTCTAGATAAAAAATTAAAAGATAATACATATATCAAAGAA ATGCATACTGCTTCTTTAGTTCAAATAACTCAATATATTCCTTATGAAGATAAAA CAATAAGTGAACTTGAGCAAGAATTTAATAATAATAATCAAAAACTTGATAATA TATTACAAGATATCAATGCAATGAATTTAAATATAAATATTCTCCAAACCTTAAA TATTGGTATAAATGCATGTAATACAAATAATAAAAATGTAGAACACTTACTTAAC AAGAAAATTGAATTAAAAAATATATTAAATGATCAAATGAAAATTATAAAAAAT GATGATATAATTCAAGATAATGAAAAAGAAAACTTTTCAAATGTTTTAAAAAAA GAAGAGGAAAAATTAGAAAAAGAATTAGATGATATCAAATTTAATAATTTGAAA ATGGACATTCATAAATTGTTGAATTCGTATGACCATACAAAGCAAAATATAGAA AGCAATCTTAAAATAAATTTAGATTCTTTCGAAAAGGAAAAAGATAGTTGGGTT CATTTTAAAAGTACTATAGATAGTTTATATGTGGAATATAACATATGTAATCAAA AGACTCATAATACTATCAAACAACAAAAAAATGATATCATAGAACTTATTTATA AACGTATAAAAGATATAAATCAAGAAATAATCGAAAAGGTAGATAATTATTATT CCCTGTCAGATAAAGCCTTAACTAAACTTAAATCTATTCATTTTAATATTGATAA GGAAAAATATAAAAATCCCAAAAGTCAAGAAAATATTAAATTATTAGAAGATAG AGTTATGATACTTGAGAAAAAGATTAAGGAAGATAAAGATGCTTTAATACAAAT TAAGAATTTATCACATGATCATTTTGTAAATGCTGATAATGAGAAAAAAAAGCA GAAGGAGAAGGAGGAGGACGACGAACAAACACACTATAGTAAAAAAAGAAAA GTAATGGGAGATATATATAAGGATATTAAAAAAAACCTAGATGAGTTAAATAAT AAAAATTTGATAGATATTACTTTAAATGAAGCAAATAAAATAGAATCAGAATAT GAAAAAATATTAATTGATGATATTTGTGAACAAATTACAAATGAAGCAAAAAAA AGTGATACTATTAAGGAAAAAATCGAATCATATAAAAAAGATATTGATTATGTA GATGTGGACGTTTCCAAAACGAGGAACGATCATCATTTGAATGGAGATAAAATA CATGATTCTTTTTTTT ATGA AG AT AC ATT AAATTATAAAGC AT ATTTTGATA AATT AAAAGATTTATATGAAAATATAAACAAGTTAACAAATGAATCAAATGGATTAAA AAGTGATGCTCATAATAACAACACACAAGTTGATAAACTAAAAGAAATTAATTT ACAAGTATTCAGCAATTTAGGAAATATAATTAAATATGTTGAAAAACTTGAGAA TACATTACATGAACTTAAAGATATGTACGAATTTCTAGAAACGATCGATATTAAT AAAATATTAAAAAGTATTC ATAATAGCATGAAGAAATCAGAAGAATATAGTAAT GAAACGAAAAAAATATTTGAACAATCAGTAAATATAACTAATCAATTTATAGAA GATGTTGAAATATTGAAAACGTCTATTAACCCAAACTATGAAAGCTTAAATGAT GATCAAATTGATGATAATATAAAATCACTTGTTCTAAAGAAAGAGGAAATATCC GAAAAAAGAAAACAAGTGAATAAATACATAACAGATATTGAATCTAATAAAGA ACAATCAGATTTACATTTACGATATGCATCTAGAAGTATATATGTTATTGATCTTT TTATAAAACATGAAATAATAAATCCTAGCGATGGAAAAAATTTTGATATTATAA AGGTTAAAGAAATGATAAATAAAACCAAACAAGTTTCAAATGAAGCTATGGAAT ATGCTAATAAAATGGATGAAAAAAATAAGGACATTATAAAAATAGAAAATGAA CTTTATAATTTAATTAATAATAACATCCGTTCATTAAAAGGGGTAAAATATGAA AAAGTTAGGAAACAAGCAAGAAATGCAATTGATGATATAAATAATATACATTCT AATATTAAAACGATTTTAACCAAATCTAAAGAACGATTAGATGAGATTAAGAAA CAACCTAACATTAAAAGAGAAGGTGATGTTTTAAATAATGATAAAACCAAAATA GCTTATATTACAATACAAATAAATAACGGAAGAATAGAATCTAATTTATTAAAT ATATTAAATATGAAACATAACATAGATACTATCTTGAATAAAGCTATGGATTATA TGAATGATGTATCAAAATCTGACCAGATTGTTATTAATATAGATTCTTTGAATAT GAACGATATATATAATAAGGATAAAGATCTTTTAATAAATATTTTAAAAGAAAA ACAGAATATGGAGGCAGAATATAAAAAAATGAATGAAATGTATAATTACGTTAA TGAAACAGAAAAAGAAATAATAAAACATAAAAAAAATTATGAAATAAGAATTA TGGAACATATAAAAAAAGAAACAAATGAAAAAAAAAAAAAATTTATGGAATCT AATAACAAATCATTAACTACTTTAATGGATTCATTCAGATCTATGTTTTATAATG AATATATAAATGATTATAATATAAATGAAAATTTTGAAAAACATCAAAATATATT GAATGAAATATATAATGGATTTAATGAATCATATAATATTATTAATACAAAAATG ACTGAAATTATAAATGATAATTTAGATTATAATGAAATAAAAGAAATTAAAGAA GTAGCACAAACAGAATATGATAAACTTAATAAAAAAGTTGATGAATTAAAAAAT TATTTGAATAATATTAAAGAACAAGAAGGACATCGATTAATTGATTATATAAAA GAAAAAATATTTAACTTATATATAAAATGTTCAGAACAACAAAATATAATAGAT GATTCTTATAATTATATTAC AGTTAAAAAAC AGTATATTAAAACTATTGAAGATG TGAAATTTTTATTAGATTCATTGAACACAATAGAAGAAAAAAATAAATCAGTAG CAAATCTAGAAATTTGTACTAATAAAGAAGATATAAAAAATTTACTTAAACATG TTATAAAGTTGGCAAATTTTTCAGGTATTATTGTAATGTCTGATACAAATACGGA AATAACTCCAGAAAATCCTTTAGAAGATAATGATTTATTAAATTTACAATTATAT TTTGAAAGAAAACATGAAATAACATCAACATTGGAAAATGATTCTGATTTAGAG TTAGATCATTTAGGTAGTAATTCGGATGAATCTATAGATAATTTAAAGGTTTATA ATGATATTATAGAATTACACACATATTCAACACAAATTCTTAAATATTTAGATAA TATTCAAAAACTTAAAGGAGATTGCAATGATTTAGTAAAGGATTGTAAAGAATT ACGTGAATTGTCTACGGCATTATATGATTTAAAAATACAAATTACTAGTGTAATT AATAGAGAAAATGATATTTCAAATAATATTGATATTGTATCTAATAAATTAAATG AAATAGATGCTATACAATATAATTTTGAAAAATATAAAGAAATTTTTGATAATGT AGAAGAATATAAAACATTAGATGATACAAAAAATGCATATATTGTAAAAAAGGC TGAAATTTTAAAAAATGTAGATATAAATAAAACAAAAGAAGATTTAGATATATA TTTTAATGACTTAGACGAATTAGAAAAATCTCTTACATTATCATCTAATGAAATG GAAATTAAAACAATAGTACAGAACTCATATAATTCCTTTTCTGATATTAATAAGA ACATTAATGATATTGATAAAGAAATGAAAACACTGATCCCTATGCTTGATGAATT ATTAAATGAAGGACATAATATTGATATATCATTATATAATTTTATAATTAGAAAT ATTCAGATTAAAATAGGTAATGATATAAAAAATATAAGAGAACAGGAAAATGAT ACTAATATATGTTTTGAGTATATTCAAAATAATTATAATTTTATAAAGAGTGATA TAAGTATCTTCAATAAATATGATGATCATATAAAAGTAGATAATTATATATCTAA TAATATTGATGTTGTCAATAAACATAATAGTTTATTAAGTGAACATGTTATAAAT GCTACAAATATTATAGAGAATATTATGACAAGTATTGTCGAAATAAATGAAGAT ACAGAAATGAATTCTTTAGAAGAGACACAAGACAAATTATTAGAACTATATGAA AATTTTAAGAAAGAAAAAAATATTATAAATAATAATTATAAAATAGTACATTTT AATAAATTAAAAGAAATAGAAAATAGTTTAGAGACATATAATTCAATATCAACA AACTTTAATAAAATAAATGAAACACAAAATATAGATATTTTAAAAAATGAATTT AATAATATCAAAACAAAAATTAATGATAAAGTAAAAGAATTAGTTCATGTTGAT AGTACATTAACACTTGAATCAATTCAAACGTTTAATAATTTATATGGTGACTTGA TGTCTAATATACAAGATGTATATAAATATGAAGATATTAATAATGTTGAATTGAA AAAGGTGAAATTATATATAGAAAATATTACAAATTTATTAGGAAGAATAAACAC ATTCATAAAGGAGTTAGACAAATATCAGGATGAAAATAATGGTATAGATAAGTA TATAGAAATCAATAAGGAAAATAATAGTTATATAATAAAATTGAAAGAAAAAGC CAATAATCTAAAGGAAAATTTCTCAAAATTATTACAAAATATAAAAAGAAATGA AACTGAATTATATAATATAAATAACATAAAGGATGATATTATGAATACGGGGAA ATCTGTAAATAATATAAAACAAAAATTTTCTAGTAATTTGCCACTAAAAGAAAA ATTATTTCAAATGGAAGAGATGTTACTTAATATAAATAATATTATGAATGAAACG AAAAGAATATCAAACACGGATGCATATACTAATATAACTCTCCAGGATATTGAA AATAATAAAAATAAAGAAAATAATAATATGAATATTGAAACAATTGATAAATTA ATAGATCATATAAAAATACATAATGAAAAAATACAAGCAGAAATATTAATAATT GATGATGCCAAAAGAAAAGTAAAGGAAATAACAGATAATATTAACAAGGCTTTT AATGAAATTACAGAAAATTATAATAATGAAAATAATGGGGTAATTAAATCTGCA AAAAATATTGTCGATAAAGCTACTTATTTAAATAATGAATTAGATAAATTTTTAT TGAAATTGAATGAATTATTAAGTCATAATAATAATGATATAAAGGATCTTGGTGA TGAAAAATTAATATTAAAAGAAGAAGAAGAAAGAAAAGAAAGAGAAAGATTGG AAAAAGCGAAACAAGAAGAAGAAAGAAAAGAGAGAGAAAGAATAGAAAAAGA AAAACAAGAGAAAGAAAGACTGGAAAGAGAGAAACAAGAACAACTAAAAAAA GAAGCATTAAAAAAACAAGAGCAAGAAAGACAAGAACAACAACAAAAAGAAG AAGCATTAAAAAGACAAGAACAAGAACGACTACAAAAAGAAGAAGAATTAAAA AGACAAGAGCAAGAAAGGCTGGAAAGAGAGAAACAAGAACAACTACAAAAAG AAGAAGAATTAAGAAAAAAAGAGCAGGAAAAACAACAACAAAGAAATATCCAA GAATTAGAAGAGCAAAAAAAGCCTGAAATAATAAATGAAGCATTGGTAAAGGG GGATAAAATACTAGAAGGAAGTGATCAGAGAAATATGGAATTAAGCAAACCTA ACGTTAGTATGGATAATACTAATAATAGTCCAATTAGTAACAGTGAAATTACAG AAAGCGATGATATTGATAACAGTGAAAATATACATACTAGTCATATGAGTGACA TCGAAAGTACACAAACTAGTCATAGAAGTAACACCCATGGGCAACAAATCAGTG ATATTGTTGAAGATCAAATTACACATCCTAGTAATATTGGAGGAGAAAAAATTA CTCATAATGATGAAATTTCAATCACTGGTGAAAGAAATAACATTAGCGATGTTA ATGATTATAGTGAAAGTAGCAACATATTTGAAAATGGTGACAGTACTATAAATA CCAGTACAAGAAACACGTCTAGTACACATGATGAATCCCATATAAGTCCTATCA GCAATGCGTATGATCATGTTGTTTCAGATAATAAAAAAAGTATGGATGAAAACA TAAAAGATAAATTAAAGATAGATGAAAGTATAACTACAGATGAACAAATAAGAT TAGATGATAATTCTAATATTGTTAGAATTGATAGTACTGACCAACGTGATGCTAG TAGTCATGGTAGTAGTAATAGGGATGATGATGAAATAAGTCATGTTGGTAGCGA CATTCATATGGATAGTGTTGATATTC ATGATAGTATTGAC ACTGATGAAAATGCT GATCACAGACATAATGTTAACTCTGTTGATAGTCTTAGTTCTAGTGATTACACTG ATACACAGAAAGACTTTAGTAGTATTATTAAAGATGGGGGAAATAAAGAAGGAC ATGCTGAGAATGAATCTAAAGAATATGAATCCCAAACAGAACAAACACATGAAG AAGGAATTATGAATCCAAATAAATATTCAATTAGTGAAGTTGATGGTATTAAATT AAATGAAGAAGCTAAAC ATAAAATTACAGAAAAACTGGTAGATATCTATCCTTC TACATATAGAACACTTGATGAACCTATGGAAACACATGGTCCAAATGAAAAATT TCATATGTTTGGTAGTCCATATGTAACAGAAGAAGATTACACGGAAAAACATGA TTATGATAAGCATGAAGATTTCAATAATGAAAGGTATTCAAACCATAACAAAAT GGATGATTTCGTATATAATGCTGGAGGAGTTGTTTGTTGTGTATTATTTTTTGCAA GTATTACTTTCTTTTCTATGGACAGATCAAATAAGGATGAATGCGATTTTGATAT GTGTGAAGAAGTAAATAATAATGATCACTTATCGAATTATGCTGATAAAGAAGA AATTATTGAAATTGTGTTTGATGAAAATGAAGAAAAATATTTTTAA

Codon optimized DNA sequence for expression of rPfRH24o_(SEO ID NO. 5):

CATATGAGCGAAGTTGTGAAACTGCAAATCAAAATCAACGAGAAAAGTAACGA GTTAGATAACGCGATAAGTCAGGTGAAAACCCTTATCATTATCATGAAATCCTTT TACGATATCATTATCTCTGAAAAAGCCTCCATGGACGAAATGGAAAAGAAAGAG TTATCGCTGAACAATTACATTGAGAAAACCGACTATATCTTACAAACCTATAACA TTTTCAAAAGCAAAAGCAATATCATTAACAACAATAGTAAGAACATAAGTTCTA AGTATATCACTATTGAAGGTCTGAAAAACGATATTGACGAACTGAATTCTTTGAT TAGCTATTTCAAAGATAGTCAGGAAACACTGATCAAAGACGACGAACTTAAGAA AAACATGAAAACGGATTATCTGAATAACGTCAAATACATTGAAGAAAACGTTAC TCACATAAACGAAATCATTCTGCTGAAAGATTCCATTACACAACGTATAGCAGAT ATTGACGAGCTGAATAGCTTGAATCTGATAAACATTAACGACTTTATCAACGAG AAAAACATTTCACAAGAAAAAGTTTCTTACAATCTTAACAAACTGTATAAGGGA TCTTTTGAAGAACTGGAATCGGAGTTATCACACTTTCTTGATACTAAGTATTTGTT TCACGAAAAGAAAAGTGTCAACGAGTTACAGACAATTCTGAATACATCGAATAA CGAGTGTGCAAAACTGAATTTCATGAAATCAGACAATAACAATAACAATAACAA TAGCAATATCATCAATCTTCTGAAAACGGAACTGAGTCATTTGCTGTCACTGAAA GAGAATATCATTAAGAAACTGCTGAATCATATTGAACAGAATATCCAGAATTCA TCCAATAAGTATACCATTACGTATACCGATATCAACAATCGCATGGAAGATTACA AAGAGGAAATTGAAAGCTTAGAAGTGTATAAGCATACGATTGGCAATATCCAGA AAGAGTATATCTTAC ATTTGTACGAAAACGATAAGAACGCTTTAGCCGTACACA ATACCTCTATGCAGATTCTTCAGTATAAGGACGCGATTCAGAATATCAAAAACA AGATATCAGACGATATTCACCACCACCACCACCACTGACTCGAG

PfRH2a/b:

P. falciparum has two PfRH2 genes - PfRH2a and PfRH2b, which have an identical 2700 amino acid sequence in their ectodomain, which is referred to as PfRH2a/b. The receptor binding region is from amino acids 495-860, which is identical in both homologues.

Protein sequence of PfRH2b (highlighted portion is Receptor binding domain of the PfRH2 protein (SEP ID NO 24 :

MKTTLFCSISFCNIIFFFLELSHEHFVGQSSNTHGASSVTDFNFSEEKNLKSFEGKNNN NDNYASINRLYRKXPYMKRSLINLENDLFP EPISYIQRYYKKNINRSDffHNKKERG SKVYSNVSSFHSFIQEGKEEVEVFSIWGSNSVLDHIDVLRDNGTVVFSVQPYYLDIYT CKEAILFTTSFYKDLDKSSITKINEDIEKFNEEIIKNEEQCLVGGKTDFDNLLIVLENAE KANVRKTLFDNTFNDYKNK SSFYNCLKN KNDYDKKIKNIKNEITKLLKNIESTGN MCKTESYVMNNNLYLLRVNEVKSTPIDLYLNRAKELLESSSKLVNPIKM LGDNKN MYSIGYIHDEIKDIIKRYNFHLKHIEKGKEYIKRITQANNIAD MKKDELIKKIFESSKF ASFKYSNEMISKLDSLFIKNEEILNNLFNNIFNIFKKKYETYVDMKTIESKYTTVMTLS EHLLEYAMDVLKANPQKPIDPKANLDSEVV LQI INEKSNELDNArSQVKl ^

SFYDlIISEf ^SMDEMEK ELSLNN

ELGLkNDipELNSLlSYFKDSQE U]^DELkl^

Figure imgf000042_0001
YENDKNALAVHNTSMQILQ

KLKDNTYIKEMHTASLVQITQYIPYEDKTISELEQEFNNNNQ LDNILQDINAMNLNI NILQTLNIGINACNTNNKNVEHLLN KIEL NILNDQMKIIKNDDIIQDNEKENFSNVL KKEEEKLEKELDDIKFNNLKMDIHKLLNSYDHTKQNIESNLKINLDSFEKEKDSWVH FKSTIDSLYVEYNICNQKTHNTI QQKNDIIELIYKRIKDINQEIIE VDNYYSLSDKAL TKL SIHFNIDKE YKNPKSQENIKLLEDRVMILEKKIKEDKDALIQIKNLSHDHFVN ADNEK KQKEKEEDDEQTHYSKKRKVMGDIYKDIKKNLDELNNKNLIDITLNEANK IESEYEKILIDDICEQITNEA KSDTI EKIES YKKDID YVDVDVSKTRNDHHLNGDKI HDSFFYEDTLNYKAYFD LKDLYENINKLTNESNGLKSDAHNNNTQVDKLKEINLQ VFSNLGNIIKYVEKLENTLHEL DMYEFLETIDINKILKSIHNSM KSEEYSNETKKIF EQSVNITNQFIEDVEILKTSINPNYESLNDDQIDDNIKSLVLKKEEISEKRKQVNKYITD IESNKEQSDLHLRYASRSIYVIDLFIKHEIINPSDGKNFDIIKVKEMIN T QVSNEAME YANKMDEKNKDIIKIENELYNLI NNIRSLKGVKYEKVRKQARNAIDDINNIHSNIKTI LTKSKERLDEIKKQPNIKREGDVLNNDKTKIAYITIQIN GmESNLLNILNMKHNIDTI LNKAMDYMNDVSKSDQIVINIDSLNMNDIYNKDKX)LLINIL EKQNMEAEYKKMNE MYNYVNETEKEIIKHKKNYEIWMEHIKKETNEKKKKFMESNNKSLTTLMDSFRSMF YNEYINDYNINENFE HQNILNEIYNGFNESYNIINTKMTEIINDNLDYNEI EIKEVA QTEYDKLNKKVDELKNYLNNIKEQEGHRLIDYIKEKIFNLYI CSEQQNIIDDSYNYIT VKKQYIKTIEDV FLLDSLNTIEEKNKSVANLEICTN EDIKNLLKHVIKLANFSGIIV MSDTNTEITPENPLEDNDLLNLQLYFERKHEITSTLENDSDLELDHLGSNSDESIDNLK VYNDIIELHTYSTQILKYLDNIQKLKGDCNDLVKDCKELRELSTALYDLKIQITSVINR ENDIS^IDIVSNKLNEIDAIQYNFEKYKEIFDNVEEYKTLDDTKNAYIVKKAEIL NV DINKTKEDLDIYFNDLDELEKSLTLSSNEMEIKTIVQNSYNSFSDINKNINDIDKEMKT LIPMLDELLNEGHNIDISLYNFIIRNIQI IGNDIKNIREQENDTNICFEYIQNNYNFIKSD ISIFNKYDDHIKVDNYISNNIDVVNKHNSLLSEHVINATNIIENIMTSIVEINEDTEMNS LEETQDKLLELYENFKKEKNIINNNYKIVHFNKLKEIENSLETYNSISTNFNKINETQNI DILKNEFN IKTKINDKVKELVHVDSTLTLESIQTFNNLYGDLMSNIQDVYKYEDINN VELKKVKLYIENITNLLGRINTFI ELDKYQDEN GIDKYIEINKENNSYIIKLKEKAN NLKENFSKLLQNIKRNETELY INNIKDDIMNTGKSVNNIKQKFSSNLPLKEKLFQME EMLLNINNIMNET ^SNTDAYTNITLQDIENNKNKENNNMNIETIDKLIDHIKIH EK IQAEILIIDDAKRKVKEITDNINKAFNEITENYNNENNGVIKSAKNIVDKATYLNNELD KFLLKLNELLSHNNNDIKDLGDEKLILKEEEERKERERLEKA QEEERKERERIEKEK QEKERLEREKQEQLKKEALKKQEQERQEQQQKEEALKRQEQERLQKEEELKRQEQE RLEREKQEQLQKEEELRKKEQE QQQRNIQELEEQK PEII EALVKGDKILEGSDQR NMELSKPNVSMDNTNNSPISNSEITESDDIDNSENIHTSHMSDIESTQTSHRSNTHGQQ ISDIVEDQITHPSNIGGE ITHNDEISITGERNNISDVNDYSESSNIFENGDSTINTSTRNT SSTHDESHISPISNAYDHVVSDN KSMDENIKDKLKIDESITTDEQIRLDDNSNIVRIDS TDQRDASSHGSSNRDDDEISHVGSDIHMDSVDIHDSIDTDENADHRHNVNSVDSLSS SDYTDTQKDFSSIIKDGGNKEGHAENESKEYESQTEQTHEEGIMNPNKYSISEVDGIK LNEEAKHKITEKLVDIYPSTYRTLDEPMETHGPNEKFHMFGSPYVTEEDYTEKHDYD KHEDFNNERYSNHNKMDDFVYNAGGVVCCVLFFASITFFSMDRSNKDECDFDMCE EVN NDHLSNYADKEEIIEIVFDENEEKYF

(3254 aa)

Expression and purification PfRH4 recombinant protein (rPfRH4m aa 328-588): 20 ul aliquot of BL21 (DE3) competent cells (Novagen) has been transformed with 1 ng RH4 plasmid (RH4-l/pETl la), few colonies around 10 are obtained. 2 single colonies are picked up and 5 ml LB-amp is inoculated for 5 hours. After 5 hours, 25 ul of each culture is inoculated into 50 ml LB-amp overnight for 13-14 hours. 10 ml of the 50 ml culture is seeded into 1 L each LB amp. Total 40 ml is seeded into 4 LLB amp (4 large spinner flasks). Started OD 600 nm = 0.05 at time O. The cultures are monitored for absorbance. After 3 hours OD 600nm = 1.45; Cultures are induced with 2 mM IPTG (1908 mg dissolved in 20 ml LB amp; 5 ml added in each flask). After 3.5 hours, culture spun in bags using Beckman rotor and bottles (MVDB). Weight of E. coli pellet: 2 pellets are 7 g and 2 pellets are 6.6 g. Pellets frozen at -80 C. Culture is resuspended in Lysisbuffer and mixed at 4 °C for 1 hour. Lysis buffer- lOmM Tris, HOmM NaCl, lOmM EDTA, Suspension is passed through microfluidizer, Equal volume of wash buffer is added (lOmM Tris-EDTA, 1% Triton X-100, 8M Urea pH8.0)and mixed (vortex) for 2 hours at room temperature. (Buffer should be cold), Spinned 25 000 x g X 30 min. at 4 C, washed pellet with H20 and spinned, Pellet are solublized in 25 ml of solubilization buffer (8 M Guanidine HC1, 10 mMTris pH 8.0) and overnight. 20 ml of RH4 protein is added in GnHCl solubilization buffer to 600 ml of cold refolding buffer. Protein is added slowly while mixing buffer. Mixed buffer is refolded overnight at 4°C.

Refolding Buffer Composition (for 1 L):

55 mM MES pH 6.5 11.92 g 264 mMNaCl 15.40 g

11 M KCll l ml

550 n MGnHCl 52.52 g

1.1 mM EDTA 2.2 ml (of 0.5 M EDTA)

550 mML-Arg 95.6 g

Equal volume of 100 mM Phosphate buffer pH 7.4 is added to2 M Ammonium sulphate, 20% glycerol. Mixedfor 1 hr at 4 C. Loading 1.2 L on a Hydrophobic interaction column (HIC) - low substitution phenyl sepharose in 2 parts (400 ml & 800 ml), protein is eluted with 50 mM Phosphate buffer pH 7.4 with 10% glycerol. Finally running protein on a gel exclusion sizing column.

DNA sequence of the PfRH4 gene (Open Reading Frame; Introns Spliced Out; Receptor binding domain marked- region marked in red is the DNA gene sequence that encodes the receptor binding region of the PfRH4 protein) (SEQ ID NO. 6):

ATGAATAAGAATATATTGTGGATAACTTTTTTTTATTTTTTATTTTTTCTCTTGGAT ATGTACCAAGGAAATGACGC AATTCCCTC AAAAGAAAAAAAAAACGATCCAGA AGCAGATTCTAAGAACTCACAGAATCAACATGATATAAATAAAACACACCATAC GAACAATAATTATGATCTGAATATTAAGGATAAAGATGAGAAAAAAAGAAAAA ATGATAATTTAATCAATAATTATGATTACTCTCTTTTAAAGTTATCTTATAATAAG AATCAAGATATATATAAGAATATACAAAATGGCCAAAAGCTTAAAACAGACATA ATATTAAACTCATTTGTTCAAATTAATTCATCAAACATATTAATGGATGAAATAG AAAATTATGTGAAAAAATATACGGAATCGAATCGTATTATGTACTTACAATTTAA ATATATATATCTACAATCCTTAAATATAACAGTATCTTTTGTACCTCCGAATTCAC CATTTCGAAGTTATTATGACAAAAATTTAAATAAAGATATAAATGAAACTTGTCA TTCCATACAAACACTTCTAAACAATCTAATATCTTCCAAAATTATATTTAAAATG TTAGAAACTACAAAAGAACAAATATTACTTTTATGGAATAACAAAAAAATTAGT CAACAAAATTATAATCAAGAAAATCAAGAAAAAAGTAAAATGATCGATTCGGA AAATGAAAAACTAGAAAAGTACACAAACAAGTTTGAACATAATATCAAACCTCA TATAGAAGATATAGAGAAAAAAGTAAATGAATATATTAATAATTCCGATTGTCA TTTAACATGTTCAAAATATAAAACAATTATCAATAATTATATAGATGAAATAATA ACAACTAATACAAACATATACGAAAACAAATATAATCTACCACAAGAACGAATT

Figure imgf000045_0001
AGAAAAGAATTAATCTATATTGAATATATTTAJTJTATTA

ATAAAATTCAAGAAAACTTTAAATTAAATCAAAATAAATATATACATTTTATTAA TTCAAATAATGCTGTTAATGCTGCTAAAGAATATGAATATATCATAAAATATTAT ACTACA rCAAATATCTACAGACATTAAATAAA CA

AACATAAAATAAATAATTATTCTC

ACATAAAATTAATAACCTAATGATTATCTCAT¾C¾

TTAATGTTAC^

TTA^ ACCTATAGC^

ACAAAAATGAATTC TAGATGATAAAATCAAAGAAATGAATAATATATACGATAATATATATAXCATAT TAAAACAAAAArrC;rTAAACAAATTAAACGAAATCATACAAAATCATAAAAATA AACAAGAAACAAAATfAAATACCACAACCATfCAAGAATTGTTACAACTTCrAA AGGATATTAA GAAATACAAACAAAACAAATCGAJ ACA^

ATATGTATrATAACGATATACAACAAATAAAAATAAAGATTAATCAAAATGAAA AAGAAATAAAAAAGGTACTCCCTC AATTATATATCCCAAAAAATGAAC AAGAAT ATATACAAATATATAAAAATGAATTAAAGGATAGAATAAAAGAAACACAAACA AAAATTAATTTATTTAAGCAAATTTTAGAATTAAAAGAAAAAGAACATTATATTA CAAACAAACATACATACCTAAATTTTACACACAAAACTATTCAACAAATATTAC AACAACAATATAAAAACAACACACAAGAAAAAAATACACTAGCACAATTTTTAT ACAATGCAGATATCAAAAAATATATTGATGAATTAATACCTATCACACAACAAA TACAAACCAAAATGTATACAACAAATAATATAGAACATATTAAACAAATACTCA TAAATTATATACAAGAATGTAAACCTATACAAAATATATCAGAACATACTATTTA TACACTATATCAAGAAATCAAAACAAATCTGGAAAACATCGAACAGAAAATTAT GCAAAATATACAACAAACTACAAATCGGTTAAAAATAAATATTAAAAAAATATT TGATCAAATAAATCAAAAATATGACGACTTAACAAAAAATATAAACCAAATGAA TGATGAAAAAATTGGGTTACGACAAATGGAAAATAGGTTGAAAGGGAAATATG AAGAAATAAAAAAGGCAAATCTTCAAGATAGGGACATAAAATATATAGTCCAA AATAATGATGCTAATAATAATAATAATAATATTATTATTATTAATGGTAATAATC AAACCGGTGATTATAATCACATCTTGTTCGATTATACTCACCTTTGGGATAATGC ACAATTTACTAGAACAAAAGAAAATATAAACAACCTAAAAGATAATATACAAAT CAACATAAATAATATCAAAAGTATAATAAGAAATTTACAAAACGAACTAAACAA TTATAATACTCTTAAAAGCAATTCCATCCATATTTATGATAAAATACACACATTA GAAGAATTAAAAATATTAACTCAAGAAATTAATGATAAAAATGTTATCAGAAAA ATATATGATATTGAAACCATATATCAAAATGATTTACATAACATAGAAGAAATT ATTAAAAATATTACAAGCATTTATTACAAAATAAATATCTTAAATATATTAATTA TTTGCATCAAACAAACATATAATAATAATAAATCCATTGAAAGCTTAAAACTTAA AATTAATAACTTAACAAATTCAACACAAGAATATATTAATCAAATAAAAGCTAT CCCAACTAATTTATTACCAGAACATATAAAACAAAAAAGTGTAAGCGAACTAAA TATTTATATGAAACAAATATATGATAAATTAAATGAACATGTTATTAATAATTTA TATACAAAATCAAAGGATTCATTACAATTTTATATTAACGAAAAAAATTATAATA ATAATCATGATGATCATAATGATGACCATAATGATGTATATAATGATATCAAAG AAAATGAAATATATAAAAATA ATAAATTATACGAATGC ATAC AAATC AAAA AGG ATGTAGACGAATTATATAATATTTATGATCAACTCTTTAAAAATATATCCCAAAA TTATAATAACCACTCCCTTAGTTTTGTACATTCAATAAATAATCATATGCTATCTA TTTTTCAAGATACTAAATATGGAAAACACAAAAATCAACAAATCCTATCCGATAT AGAAAATATTATAAAACAAAATGAACACACAGAATCATATAAAAATTTAGACAC AAGTAATATAC AACTAATAAAAGAAC AAATTAAATATTTCTTAC AAATATTTC AT ATACTTCAAGAAAATATAACCACTTTCGAAAATCAATATAAAGATTTAATTATCA AAATGAACCATAAAATTAATAATAATCTAAAAGATATTACACATATTGTCATAA ACGATAACAATACATTACAAGAACAAAATCGTATTTATAACGAACTTCAAAACA AAATTAAACAAATAAAAAATGTCAGTGATGTATTCACACATAATATTAATTACA GTCAACAAATATTAAATTATTCTCAAGCACAAAATAGTTTTTTTAATATATTTAT GAAATTTCAAAACATTAATAATGATATTAATAGCAAACGATATAATGTACAAAA AAAAATTACAGAGATAATCAATTCATATGATATAATAAATTATAACAAAAATAA TATCAAAGATATTTATCAACAATTCAAAAATATACAACAACAATTAAATACAAC AGAAACGCAATTGAATCATATAAAACAAAATATTAATCATTTCAAATATTTTTAT GAATCTCATCAAACCATATCTATAGTAAAGAATATGCAAAATGAAAAACTAAAA ATTCAAGAATTCAACAAAAAAATACAACACTTCAAGGAAGAAACACAAATTATG ATAAACAAGTTAATACAACCTAGCCACATACATTTACATAAAATGAAATTGCCT ATAACTCAACAGCAACTTAATACAATTCTTCATAGAAATGAACAAACAAAAAAT GCTACAAGAAGTTACAATATGAATGAGGAGGAAAATGAAATGGGATATGGCAT AACTAATAAAAGGAAAAATAGTGAGACAAATGACATGATAAATACCACCATAG GAGACAAGACAAATGTCTTAAAAAATGATGATCAAGAAAAAGGTAAAAGGGGA ACTTCCAGAAATAATAATATTCATACAAATGAAAATAATATAAATAATGAACAT ACAAATGAAAATAATATAAATAATGAACATACAAATGAAAAGAATATAAATAAT GAACATGCAAATGAAAAGAATATATATAATGAACATACAAATGAAAATAATATA AATTATGAACATCCAAATAATTATCAACAAAAAAATGATGAAAAAATATCACTA CAACATAAAACAATTAATACATCACAACGTACCATAGATGATTCGAATATGGAT CGAAATAATAGATATAACACATCATCACAACAAAAAAATAATTTGCATACAAAT AATAATAGTAATAGTAGATACAACAATAACCATGATAAACAAAATGAACATAAA TATAATCAAGGAAAATCTTCAGGGAAAGATAACGCATATTATAGAATTTTTTATG CTGGAGGAATTACAGCTGTCTTACTTTTATGTTCAAGTACTGCATTCTTTTTTATA AAAAACTCTAATGAACCACATCATATTTTTAATATTTTTCAAAAGGAATTTAGTG AAGCAGATA ATGC AC ATTC AGAAGAAAAAGAAGA ATATCTACCTGTCTATTTTG ATGAAGTTGAAGATGAAGTTGAAGATGAAGTTGAAGATGAAGATGAAAATGAA AATGAAGTTGAAAATGAAAATGAAGATTTTAATGACATATGA

The highlighted portion is the DNA gene sequence that encodes the receptor binding region of the PfRH4 protein; Sequence Length: 5151 bp

Protein sequence of PfRH4 (the erythrocyte binding domain (aa 328-588) of PfRH4 is highlighted which has been expressed as a recombinant protein) (SEQ ID NO. 7):

MNIO^ILWITFFYFLFFLLDMYQGNDAIPSKEKKNDPEADSKNSQNQHDINKTHHTN NNYDLNIKDKDE KJ KNDNLINNYDYSLLKLSYNKNQDIYKNIQNGQKLKTDIILNS. FVQINSSNILMDEIENYVKKYTESNRIMYLQFKYIYLQSLNITVSFVPPNSPFRSYYDK NLNKDINETCHSIQTLLNNLISSKIIFKMLETTKEQILLLwT NKKISQQNYNQENQEKS miDSENEKLEKYTO TEHNIKPHIEDIEKKVNEYINNSDCHLTCSKYKTIINNYIDEII TTKTOIYENKYNLPQERIIKNYNHNGINNDDNFIEYNlLNADP¾LRSHfTTL

IY¾YIYEINi HIWKIQENFKLNQNKYI

NKSLYDSIYKHKI^YSHNIEDLINQLQH ^

DlCLSYKP ALEVEYLRNiNKH

IL QKFLNKLNEIIQN NKQETKLNTTTIQELLQLLK^

¾QQI IKINQNEKERKKVLPQLYIPKNEQEYIQIYKNELKDRIKETQTKINLFKQILE LKEKEHYITNKHTYLNFTHKTIQQILQQQYKNNTQEKNTLAQFLYNADIKKYIDELIP ITQQIQTKMYTTNNIEHIKQILINYIQECKPIQNISEHTIYTLYQEIKTNLENIEQKIMQNI QQTTTSFRLKINIKKIFDQINQKYDDLTKNINQMNDEKIGLRQMENRLKGKYEEIKKAN LQDRDIKYIVQNNDANNNNNNIIIINGNNQTGDYNHILFDYTHLWDNAQFTRTKENI NNLKDNIQININNIKSIIRNLQNELNNYNTLKSNSIHIYDKIHTLEELKILTQEINDKNVI RKIYDIETIYQNDLHNIEEIIKNITSIYYKINILNILIICI QTYNNNKSIESLKLKIN LTN STQEYINQIKAIPTNLLPEHIKQKSVSELNIYMKQIYDKLNEHVINNLYTKSKDSLQFY INEKNYNNNHDDH DDHNDVYNDIKENEIYKN KLYECIQIKKDVDELYNIYDQLF KNISQNYNNHSLSFVHSINNHMLSIFQDTKYGKHKNQQILSDIENIIKQNEHTESYKN LDTSNIQLIKEQIKYFLQIFHILQENITTFENQYKDLIIKMNHKINIWLKJDITHIVINDNN TLQEQNRIYNELQNKI QIKNVSDVFTHNINYSQQILNYSQAQNSFFNIFMKFQNINN DINSKRYNVQ KITEIINSYDIINYNKNNIKDIYQQFKNIQQQLNTTETQLNHIKQNIN HFKYFYESHQTISIVKNMQNEKLKIQEFNKXIQHFKEETQIMINKLIQPSHIHLHKMKL PITQQQLNTILHRNEQTKNATRSYNM EEENEMGYGITOKRKNSETNDMINTTIGDK TNVLKNDDQEKGKRGTSRNNNIHTNENNINNEHTNENNINNEHTNEKNINNEHANE KNIYNEHTNEi ilNYEHPNNYQQKNDEKISLQHKTINTSQRTIDDSNMDR NRYNTS SQQKNNLHTNNNSNSRYNNNHDKQNEHKYNQGKSSGKDNAYYRTFYAGGITAVL LLCSSTAFFFIKNSNEPHHIFNIFOKEFSEADNAHSEEKEEYLPVYFDEVEDEVEDEV EDEDENENEVENENEDFNDI

(1715 aa)

Region highlighted (aa 327-587) are expressed in E.coli(~ 30 kDa).

Region bold and underlined is the trans-membrane domain.

Expression & Purification of the rPfRH4m (SEP ID NO. 8):

Cloning of rPfRH43o in pET- 11a expression plasmid:

Restriction Enzymes: Nhe I (GCTAGC) / Bam HI (GGATCC)

Nhel Start

( id A( IAATATTCTTAATGCAGATCCTGATTTAAGATCTCATTTTATAACACTTCT

TGTTTCAAGAAAACAATTAATCTATATTGAATATATTTATTTTATTAACAAACAT ATTGTAAATAAAATTCAAGAAAACTTTAAATTAAATCAAAATAAATATATACATT TTATTAATTCAAATAATGCTGTTAATGCTGCTAAAGAATATGAATATATCATAAA ATATTATACTACATTCAAATATCTACAGACATTAAATAAATCATTATACGACTCT ATATATAAACATAAAATAAATAATTATTCTCATAACATTGAAGATCTTATAAACC AACTACAACATAAAATTAATAACCTAATGATTATCTCATTCGATAAAAATAAATC ATCAGATTTAATGTTACAATGTACAAATATAAAAAAATATACCGATGATATATGT TTATCCATTAAACCTAAAGCATTAGAAGTCGAATATTTAAGAAATATAAATAAA CACATCAACAAAAATGAATTCCTAAATAAAT†CATGCAAAACGAAACATTTAAA AAAAATATAGATGATAAAATCAAAGAAATGAATAATATATACGATAATATATAT ATCATATTAAAACAAAAATTCTTAAACAAATTAAACGAAATCATACAAAATCAT AAAAATAAACAAGAAACAAAATTAAATACCACAACCATTCAAGAATTGTTACAA CTTCTAAAGGATATTAAAGAAATACAAACAAAACAAATCGATACAAAAATTAAT ACTTTTAATATGTATTATAACGATCTAGAGCATCACCATCACCATCACTAAGGAT H

LeuGlu HisHisHisHisHisHisStopBamHI

Translated protein sequence of rPfRH4 (SEQ ID NO. 9):

i¾NILNADPDLRSHFITLLVSRKQLIYIEYIYFINKMIWKIQENFKLNQNKYIHFINS NNAWAAKEYEYII YYTTFKYLQTLNKSLY 3SIYKHKINNYSHNIEDLINQLQHKIN NLMIISFDK KSSDLMLQCTNIKKYTDDICLSIKPKALEVEYLRNINKHINKNEFLNKF MQNETFKKNIDDKIKEMNNIYDNIYIIL Q FLNKLNEIIQNH N QETKLNTTTIQEL LQLLKDIKEIQTKQIDTKINTFNMYYNDLEHHHHHH.GS

PfRH5:

PfRH5 is a unique member of the family in terms of its smaller size, 66 kDa, compared to the other members [Hayton K et al, (2008) Cell Host Microbe 4(1):40-51] (Fig. 1), and thus we expressed the full length native PfRH5 protein. All recombinant proteins are expressed in E. coli in an insoluble form in inclusion bodies, are refolded in vitro and further purified to homogeneity by ion-exchange and/or gel filtration chromatography (Fig. 1). All recombinant proteins bind erythrocytes with the same specificity as that of the native parasite proteins from which they are derived (Fig. 2,3). Specific binding proves that the recombinant proteins constituting the erythrocyte binding domains of the PfRH proteins are refolded in their correct three dimensional conformations (Fig. 15B) and recognizes the same erythrocyte receptor molecule as the native PfRH parasite proteins. Figure 6 is a schematic representation of Recombinant PfRH5

Expression & Purification of the full length rPfRH5(rPfRH5*n): (Figure 7A-7C

Full length PfRH5 gene (Asn27-Gln526) excluding the sequence for signal peptide, is codon optimized, and inserted downstream of the T7 promoter in the E. coli expression vector, pET-24b (Novagen, San Diego, CA), with a C-terminal 6-histidine (6-His) tag. E. coli BL21(DE3) are transformed with pPfRH5-pET24b and are used to produce the recombinant protein. Transformed E. coli BL21(DE3) are cultured in superbroth (Tryptone- 12g, yeast extract-24g, Glycerol-5ml, KH2P04(monobasic) 3.8g, K2HP04-12.5g, MgS04- 0.4g, glucose-4g per litre) at 37°C and later induced with ImM IPTG at OD600 0.8-0.9. Cells are harvested by centrifugation at 3000g, after 4 hours of induction at 37°C.

Cell pellets are lysed by sonication and full length rPfRH5 are found to be expressed as inclusion bodies. The inclusion bodies are washed first with WB1 (lOmM Tris, 10 mM EDTA, l lOmM NaCl, 1% TRITON X-100, pH-8), followed by WB2(10mM Tris, 10 mM EDTA, l lOmM NaCl, 1% TRITON X-100, 4M Urea, pH-8) and WB3 (lOmM Tris, 10 mM EDTA, 1 lOmM NaCl, 4M Urea, pH-8) and then collected by centrifugation at 15000g. The washed IB's are solubilised in Guanidium-HCl containing buffer (6M GnHCl, 150mM NaCl, 50mM Tris, 2mM pMercaptoethanol, pH-7.5).rPfRH5 is purified from solubilized inclusion bodies by Ni-NTA (nitrilotriacetic acid) affinity chromatography.

Metal affinity purified rPfRH5 is refolded by rapid dilution method in a MES based buffer (55mM MES pH 6.5, 440 mM sucrose, 264mM NaCl, l lmM KC1, 2.2mM MgC12, 2.2mM CaC12,440 mM Sucrose, ImM GSH and O.lmM GSSG). The refolded protein is dailysed (55mM MES pH 6.5, 440 mM sucrose) and concentrated.

The purified recombinant protein with a C- terminal HIS Tag is of around 60kDa. rPfRH5 is identified in immunoblots using an anti-His-tag specific antibody confirming expression of the full length protein

Coding DNA Sequence of PfRH5 (The highlighted recombinant protein sequence represents DNA gene sequence that encodes the recombinant PfRH5 protein ) (SEP ID NO. 10)

ATGATAAGAATAAAAAAAAAATTAATTTTGACCATTATATATATTCATCTGTTT^ TATTAAATAGATTAAGTTTTGAAAATGCAATAAAAAAAA ATAATCTGACGTfACTACCAATAAAGAGCACTGAAG

ACAAATAATGCT GTA TGA^

AT TTCTGT ATTTAATCA

ΑΑΤΛ fCA 1 Γ I rAAAGAGTTA-I CAAA l ATAACATTGCAAA'l TC ΓΛΤ I GA'I ΑΤΤΊ Ί A

Figure imgf000052_0001

Figure imgf000052_0002

PfAARP: DNA sequence of the PfAARP gene (SEP ID No. 12):

ATGTGGAAGTTTATTACCATAATAATATTTTCCATATATTATATTGACGGAAAA AGTATATTGAGGAATAACAAAAGTCATAACAATTTGCCTATATCAAAAACGAA TGAAGAAGAGGAAGGTAAAATAAATATAAATAATTTAAAGCCTATAAAACAA CATGATAATATTATAGAAGATGTACATATTAAGGAGAACAAATTTATTTCCAT AAAAAATAAAGACAAAAATGGTTCCTTTATTGATTTAAGTATGAGATATAATG AAAAAGAATCTGATAATGATGAGGAAGAAGAAGAAGATGAAGAAGACAATG AAGATAATAACACAAATAATAATAATAATAATAATAATAATAATAACAATGAT GATGATCACCATAATGATGATGATCATCATGATAATAATGATAATCATGATAA TGAT AATA ATGATA ATAAT AATAAT AAT AATG AT A AT AATAAT AATAAT A ATG ATAATAATAATAATAATAATGATAATAATAATAATTCCTCTGCTGCATTCACA GCTCTTCCTCCACCACCACCTCCTGTACCTCCCCCACCTCCACCAACATTAACA CCTTCAGGTATTGTAGGTAATGTTCTTTCAACTTTTGTATCACATGGTTTAAAA TTAATCGGAGTACCCTAA

Codon optimized DNA sequence for expression of rPfAARPfSEQ ID NO. 13):

ATTCTGCGTAATAATAAAAGCCATAATAATCTGCCTATTTCTAAAACGAATGAAG AAGAGGAAGGTAAAATTAATATTAATAATCTGAAGCCTATTAAACAACATGATA ATATTATTGAAGATGTACATATTAAGGAGAATAAATTTATTTCCATTAAAAATAA AGACAAAAATGGTTCCTTTATTGATCTGAGCATGCGCTATAATGAAAAAGAATCT GATAATGATGAGGAAGAAGAAGAAGATGAAGAAGACAATGAAGATAATAATAC CAACAATAACAATAACAATAATAACAATAATAACAATGATGACGACCACCATAA CGATGAC

Protein Sequence of PfAARP (The highlighted portion represents the erythrocyte binding region of AARP that has been expressed as a recombinant protein) (SEQ ID NO. 14)

MWKTITIIIFSIYYIDGKSILRNNKSHNNLPISKTNEEEEGKININNLKPIKQHDNIIEDVI KENKFISIKNK KNGSFIDLSMRYNEKESD NNNDDDHHNDDDHHDNNDNHDNDNNDNNNNNNDNNNNTW SAAFTALPPPPPPVPPPPPPTLTPSGIVGNVLSTFVSHGLKLIGVP

(217aa)

Figure imgf000053_0001
catatgattctgcgtaataat M I L N N

aaaagccataataatctgcctatttctaaaacgaatgaagaagaggaaggtaaaattaat

K S H N N L P I S K T N E E E E G K I N

attaataatctgaagcctattaaacaacatgataatattattgaagatgtacatattaag

I N N L K P I K Q H D N I I E D V H I K

gagaataaatttatttccattaaaaataaagacaaaaatggttcctttattgatctgagc

E N K F I S I K N K D K N G S F I D L S

atgcgctataatgaaaaagaatctgataatgatgaggaagaagaagaagatgaagaagac

M R Y N E K E S D N D E E E E E D E E D

aatgaagatctcgagcaccaccaccaccaccactga

N E D L E H H H II H H -

(Methodology to express PfAARP is described in it's individual patent application and in the publication (Wikramachi et al ; PloS One 2008)

PfF2 (PfEBA-175 :

PfF2 can be expressed and purified using a protocol known in the art. A method of expression and purification is well described K.C. Pandey et al. I Molecular & Biochemical Parasitology 123 (2002) 23/33. This process is described below:

Plasmid construct and E.coli strain used for recombinant expression of PfF2:

DNA fragments encoding PfF2 (amino acids 447-795of P. falciparum EBA-175) fused to hexa-histidine at the C-terminal end are amplified by polymerase chain reaction (PCR) using primers 5 ' -TCT AGTCCATGGA A A AGCGTGA AC ATATT-3 ' and 5'-ACG AGTGTC GACTCAGTGATGGTGATGGTGATGATCGTCATCACGTTCTT-3' and a plasmid containing the gene encoding EBA-175 of P. falciparum CAMP strain as template [5]. The PCR product is digested with Ncol and Sail and the resulting Ncol/Sallfragment is cloned downstream of the T7 promoter in the E. coli expression vector pET28a (Novagen) to yield plasmid pF2PETl. The N-terminal methionine in recombinantPfF2 expressed by this construct is provided by the expression vector. The insert as well as junctions between vector and insert sequences are sequenced inboth directions using an Automated DNA sequencer ABI 310 (Applied Biosystems). E. coli BL21(DE3) cells(Novagen) are transformed with plasmid pF2PETl and used for expression of recombinant PfF2.

Expression of recombinant PfF2 in E. coli Luria broth containing kanamycin (25 mg mi l) is inoculated with an overnight culture of E. coli BL21(DE3) pF2PETl at a dilution of 1 :50 and cultured to an OD600 nm of 0.6/0.8 at 37 8C. Expression of recombinant PfF2 is induced by addition of isopropyl-l-thio- b-galactopyranoside to the culture at a final concentration of 1 mM. Induced cultures are harvested after 4 h of growth at 37 8C.

Purification and refolding of recombinant PfF2

E. coli cells are harvested by centrifugation and lysed by sonication. Inclusion bodies are collected by centrifugation and solubilized in 10 mMTrispH 8.0 containing 6 M guanidine hydrochloride (GdnHCl). Recombinant PfF2 was purified from solubilized inclusionbodies under denaturing conditions by metal affinity chromatography using a nickel nitrilo-triaceticacid (Ni-NTA) column as described by the manufacturer (Qiagen). Solubilized inclusion bodies are loaded on a Ni NTA column previously equilibrated with equilibration buffer (10 mMTris pH 8.0, 100 mMNaH2P04, 6 M GdnHCl). The column is washed with equilibration buffer at pH 6.3 and bound protein is eluted using a pH gradient starting at pH 6.3 and ending at pH 4.3. The final concentration of the purified protein is adjusted to 4.5 mg ml/1 with equilibration buffer.

Purified, denatured PfF2 is refolded by 100-folddilution in refolding buffer containing 50 mM phosphate buffer, pH 5.8, 2 mM reduced glutathione, 0.2mM oxidized gluthathione, 1 M urea and 0.5 M arginine so that the final concentration of PfF2 is 45 mg/nil. Refolding is allowed to proceed for 36 h at 8-10°C. At the end of 36 h, the refolding solution is dialyzed for 48 h against dialysis buffer (50 mMphosphate buffer, pH 5.8, 1 M urea) to remove arginine. Following removal of arginine, refolded PfF2 is loaded on a SP- Sepharose column (Pharmacia) equilibrated with 50 mM phosphate buffer, pH 5.8. Thebound protein is eluted with a linear gradient of NaCl (100 mMNaCl to 1 M NaCl). Fractions containing refolded PfF2 are pooled and recombinant PfF2 is further purified by gel filtration chromatography using apreparative grade Superdex 75 column (Pharmacia) with 50 mM phosphate buffer, pH 6.0, containing 150 mMNaCl as running buffer.

Sequence of synthetic gene encoding PfF2 of P. falciparum CAMP strain EBA-175 (SEQ ID No 16):

ATGGAAAAACGTGAACACATTGATCTGGACGACTTTTCTAAATTTGGGTGTGATA AGAACTCCGTCGACACCAACACTAAAGTGTGGGAGTGCAAAAACCCGTATATCC TTTCGACTAAAGATGTGTGCGTACCGCCGCGTCGCCAGGAACTGTGTCTGGGTAA CATCGATCGCATCTATGACAAAAACCTGCTGATGATCAAAGAGCACATCCTCGC TATCGCAATCTACGAATCCCGCATCCTGAAACGTAAATACAAGAACAAAGACGA CAAAGAGGTTTGCAAAATCATCAACAAAACATTCGCGGACATCCGTGACATTAT CGGCGGTACTGACTACTGGAACGACCTGAGTAACCGCAAACTGGTAGGTAAAAT CAACACCAACTCCAAATATGTTCACCGTAACAAGAAAAACGACAAGCTCTTTAG GGATGAATGGTGGAAAGTGATCAAGAAAGACGTTTGGAACGTGATCTCCTGGGT ATTCAAGGACAAAACCGTTTGTAAGGAAGATGACATTGAAAACATCCCACAGTT CTTCAGATGGTTCAGCGAATGGGGTGACGACTACTGCCAAGACAAAACCAAAAT GATCGAGACCCTGAAGGTTGAATGCAAGGAGAAGCCTTGTGAAGACGACAACTG CAAAAGC AAATGC AATTCCTAC AAAGAATGGATCTCC AAAAAGAAAGAAG AGT ACAACAAACAGGCGAAACAGTACCAGGAATACCAAAAAGGTAACAACTACAAA ATGTATTCTGAATTTAAATCTATCAAACCGGAAGTTTACCTGAAGAAATACAGCG AAAAATGTTCTAACCTGAACTTCGAAGACGAATTCAAGGAAGAACTGCACTCCG ACTACAAAAACAAGTGTACGATGTGTCCAGAAGTAAAAGATGTACCAATCTCTA TCATCCGTAACAATGAACAGACTAGCCAGGAAGCAGTTCCGGAGGAAAACACTG AAATCGCTCACCGTACCGAGACCCCGTCTATCTCTGAAGGCCCGAAAGGCAACG AACAGAAAGAACGTGATGACGATCACCATCACCATCATCACTGA

Protein Sequence of PfF2 from P. falciparum CAMP strain EBA-175 (SEQ ID No 17): MEKREHIDLDDFSKFGCDKNSVDTNTKVWECKNPYILST DVCVPPRRQELCLGNID RIYDKNLLMIKEHILAIAIYESRILKRKYKNKDDKEVCKIIN TFADIRDIIGGTDYWN DLSNRKLVGKmTNSKYVHRNKKNDKLFRDEWWKVIKKDVWNVISWVFKDKTVC KEDDIENIPQFFRWFSEWGDDYCQDKTKMIETL VECKE PCEDDNCKSKCNSYKE WISKKKEEYNKQAKQYQEYQKGNNYKMYSEFKSI PEVYLKKYSE CSNLNFEDEF KEELHSDYKNKCTMCPEVKDVPISIIRNNEQTSQEAVPEENTEIAHRTETPSISEGPKG NEQKERDDDHHHHHH.

MSP-Fusion (MSP-Fu: MSP3-MSP1 IQ):

Construction of plasmids expressing PfMSP-311, PfMSP-l 19. and MSP-Fu24

To design a synthetic gene corresponding to the C-terminal 19-kDa fragment of PfMSP-l, the amino acid sequence of PfMSP-l 19 corresponding to. residues 1526to 1619 of the P. falciparum Welcome strain (GenBank accession no. P04933) is back-translated to the nucleotide sequence based on the E. coli codon frequency table (available at http://www.kazusa.or.jp/codon). This synthetic gene is used as a template to amplify PfMSP- 119 with the Ncol-Xhol site with the following primer set (A & B): forward primer (A) 5'- GTG ACA CCA TGG GTA ACA TTTCTC AGC ATC AGT G-3' (SEQ ID No. 20) and reverse primer (B) 5'-GCC CTC GAG TTA GTGGTG GTG GTGGTGGTG GGA ACT GCA GAA AAT ACC ATC-3' (SEQ ID No. 21). The amplified product is cloned into the Ncol and Xhol sites of pET28a, akanamycin-based vector (Novagen), in frame with the coding sequence of the 6_His tag at its C terminus, to obtain the pET28a-PfMSP-l 19 construct. The conserved 11-kDa fragment of PfMSP-3 corresponding to 70 amino acids(163 to 230 amino acids) was amplified from a PfMSP-3 synthetic gene with the following set of primers (C & D): forward primer (C) 5'-GGC GGC CAT GGC AAA GAATGC TTA CGA AAA GGC C-3' (SEQ ID No. 22) and reverse primer (D) 5'-GGC CTC GAG TTAGTG GTG GTGGTGGTGGTG GTC GTT TTC CTT AGA GAT GTTTTC-3' (SEQ ID No. 23). The amplified product is cloned into the Ncol and Xhol sites of pET28a, in frame with the coding sequence of the 6-His tag at its C terminus, to obtain the pET28a-PfMSP-3u construct.To generate a fusion construct consisting of PfMSP-3n and PfMSP-11 , thel 1-kDa fragment of PfMSP-3 is amplified again with different sets of primers: forward primer 5'- GGC GGC CAT GGC AAA GAA TGC TTA CGA AAAGGC C-3' and reverse primer 5'- GCC GCC CAT GGC GTC GTT TTC CTTAGA GAT GTT TTC-3'. The amplified PCR product is purified and digested with Ncol. The excised fragment is cloned into the Ncol site of pET28a-PfMSP-l19 to generate the pET28a-MSP-Fu24 construct. All constructs are sequenced from both ends to confirm the orientation andsequence of the inserts and transformed into E. coli BLR(DE3) cells (Novagen) for the expression of recombinant proteins with 6-His tags.

Expression and purification of recombinant proteins

E. coli BLR(DE3) cells containing recombinant plasmids pET28a-PfMSP-li9, pET28a-PfMSP-3n, and pET28a-MSP-Fu24 are grown in Luria broth containing kanamycin (30 g/ml)at 37°C until an optical density at 600 ran (OD600) of 0.6 to 0.7 was reached. Theexpressions of the respective recombinant proteins are induced with 1 mM isopropyl-D- thiogalactopyranoside (IPTG) for 3 h at 37°C, and the expressed proteins are analyzed and localized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting of the soluble and insoluble fractions of the E. coli cells after disruption. For the purification of recombinant MSP-Fu24 and PfMSP-119, the E. coli cell pellets from the respective 6-liter shake flask cultures are washed with phosphate buffer saline (PBS) and resuspended in lysis buffer (20 niMTris-HCl [pH 8.0],500 mMNaCl, 10 mM imidazole, 1% Triton X-100, 25 mg per liter lysozyme, 5mMbenzamidineHCl). The bacterial cells are lysed on ice by sonication, and the lysate is centrifuged at 12,000 rpm for 45 min at 4°C. The clarified supernatant is loaded onto a column containing pre-charged streamline chelating matrix (GE Healthcare). The column is subsequently washed with 10 column volumes of equilibration buffer (20mMTris-HCl [pH 8.0], 500mMNaCl, lOmMimidazole), followed by 10 column volumes each of wash buffer 1 (20mMTris-HCl [pH8.0], 500 mMNaCl, 40 mM imidazole) and wash buffer 2 (20 mMTris-HCl [pH8.0], 10 mMNaCl, 40 mM imidazole). Bound protein is eluted with a lineargradient of imidazole (40 mM to 1 M) in 20 mMTris-10 mMNaCl (pH 8.0)buffer. The eluted fractions a re analyzed by SDS-PAGE, and fractions containing the recombinant protein are pooled. The pooled protein is further purified by anion-exchange chromatography on a column of Q-Sepharose resin(GE Healthcare) equilibrated with equilibration buffer (20 mMTris-HCl [pH8.0], 10 mMNaCl). The bound proteins are eluted with a linear gradient ofNaCl (10 mM to 1 M) in Tris-HCl buffer (pH 8.0). Eluates are analyzed bySDS-PAGE, fractions containing a single protein band of MSP- Fu24 or PfMSP-l i9 are pooled, and the protein concentration is determined by a bicinchoninic acid assay (BCA).

The homogeneity of purified MSP-Fu24, PfMSP-3n, and PfMSP- l i9 is assessed by SDS-PAGE under reducing and non-reducing conditions, on an analytical gel permeation chromatography column, and by reverse-phase chromatography on an analytical C8 column (Supelcosil; 5 by 4.9 cm, 5 _m). Endotoxin contents in the protein samples are estimated by using a Limulus amebocytelysate (LAL) gel clot assay (Charles River Endosafe), and host cell protein contamination is estimated by immunoblotting as well as by enzyme-linked immunosorbent assay (ELISA) using anti-E.coli antibodies (Cygnus Technologies). The reactivity of the recombinant proteins with monoclonal and polyclonal antibodies is analyzed by immunoblotting according to standard protocols.

Recombinant proteins are separated by SDS-PAGE under reducingor non-reducing conditions and blotted onto a nitrocellulose membrane, followed by the blocking of the membrane using 5% nonfat milk in PBS (pH 7.4). The blots are sequentially incubated with the respective monoclonal or polyclonal antibodies in PBS (pH 7.4) containing 0.5% milk and 0.05% Tween 20, followed by the respective horseradish peroxidase-conjugated secondary antibody, after prior washing with PBS containing 0.05% Tween 20 (PBS-T). The protein bands are detected after developing the reaction mixture with 3,3- diaminobenzidinetetrahydrochloride (DAB) in PBS and hydrogen peroxide (H202). DNA sequence MSP-Fu sequence (macrogen) - subcloned in pET-28a - Ncol/Xhol (SEQ ID NO.18):

Figure imgf000059_0001

TGiACGTTTCTTCCAAGGACAAGGAAAACATCTCTAAGGAAAACpAC

GTAACATTTCTCAGCATCAGTGCGTAAAAAAGCAGTGTCCGCAAAATTCTGGCTG CTTCCGTCATCTCGATGAACGTGAGGAATGTAAATGCCTGCTGAACTACAAACA GGAGGGTGACAAATGCGTTGAAAACCCAAATCCTACCTGTAACGAGAACAACGG TGGCTGCGACGCAGACGCCAAATGCACCGAAGAAGACTCCGGTAGCAACGGCA AG AAAATCACGTGCG AATGTACTAAACCGGATTCTTATCCGCTGTTCG ATGGTAT CTTCTGCAGCTCCCAfCACGA'rc:ACC\ATCACTA^GTCGACAAGCTTGCGGCCGC CTCGAG

Protein Sequence of MSP-Fu (SEP ID NO.19):

MAKNAYEKAKNAYQKANQAVLKAKEASSYDYILGWEFGGGVPEHKKEENMLSHL YVSSKDK£NISK£NDAMGNlg§HQCVKKQ

D C VEN I'M CM \ sfGGC RApAKC r EDSGSNGKKriCLCTKPDS YPLFDGIFCf ¾H HHHHH

Number of amino acids: 172

Molecular weight: 19320.3

Theoretical pi: 6.05

Figure 3 depicts erythrocyte binding phenotypes of the native parasite proteins and their recombinant receptor binding regions.

To confirm whether the purified recombinant receptor binding domains has the correct conformation as the native parasite proteins, erythrocyte-binding assays (EBA) are performed to assess the binding specificity of the different recombinant proteins. Consistent with previous reports, the recombinant proteins rPfRHHO, rPfRH240, rPfRH430, PfF2 bind erythrocytes with the same specificity as the native parasite proteins they are derived from confirming that they are structurally similar (Murray CJ et al., Lancet 379:413-431; Crompton PD et al., J. Clin. Invest. 120:4168^1178; Ogutu BR et al., PLoS One 4:e4708; Spring MD et al., PLoS One 4:e5254; Crompton PD et al., Mali. Infect. Immun. 78:737-745) ( Recombinant rPfRH140 and PfF2 bind in a sialic acid dependent manner, whereas the binding of rPfRH240 and rPfRH430 is sialic acid independent. Similarly, rPfAARP20-107 bind erythrocytes in a sialic acid dependent manner (Thera MA et al., N. Engl. J. Med. 365:1004-1013) The erythrocyte binding specificities of all recombinant proteins prepared against the receptor binding domains matched the specificities of their respective parasite proteins, confirming that the recombinant proteins are expressed in a 3 -dimensional conformation similar to the native parasite proteins.

Methodology of Erythrocyte Binding Assays (EB A)

Erythrocyte binding assays are performed as described previously (Sahar T et al., PLOS One 6:el7102; Gaur D et al., Proc. Natl. Acad. Sci. USA. 104:17789-17794) () Soluble parasite proteins are obtained from P. falciparum 3D7 culture supernatant. Culture supernatants (500 □!) or recombinant protein (0.5 μg) are incubated with the different enzymatic treated human erythrocytes (100 Dl) at 370°C for 1 hour. After incubation, the suspension is centrifuged through dibutyl phthalate (Sigma, St. Louis, MO). The supernatant and oil are removed by aspiration. Bound parasite proteins are eluted from the erythrocytes with 1.5 M NaCl. The eluate fractions are analyzed for the presence of the proteins of interest using specific antibodies in immunoblots (Sahar T et al., PLOS One 6:el7102; Gaur D et al., Proc. Natl. Acad. Sci. USA. 104:17789-17794; Jiang L et al., Proc. Natl. Acad. Sci. USA. 108:7553-7558).

Figure 4 depicts PfRH antibodies block erythrocyte binding of parasite and recombinant PfRH proteins.

Antibodies against the purified recombinant proteins are specific as they detected the native parasite proteins of the expected molecular mass in immunoblots using parasite culture supernatants (Fig. 4). They also specifically recognize the recombinant proteins in immunoblots (Fig. ,4) and ELISA. Purified IgG against both rPfRH140 and rPfRH240 also inhibited erythrocyte binding of recombinant proteins as well as native PfRHl and PfRH2 respectively in a dose dependent manner with complete binding inhibition observed at 40 μg/ml IgG (Fig. S3). In contrast, even at 320 μg/ml, the IgG against PfRH has no effect on the binding of EB A- 175 (Fig. 4), thus confirming their specific binding inhibitory activity against the PfRH proteins only.

Methodology of Raising Antibodies and IgG Purification

Specific polyclonal antibodies are raised in rabbits separately against PfRH 140, PfRH24o, PfRH430 and PfRH5 recombinant proteins. Rabbits are immunized intramuscularly once with 100 g of each recombinant protein emulsified with complete Freund's adjuvant (CFA) [Sigma, St. Louis, MO] on day 0 followed by two boosts emulsified with 100μg of each protein formulated with incomplete Freund's adjuvant (IF A) [Sigma, St. Louis, MO] on day 28 and 56. Sera are tested for antibody titers and specific recognition of each recombinant protein by ELISA. Analysis of sera by ELISA shows that high titre specific antibodies are generated with end point titres of -1:320,000 (data not shown) [Sahar T et a/.,(2011) PLOS OneFeb;6 (2): el7102].

The anti-PfRH antibodies exhibit a fine specificity as they detect the native parasite proteins of the expected sizes in the parasite lines in which they have been reported to be expressed. Further, anti-PfRH purified IgG block the binding of both their corresponding recombinant and native parasite proteins in a dose dependent manner (Fig. 4) further corroborates their specificity.

For co-immunogenicity experiments, a group of six mice (BALB/c) are immunized on day 0 with each immunogen formulation emulsified with complete Freund's adjuvant followed by two boosts emulsified with incomplete Freund's adjuvant on days 28 and 56. Both antigen mixtures and individual antigens are immunized separately in mice. Terminal bleeds are collected on Day 70. Sera are tested for antibody titers and specific recognition of each recombinant protein by ELISA.

Total IgG is purified from rabbit and pooled mice sera using a Protein G affinity column (GE Healthcare, Uppsala, Sweden) in accordance with the manufacturer's instructions. The purified IgGs are dialyzed with modified RPMI 1640 medium (as above) and used in invasion inhibition assays.

Figure 8 shows Erythrocyte invasion inhibitory activity of PfRH antibodies in combination against P. falciparum 3D7 clone.

Total IgG purified from rabbit sera raised against the receptor binding regions of

RH1, RH2, RH4 are tested for its invasion inhibitory activity individually (2.5-10 mg/ml) and in combination. Combinations of two RH IgG are assessed at two concentrations (2.5 + 2.5 mg/ml, 5.0 + 5.0 mg/ml) and combinations of three RH IgG were tested at 3.3 mg/ml each. AMA-1 IgG (5 mg/ml) is used as a positive control. Negative control is an average of inhibition of purified IgG from pre-immune rabbit sera and a control rabbit immunized with a non-related peptide. Three independent assays are performed in duplicate. The error bars show the standard error of the mean.

The purified IgGs are tested individually at four different final concentrations (2.5 mg/ml, 3.3 mg/ml. 5.0 mg/ml, 10.0 mg/ml) in invasion assays. The highest total IgG concentration tested is 10 mg/ml as this is the IgG concentration observed in human sera. The antibodies exhibit a dose dependent increase in invasion inhibition confirming that the inhibitory effect observed is specific (Fig. 8). Anti-PfRH24o antibodies exhibited maximum efficacy in inhibiting invasion with an inhibition efficiency of 52% (10 mg/ml) and 25% (5 mg/ml) (Fig. 8). This inhibition of invasion is reversed in the presence of the PfRH2 recombinant protein further proving that the inhibition observed is specific. The anti-PfRHUo and anti-PfRH430 antibodies are less potent in inhibiting invasion with efficiency of 25-30% at concentration of 10 mg/ml (Fig. 8).

Methodology of Invasion inhibition Assay

Invasion inhibition assays are done at ICGEB by the methodology previously described in Sahar T et al, PLOS One 6:el7102. Briefly, schizont stage 3D7 parasites at an initial parasitemia of 0.3% at 2% hematocrit are incubated with purified IgG and incubated for one cycle of parasite growth (40 hours post-invasion). The parasite infected erythrocytes are stained with ethidium bromide dye and measured by a FACS based assay as described previously (Sahar T et al, PLOS One 6:el7102) Invasion inhibition is calculated with respect to purified pre-immune IgG as well as immune IgG generated against a non-related peptide formulated with the same adjuvant (CFA/IFA) used for raising the other antibodies. P. values are calculated using the students t-test.

The invasion inhibitory activity of different combinations of purified total IgG against each of the three PfRH proteins is assessed over one cycle against the P. falciparum clone, 3D7 (Sahar T et al, PLOS One 6:el7102; Gaur D et al, Proc. Natl. Acad. Sci. USA. 104:17789-17794) that invades erythrocytes using both sialic acid dependent and independent pathways (Fig. 8). The purified total IgGs are tested individually (2.5 mg/ml, 3.3 mg/ml. 5.0 mg/ml, 10.0 mg/ml) as well as in double (2.5 mg/ml and 5 mg/ml each) and triple antibody combinations (3.3mg/ml each) (Fig. 8). The maximum total IgG concentration tested is 10 mg/ml as this is close to the physiological concentration of IgG in human sera (Walliker D et al., Science 236: 1661-1666) (. Individual PfRH antibodies exhibited a dose dependent invasion inhibition confirming a specific effect (Fig. 8). Anti-PfRH240 IgG exhibited maximum inhibition of 54% (10 mg/ml) and 29% (5 mg/ml) (Fig. 8). Anti- PfRHl40 and anti-PfRH430IgG were less potent with an inhibition of 22-30% at 10 mg/ml (Fig. 8).

PfRHIgG combinations at 2.5 mg/ml each do not produce any significant increase in invasion inhibition, however, the combinations at 5.0 mg/ml each, produced an additive effect (Fig. 8). Individually, the three PfRHIgGs at 5 mg/ml blocked invasion by 17-29% (Fig. 8). Among the double combinations, RH2+RH4 IgG inhibited invasion by 54%, while the inhibition of RH1+RH2 and RH1+RH4 is 30-37% (Fig. 8).

PfRHl + PfRH2 IgG inhibited invasion by 50% compared to 18-25% inhibition by the two antibodies individually (Fig. 8). PfRHl + PfRH4 and PfRH2 + PfRH4 combinations also inhibit invasion at a rate of 40-48% (Fig. 8).

The most potent inhibition is observed with the combination of three antibodies

(RH1+RH2+RH4 3.3 mg/ml each) that produced 66% inhibition compared to the low inhibition (< 12%) exhibited by each individual IgG at 3.3 mg/ml (Fig. 8).

These results clearly prove that PfRH antibodies efficiently blocks erythrocyte invasion in combination rather than individually. This inhibition may be improved by inclusion of antibodies against PfRH5 and other blood stage antigens.

Figure 9 shows Invasion inhibitory activity of antibody combinations against the P. falciparum clones 3D7 & Dd2.

Total IgGs purified from rabbit sera against the six proteins (PfRHl, PfRH2, PfRH4, PfF2, PfAARP, and PTRAMP) are assayed individually at a concentration of 3.3 mg/ml and in combinations of three IgGs (3.3 mg/ml each) against sialic acid-independent clone 3D7 (A) and sialic acid-dependent clone Dd2 (B). Three independent assays are performed in duplicate. The error bars show the standard errors of the means. P values are calculated by using the Student t test.

The invasion-inhibitory activities of all 20 possible triple-antibody combinations of purified total IgG (3.3 mg/ml each) against a pool of 6 antigens (RH1, RH2, RH4, PfF2, AARP, and PTRAMP) are tested. Invasion inhibition is assayed against P. falciparum clones 3D7 and Dd2. Against 3D7, individual IgGs against each of the 6 antigens produced 8 to 25% inhibition, with AARP IgG being the most potent (25%) at 3.3 mg/ml. Different combinations of three IgGs displayed a potent inhibition of erythrocyte invasion, with the maximum inhibition (79%) being elicited by PfF2+RH2+AARP IgGs, which is higher than the 66% observed with RH1 +RH2+RH4 IgGs.

Against the sialic acid-dependent clone Dd2, AARP IgG inhibited erythrocyte invasion with the same efficiency as that observed with 3D7. Total IgG against PfF2 and PfRHl, which are sialic acid binding proteins, exhibited higher-level inhibition against Dd2 than against 3D7. PfF2+PfRHl+AARP yielded the maximum invasion inhibition (75%) against Dd2, consistent with the three antigens being involved in sialic acid-dependent invasion. However, this combination yielded only 48% inhibition against clone 3D7. The most effective combination against 3D7, PfF2+PfRH2+AARP, exhibited the second highest level of inhibition (68%) against Dd2 and is therefore most efficacious against both parasite clones.

Figure 10 shows combination GIAs with five diverse P. falciparum clones.

The invasion inhibition of three antibody combinations (PfF2+RH2+AARP, RH1+RH2+RH4, RHl+RH2+PfF2) is assayed with five P. falciparum clones: 3D7, 7G8, HB3, Dd2, and Mcamp (MC). 3D7, 7G8, and HB3 are sialic acid-independent clones; Dd2, MCamp, are sialic acid-dependent clones.

The efficacy of PfF2+RH2+AARP is further analyzed against three other diverse P. falciparum clones. In addition to 3D7 and Dd2, the inhibition of invasion by sialic acid- independent clones (7G8 and HB3) and a sialic acid-dependent clone (MCamp) is tested. PfF2+PfRH2+AARP IgGs inhibited the invasion of all five clones with an invasion inhibition efficiency ranging between 67 and 79%. Two other IgG combinations (PfRH 1 +PfRH2+PfRH4 and PfRHl +PfRH2+PfF2) are also tested for the inhibition of invasion by multiple clones. The invasion inhibitory efficiencies of these combinations are not similar for all clones, with broad inhibition efficiencies of between 36 and 67%. Thus, the PfF2+PfRH2+PfAARP antibody combination exhibited the most potent, strain-transcending, invasion inhibitory activity.

Figure 11 is graphical representation of Immunogenicity of the antigens in mice when used alone and in combination. Antigen combinations (PfF2+PfRH2+Pf AARP and PfRH 1 +PfRH2+PfRH4) as well as the individual corresponding antigens are used to immunize mice. Day 70 sera from the immunized animals are probed against the respective antigens to determine their immunogenicity. (A) Immunogenicity of antibodies against PfF2+PfRH2+PfAARP (purple) compared with that of the sera raised against the individual antigens (green). (B) Immunogenicity of antibodies against PfRHl+PfRH2+PfRH4 sera (purple) is compared with that of antibodies against sera raised against the individual corresponding antigens (green). For ELISAs, each individual antigen is coated separately onto 96-well plates for both the co- immunized as well as the individual antigens. Pre-immune or Pre-bleed (PB) sera are used as controls. The data points represent average values for the six mice included in each group. Two independent experiments are done in duplicate. The error bars represent the standard errors of the means.

After evaluating antibody combinations that are mixed in vitro for their invasion- inhibitory activities, it is tested whether the most potent combination identified would elicit similar invasion-inhibitory antibodies when co-immunized together as a single formulation. The ELISA (OD492) showed that the antibody titers (endpoint, 1 :320,000) against each protein immunized individually are not significantly altered when immunized as a mixture with the two other antigens. The recombinant antigens of present invention are immunogenic and do not elicit any significant immune interference when immunized in combination.

Figure 12 shows invasion inhibitory activity of antibodies raised against antigen mixtures against the P. falciparum clones 3D7 & Dd2.

Total IgGs from mouse sera raised against the immunogens (PfRHl, PfRH2, PfRH4, PfF2, PfAARP, PfRHl+PfRH2+PfRH4, and PfF2+PfRH2+PfAARP) are evaluated for their invasion-inhibitory activities (at concentrations of 1, 3.3, 5, and 10 mg/ml) against sialic acid-independent clone 3D7 (A) and sialic acid-dependent clone Dd2 (B). Two independent assays are performed in duplicate. The error bars show the standard errors of the means.

Consistent with the invasion inhibition observed with the antibody combinations mixed in vitro, the antibodies raised against the antigen mixtures are highly potent and equally efficient in inhibiting erythrocyte invasion. Against the 3D7 clone, antibodies against the PfF2+PfRH2+PfAARP antigen mixture displayed 69% and 85% inhibitions at concentrations of 5 and 10 mg/ml, respectively. PfF2 IgG, PfRH2 IgG, and PfAARP IgG individually exhibited 23%, 52%, and 56% inhibitions, respectively, at 10 mg/ml. Thus, the PfF2+RH2+AARP formulation induced antibodies that are much more potent than the individual antibodies at the same IgG concentrations, clearly suggesting synergistic inhibitory effect. The invasion inhibition exhibited by all antibodies is observed to be dose dependent. A similar trend is observed with P. falciparum clone Dd2, which is inhibited by the antibodies raised against the PfF2+PfRH2+PfAARP mixture by 62% and 80% at 5 and 10 mg/ml, respectively. The antibodies raised against the PfRHl+PfRH2+PfRH4 mixture inhibited the invasion of 3D7 with lower efficiencies of 58% and 69% at 5 and 10 mg/ml, respectively . On the other hand, these antibodies exhibited a much lower inhibitory activity (42% at 10 mg/ml) with the Dd2 clone, which expresses lower levels of PfRH2 and PfRH4. These results are consistent with the inhibition observed for the same antibody combinations in the first screen.

Figure 13 illustrates Antibodies raised against the antigen mixtures exhibit potent invasion inhibition.

The strain-transcending neutralizing activities of purified total IgGs raised against the co-immunized PfF2+PfRH2+PfAARP and PfRHl+PfRH2+PfRH4 antigen formulations are evaluated against three sialic acid-independent (3D7, 7G8, and HB3) and two sialic acid- dependent (Dd2 and MCamp) clones at a concentration of 10 mg/ml. Two independent assays are performed in duplicate. The error bars show the standard errors of the means.

The antibodies against the PfF2+RH2+AARP antigen mixture displayed strain- transcending inhibition efficiencies of around 80 to 87% against five diverse P. falciparum clones, consistent with those observed when antibodies against these antigens are tested in combination. The invasion inhibition observed with the antibodies against the PfRHl+PfRH2+PfRH4 antigen mixture against the five clones varied over a broader range of 40 to 70%, similar to that observed for the antibody combination.

Figure 14 illustrates additive invasion inhibitory effect observed with two antibody combinations against P. falciparum 3D7 clone.

Additive effect of MSP-Fu antibodies in double combination with antibodies raised against other merozoiteadhesins - The combination of fixed concentration of MSPFu antibodies (1 mg/ml) are accessed with increasing concentration of PfAARP, PfRHl, PfRH2 and PfF2 antibodies in double antibody combination. MSP-Fu+RH2 and MSP-Fu+AARP combinations displayed more potent invasion inhibition activity as compared with the MSP- Fu+PfRHl and MSP-Fu+PfF2 combination. The inhibition effect observed is majorly subadditive for most of the combinations.

Figure 16 illustrates rPfRH5 antibodies specifically block erythrocyte binding of the native PfRH5 protein.

The rPfRH5 antibodies recognized native RH5 in P. falciparum culture supernatant and specifically abrogated the binding of native as well as rPfRH5 to erythrocyte, but did do not effect the binding of EB A- 175.

To further validate the specificity and inhibitory activity of the antibodies, the inventors performed erythrocyte binding with both the culture supernatant as well as the recombinant protein in presence of anti-rPfRH5 antibodies is perfomed. The anti-rPfRH5 antibodies blocked the binding of the native PfRH5 protein, and the inhibition of binding increased with increasing antibody concentration. In a parallel experiment with the same erythrocyte binding assay eluates, anti-rPfRH5 antibodies failed to block the binding of EBA 175 even at a higher concentration of antibodies (Figure 4B); further corroborating the inhibitory specificity of anti-rPfRH5 antibodies. Consistent with that of the native PfRH5, the anti-rPfRH5 antibodies blocked the binding of rPfRH5 in a dose dependent manner

Figure 17 shows Anti-BSG monoclonal antibodies block erythrocyte binding activity ofPfRH5.

TRA-1-85 antibodies abrogates invasion into human erythrocytes. TRA-1-85 antibodies reduce binding of the native PfRH5 to human erythrocytes in a dose dependent manner validating the importance of PfRH5-BSG interaction.

PfRH5 interacting with the core protein backbone of Basigin on the surface of human erythrocytes have been reported recently through an in vitro AVEXIS screening, using a mutated rPfRH5 protein. Monoclonal antibodies against human Basigin (BSG) abrogates P. falciparum invasion into human erythrocytes. To further validate this interaction, anti- TRA- 1-85 (anti-BSG), commercially available was tested for its invasion inhibitory potential of 3D7 into human erythrocytes. Consistent with previous reports, complete abrogation of invasion into human erythrocytes was observed at a concentration of 5μg/ml. To corroborate the specificity of anti TRA-1-85, that it specifically blocks the RH5-BSG interaction on human erythrocyte surface, the inventors performed erythrocyte binding in presence of increasing concentration of Anti TRA-1-85 antibodies is performed. The anti TRA-1-85 antibodies did reduce the binding of native Pf H5 to human erythrocytes in a dose dependent way, validating the importance of PfRH5-BSG interaction for human erythrocyte invasion.

Figure 18 depicts rPfRH5 antibodies exhibit strain transcending activity.

rPfRH5 antibodies display highly potent and cross strain neutralising activity when evaluated against sialic acid independent and trypsin resistant clone- 3D7 and HB3; sialic acid independent and trypsin sensitive clone- 7G8, sialic acid dependent and trypsin resistant clone Mcamp

After having validated that the antibodies have been raised against a correctly folded rPfRH5 immunogen and corroborating the specificity of the anti-rPfRH5 antibodies, we proceeded to test the invasion inhibitory potential of the antibodies is tested. Anti-rPfRH5 IgG from rabbit exhibited a dose dependent inhibition of erythrocyte invasion of P. falciparum 3D7 with maximum inhibition of 75±3% at a concentration of lOmg/ml.

Though PfRH5 is now believed to be an indispensible ligand for erythrocyte invasion, amino acid polymorphisms in,PfRH5 protein has been reported for multiple strains. These polymorphisms alter its receptor binding specificity and also dictate the species specific invasion in Aotus erythrocytes. Since polymorphisms have been accounted to be the major obstacle for the development of AMA 1, as a cross strain neutralizing vaccine candidate, the erythrocyte invasion inhibitory activity of the anti-PfRH5 IgG in different strains has been tested for which polymorphisms have been reported, and invaded erythrocytes using different invasion pathways. Interestingly, polymorphisms do not have any major impact on the invasion inhibitory activity of anti-rPfRH5 IgG, abrogating erythrocyte invasion by different strains of P. falciparum i.e. anti-rPfRH5 antibodies were cross strain neutralizing

The present invention also provides compositions comprising two or more erythrocyte binding merozoite antigens as described herein. Compositions of the invention can be combined with conventional pharmaceutically acceptable excipient and optionally a vaccine adjuvant also the US patent application 20090175895.

Such excipients include any excipient that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate-buffered physiologic saline is a typical carrier.

The vaccine composition may also contain diluents, such as water, saline, glycerol, etc.

Auxiliary substances such as wetting or emulsifying agents, pH buffering substance, and the like may be present.

The pH of the composition is preferably between 6 and 8, preferably about 7. The pH may be maintained by the use of a buffer. A phosphate buffer is typical. The composition may be sterile and/or pyrogen-free. The composition may be isotonic with respect to humans. Compositions may include sodium salts (e.g. sodium chloride) to give tonicity.

Compositions may comprise a sugar alcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml (e.g. 25 mg/ml), particularly if they are to be lyophilised or if they include material which has been reconstituted from lyophilised material. The pH of a composition for lyophilisation may be adjusted to around 6.1 prior to lyophilisation.

Antimalarial:

The composition may further comprise an antimalarial that is useful for the treatment of Plasmodial infection. Preferred antimalarials for use in the compositions include the chloroquine phosphate, proguanil, primaquine, doxycycline, mefloquine, clindamycin, halofantrine, quinine sulphate, quinine dihydrochloride, gluconate, prim aquine phosphate and sulfadoxine.

The compositions of the invention may also comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents include(s) an adjuvant.

The adjuvents suitaible for use in the invention include mineral salts, such as aluminium salts and calcium salts, mineral salts such as hydroxides (for example, oxyhydroxides), phosphates (for example, hydroxyphosphates, orthophosphates), sulphates, etc. (for example, see chapters 8 & 9 of Powell & Newman (eds.) Vaccine Design (1995) Plenum), or mixtures of different mineral compounds, with the compounds taking any suitable form (for example, gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt (WO00/23105). A typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with P04/AI molar ratio between 0.84 and 0.92, included at 0.6 mg AI3+/ml. Adsorption with a low dose of aluminium phosphate may be used for example, between 50 and 100 g AI3+ per conjugate per dose. Where an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution (for example, by the use of a phosphate buffer).

The invention also includes oil emulsion compositions, which include oil-in- water emulsions and water-in-oil emulsions.

A submicron oil-in-water emulsion may include squalene, Tween 80, and Span 85 for example, with a composition by volume of about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85 (in weight terms, 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85), known as 'MF595' (57-59 chapter 10 of Powell & Newman (eds.) Vaccine Design (1995) Plenum; chapter 12 of OHagen (ed.) Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series)). The MF59 emulsion advantageously includes citrate ions for example, 10 mM sodium citrate buffer.

An emulsion of squalene, a tocopherol, and Tween 80 can be used. The emulsion may include phosphate buffered saline. It may also include Span 85 (for example, at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably <1 as this provides a more stable emulsion. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5 g of DL-a- tocopherol and 5ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets for example, with an average diameter of between 100 and 250nm, preferably about 180nm. An emulsion of squalene, a tocopherol, and a Triton detergent (for example, Triton X- 100) can be used (WO2010127398).

An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluronic™ L 121") can be used. The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-I" adjuvant, (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant (Hariharan et al. (1995) Cancer Res 55:3486-9) (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred(WO2010127398) . Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A) may also be used (WO2010127398).

Saponin formulations may also be used as adjuvants in the invention (see for example, Chapter 22 of Powell & Newman (eds.) Vaccine Design (1995) Plenum). Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). (WO2010127398)

Saponin adjuvant formulations include purified formulations, such as QS2T, as well as lipid formulations, such as ISCOMs. QS21 is marketed as Stimulon™.(WO2010127398)

Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs) (see for example, Chapter 23 of Powell & Newman (eds.) Vaccine Design (1995) Plenum). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA and QHC. ISCOMs are further described in W096/33739, . EP-A-0109942, W096/11711). Optionally, the ISCOMS may be devoid of additional detergent WOOO/07621. A review of the development of saponin based adjuvants can be found in Barr et" al. (1998) Advanced Drug Delivery Reviews 32:247-271 and Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32:321-338.

Virosomes and virus-like particles (VLPs) can also be used as adjuvants. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins . may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qp-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi). VLPs are discussed further in (Niikura et al. (2002) Virology 293:273-280, Lenz et al. (2001) J Immunol 166:5346-5355, Pinto et al. (2003) J Infect Dis 188:327-338, Gerber et al. (2001) Virol 75:4752-4760, WO03/024480 and WO03/024481). Virosomes are discussed further in, for example, Gluck et al. (2002) Vaccine 20:B10-B16.

Adjuvants which may be used also include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostiinulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof (WO2010127398) .

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-0- deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 77. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 μιη membrane (EP-A-0689454v). Other nontoxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosamine de phosphate derivatives for example, RC-529 (Johnson et al (1999) Bioorg Med Chem Lett 9:2273-2278, Evans et al. (2003) Expert Rev Vaccines 2:219-229).

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM- 174. OM- 174 is described for example, in Meraldi et al. (2003) Vaccine 21:2485-2491 , Pajak et al. (2003) Vaccine 21 :836-842. Immunostimulatory oligonucleotides can also be used as adjuvants in the invention and include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory. The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double- stranded or single-stranded. Kandimalla et al (2003) Nucleic Acids Research 31 : 2393-2400, WO02/26757 and W099/62923 disclose possible analog substitutions for example, replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg (2003) Nature Medicine 9:831-835, McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185, WO98/40100, US patent 6,207,646, US patent 6,239,116 and US patent 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or

TTCGTT (Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658). The CpG sequence may be specific for inducing a THl immune response, such as a CpG- A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in refs. Blackwell et al. (2003) J Immunol 170:4061-4068, Krieg (2002) Trends Immunol 23:64-65. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658, Kandimalla et al (2003), BBRC 306:948-953, Bhagat et al. (2003) BBRC 300:853-861 and WO03/035836. Other immunostimulatory oligonucleotides include a double-stranded RNA or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E.coli (E.coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP- ribosylating toxins as mucosal adjuvants is described in W095/17211 and as parenteral adjuvants in W098/42375. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in Beignon et al. (2002) Infect Immun 70:3012-3019, Pizza et al. (2001) Vaccine 19:2534-2541 , Pizza et al. (2000) lnt J Med Microbiol 290:455-461 , Scharton-Kersten et al. (2000) Infect lmmun 68:5306-5313, Ryan et al. (1999) Infect lmmun 67:6270-6280, Partidos et al. (1999) Immunol Lett 67:209-216, Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293, Pine et al. (2002) J Control Release 85:263-270. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al. (1995) Mol Microbiol 15:1165-1167, specifically incorporated herein by reference in its entirety.

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (for example, IL-I5 IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-17, IL-18 (WO99/40936), IL-23, IL27 (Matsui M. et al. (2004) J. Virol 78: 9093) etc.) (W099/44636), interferons (for example, interferon-γ), macrophage colony stimulating factor, tumor necrosis factor and macrophage inflammatory protein- 1 alpha (MIP-1 alpha) and MIP-1 beta (Lillard JW et al, (2003) Blood 101(3):807-14).

Bioadhesives and mucoadhesives may also be used as adjuvants. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et ah (2001) JCont Release 70:267-276) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention (WO99/27960). Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of -lOOnm to ~150μιη in diameter, more preferably ~200nm to ~30μπι in diameter, and most preferably ~500nm to ~10μπι in diameter) formed from materials that are biodegradable and non-toxic (for example, a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (for example, with SDS) or a positively-charged surface (for example, with a cationic detergent, such as CTAB).

Examples of liposome formulations suitable for use as adjuvants are described in US patent 6,090,406, US patent 5,916,588, EP-A-0626169.

Polyoxyethylene ethers and polyoxyethylene esters are also suitable for use as adjuvents(W099/52549). Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WOO 1/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152). Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Phosphazene adjuvants include poly(di(carboxylatophenoxy)phosphazene) ("PCPP") as described, for example, in references Andrianov et al. (1998) Biomaterials 19:109-115 and Payne et al. (1998) Adv Drug Delivery Review 31 : 185-196.

Muramyl peptides which may be used as adjuvents in the invention include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D- isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE) (WO2010127398) . Imidazoquinoline adjuvants include Imiquimod ("R-837") (US 4,680,338 and US 4,988,815), Resiquimod ("R-848") (W092/15582), and their analogs;; and salts thereof (for example, the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in references Stanley (2002) Clin Exp Dermatol 27:571-577, Wu et al. (2004) Antiviral Res. 64(2):79-83, Vasilakos et al. (2000) Cell Immunol. 204(1):64- 74, US patents 4689338, 4929624, 5238944, 5266575, 5268376, 5346905, 5352784, 5389640, 5395937, 5482936, 5494916, 5525612, 6083505, 6440992, 6627640, 6656938, 6660735, 6660747, 6664260, 6664264, 6664265, 6667312, 6670372, 6677347, 6677348, 6677349, 6683088, 6703402, 6743920, 6800624, 6809203, 6888000 and 6924293 and Jones (2003) Curr Opin Investig Drugs 4:214-218.

Thiosemicarbazone adjuvants include those disclosed in WO2004/060308.

Methods of formulating, manufacturing, and screening for active compounds are also described in WO2004/060308. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-a.

Tryptanthrin adjuvants include those disclosed in WO2004/064759. Methods of formulating, manufacturing, and screening for active compounds are also described in WO2004/064759. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-a.

Various nucleoside analogs can be used as adjuvants, such as (a) lsatorabine (ANA-

245; 7-thia- 8-oxoguanosine) and prodrugs thereof; (b) ANA975; (c) ANA-025-1 ; (d) ANA380; (e) the compounds disclosed in US 6,924,271 , US2005/0070556 and US 5,658,731 , or (f) a pharmaceutically acceptable salt of any of (a) to (g), a tautomer of any of (a) to (g), or a pharmaceutically acceptable salt of the tautomer (WO2010127398) .

Lipids linked to a phosphate-containing acyclic backbone, such as adjuvants containing lipids linked to a phosphate-containing acyclic backbone include the TLR4 antagonist E5564 (Wong et al. (2003) J Clin Pharmacol 43(7):735-42 and US2005/0215517).

Small molecule immunopotentiators which may be useful adjuvants include N2- methyl-l-(2-methylpropyl)-l H-imidazo(4,5-c)quinoline-2,4-diamine; N2,N2-dimethyl-l-(2- methylpropyl)-l H-imidazo(4,5-c)quinoline-2,4-diamine; N2-ethyl-N2-methyl-l -(2- methylpropyl)-l H-imidazo(4,5-c)quinoline-2,4-diamine; N2-methyl-l -(2-methylpropyl)- N2 -propyl- 1 H-imidazo(4,5-c)quinoline-2,4-diamine; 1 -(2-methylpropyl)-N2-propyl-l H- imidazo(4,5-c)quinoline-2,4-diamine; N2-butyl-l -(2-methylpropyl)-l H-imidazo(4,5- c)quinoline-2,4-diamine; N2-butyl-N2-methyl-l-(2-methylpropyl)-l H-imidazo(4,5- c)quinorme-2,4-diamine; N2-methyl-l-(2-methylpropyl)-N2-pentyl-l H-imidazo(4,5- c)quinoline-2,4-diamine; N2-methyl-l -(2-methylpropyl)-N2-prop-2-enyl-l H-imidazo(4,5- c)quinoline-2,4- diamine; 1 .-(2-methylpropyl)-2-((phenylmethyl)thio)-lH-imidazo (4,5- c)quinolin-4-amine; 1 -(2-methylpropyl)-2-(propylthio)-l H-imidazo(4,5-c)quinolin-4-amine; 2-((4-amino-l -(2-methy I propyl)- 1 H-imidazo(4,5-c)quinolin-2-yl)(methyl)amino)ethanol; 2-((4-amino-l-(2-methylpropyl)-l H-imidazo(455-c)quinolin-2-yl)(methyl)amino)ethyl acetate; 4-amino-l -(2-methylpropyl)-l ,3-dihydro-2H-imidazo(4,5-c)quinolin-2-one; N2- butyl- 1 -(2-methylpropyl)-N4,N4-bis(phenylmethyl)-lH-imidazo(4,5-c)quinoline-2,4- diamine; N2-butyl-N2-methyl-l-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-l H- imidazo(4,5- c)quinoline-2,4-diamine; N2-methyl-T-(2-methylpropyl)-N4,N4- bis(phenylmethyl)- 1 H-imidazo(4,5-c)quinolne-2,4-diamine; N2,N2-dimethyl- 1 -(2- methylpropyI)-N4,N4-bis(phenylmethyl)-l H-imidazo(4,5- c)quinoline-2,4-diamine; 1- (4- amino-2-(methyl(propyl)amino)-l H-imidazo(4,5-c)quinolin-l -yl}-2-methylpropan-2-ol; 1 - (4-amino-2-(propylaniino)-l H-imidazo(4,5-c)quinolin-l-yl)-2-methylpropan-2-ol; N43N4- dibenzyl- 1 -(2-methoxy-2-methylpropyl)-N2propyl- 1 H-imidazo(4,5-c)quinoline-2,4-diamine. One potentially useful adjuvant is an outer membrane protein proteosome preparation prepared from a first Gram- negative bacterium in combination with a liposaccharide preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteosome and liposaccharide preparations form a stable non-covalent adjuvant complex. Such complexes include "IVX-908", a complex comprised of Neisseria meningitidis outer membrane and lipopolysaccharides. They have been used as adjuvants for influenza vaccines (WO02/072012).

Other substances that act as immunostimulating agents are disclosed in Vaccine

Design ((1995) eds. Powell & Newman. ISBN: 030644867X. Plenum) and Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series) (ISBN: 1-59259-083-7. Ed. O'Hagan). Further useful adjuvant substances include: Methyl inosine 5 '-monophosphate ("MIMP") Signorelli & Hadden (2003) lnt lmmunopharmacol 3(8): 1177); a polyhydroxlated pyrrolizidine compound (WO2004/064715), examples include, but are not limited to: casuarine, casuarine-6-cc-D- glucopyranose, 3-epz-casuarine, 7-epz-casuarine, 3,7-diepz-casuarine, etc; a gamma inulin (Cooper (1995) Phar Biotechnol 6:559) or derivative thereof, such as algammulin; compounds disclosed in PCT/US2005/022769; compounds disclosed in WO2004/87153, including: Acylpiperazine compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ) compounds, Benzocyclodione compounds, Aminoazavinyl compounds, Aminobenzimidazole quinolinone (ABIQ) compounds (US6,606617, WO02/018383), Hydrapthalamide compounds, Benzophenone compounds, Isoxazole compounds, Sterol compounds, Quinazilinone compounds, Pyrrole compounds (WO/04/018455), Anthraquinone compounds, Quinoxaline compounds, Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole compounds (WO03/082272); loxoribine (7- allyl-8-oxoguanosine) (US 5,011,828); a formulation of a cationic lipid and a (usually neutral) co-lipid, such as aminopropyl- dimethyl-myristoleyloxy-propanaminium bromide- diphytanoylphosphatidyl- ethanolamine ("Vaxfectin™") or aminopropyl-dimethyl-bis- dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine ("GAP- DLRIE:DOPE"). Formulations containing (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn- 9-tetradeceneyloxy)-l- propanaminium salts are preferred (US6, 586,409).

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in- water emulsion (W099/ 11241); (2) a saponin (for example, QS21) + a nontoxic LPS derivative (for example, 3dMPL) (WO94/00153); (3) a saponin (for example, QS21) + a non-toxic LPS derivative (for example, 3dMPL) + a cholesterol; (4) a saponin (for example, QS21) + 3dMPL + IL-12 (optionally + a sterol) (W098/57659); (Thera MA et al, N. Engl. J. Med. 365:1004-1013)( combinations of 3dMPL with, for example, QS21 and or oil-in-water emulsions (EP0835318, EP0735898, EP0761231); (6) RibiTM adjuvant system (RAS), (Ribi Imrnunochern) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); and (Rono J et al, Infect. Immun. 80:1900-1908) ( one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3d M PL).

In a further aspect, the present invention provides a method for treating or preventing

Plasmodium infection, comprising administering to a subject in need thereof an effective amount of a composition as described herein. The administration may be by any conventional means. The antibodies can be identified and isolated by any conventional means well known to those of ordinary skill in the art.

The vaccine may be administered using a variety of vaccination regimes familiar to the skilled person. In one form of the invention, the vaccine composition may be administered post antimalarial treatment. Preferred antimalarials for use include the chloroquine phosphate, proguanil, primaquine, doxycycline, mefloquine, clindamycin, halofantrine, quinine sulphate, quinine dihydrochloride, gluconate, primaquine phosphate and sulfadoxine. For example, blood stage parasitaemia may be cleared with Fansidar (25 mg sulfadoxine/0.75 mg pyrimethamine per kg body weight) before each vaccination. In another form of the invention antimalarial (for example, Fansidar) treatment is given 1 to 2 weeks before the doses (for example, first and third doses).

EXAMPLES

The inhibitory activity of the preferred combinations of antibodies in accordance with the present invention is given as examples. The example illustrates only the preferred embodiments and should not be construed to limit the scope of the invention.

Example 1

Total IgGs purified from rabbit sera against the six proteins (PfRHl, PfRH2, PfRH4, PfF2, PfAARP, and PTRAMP) are assayed individually at a concentration of 3.3 mg/ml and in combinations of three IgGs (3.3 mg/ml each) against sialic acid-independent clone 3D7 (A) and sialic acid-dependent clone Dd2 (B). Three independent assays are performed in duplicate. The error bars show the standard errors of the means. P values are calculated by using the Student t test.

The invasion-inhibitory activities of all 20 possible triple-antibody combinations of purified total IgG (3.3 mg/ml each) against a pool of 6 antigens (RH1, RH2, RH4, PfF2, AARP, and PTRAMP) are tested. Invasion inhibition is assayed against P. falciparum clones 3D7 and Dd2.

Against 3D7, individual IgGs against each of the 6 antigens produced 8 to 25% inhibition, with AARP IgG being the most potent (25%) at 3.3 mg/ml. Different combinations of three IgGs displayed a potent inhibition of erythrocyte invasion, with the maximum inhibition (79%) being elicited by PfF2+RH2+AARP IgGs, which is higher than the 66% observed with RH 1 +RH2+RH4 IgGs. Against the sialic acid-dependent clone ; Dd2, AARP IgG inhibited erythrocyte invasion with the same efficiency as that observed with 3D7. Total IgG against PfF2 and PfRHl, which are sialic acid binding proteins, exhibited higher-level inhibition against Dd2 than against 3D7. PfF2+PfRH 1 +AARP yielded the maximum invasion inhibition (75%) against Dd2, consistent with the three antigens being involved in sialic acid-dependent invasion.

Example 2

The invasion inhibitory efficacy of MSP-Fu antibodies are accessed with PfAARP, PfRHl, PfRH2 and PfF2 antibodies in triple antibody combinations (1 and 2 mg/ml each) against P. falciparum clone 3D7 (Fig.19). As opposed to the sub-additive effect observed with dual antibody combinations, the inhibitory activity of the triple antibody combinations are synergistic. All combinations exhibited synergism, however, MSP-Fu+AARP+RH2 combination displayed the maximum potency, whereas MSP-Fu+AARP+PfF2 combination was least potent. Synergistic effect of combinations of MSP-Fu antibodies along with two other merozoites antibodies between concentration of 0.5 and 1.0 mg/ml each is shown in Figure 20.

Example 3

Figure 21 shows GIA with triple antibody combinations involving MSP-fu antibodies and two other Merozoite antibodies.

Combinations of MSP-Fu antibodies were evaluated with PfAARP, PfRH2 and

PfRH5 antibodies in triple antibody combinations (0.5 and 1 mg/ml each) against P. falciparum clone 3D7. The combination of antigens with MSP-Fu exhibit synergistic effect. However, MSP-Fu+AARP+PfRH5 antibody combination was more potent followed by MSP-Fu+PfRH2+PfRH5, since both MSP-1 and PfRH5 ligands are reported to be indispensible for erythrocyte invasion.

Example 4:

Total IgGs purified from rabbit sera against the six proteins (RH1, RH2, RH4, RH5. AARP and PfF2) are assayed individually at a concentration of 3.3 mg/ml and in combinations of three IgGs (3.3 mg/ml each) against sialic acid-independent clone 3D7 (Figure 23) and sialic acid-dependent clone Dd2 (Figure 26). It is observed that the combination of antibodies as compared to individual antibodies exhibit strong synergistic inhibition of erythrocyte invasion with the P. falciparum clone 3D7 and Dd2.

Claims

We claim:
1. A blood stage malaria vaccine comprising a combination of merozoite antigens.
2. The blood stage malaria vaccine as claimed in claim 1, wherein the merozite antigens are selected from the group consisting of PfF2, PfRHl , PfRH2, PfRH4, Pf H5, AARP and PfMSP-fusion.
3. The blood stage malaria vaccine as claimed in claim 2, wherein the PfMSP-fusion antigen encodes a protein having SEQ ID 19 comprising a conserved 19 kda C- terminal region of MSP-1 and a functional 24 kDa region of MSP-3.
4. The blood stage malaria vaccine as claimed in claim 2, wherein the PfRHl encodes a protein having SEQ ID 3 comprises an amino acid region 500-833 of the 350 kDa native parasite protein having 2971 amino acids.
5. The blood stage malaria vaccine as claimed in claim 2, wherein the PfRH2 encodes a protein having SEQ ID 24 comprises the amino acid region 495-860 of the 375 kDa native parasite protein having 3310 amino acids.
6. The blood stage malaria vaccine as claimed in claim 2, wherein the PfRH4 encodes a protein having SEQ IDs 7 and 9 comprises the amino acid region 328-588 of the 250 kDa native parasite protein having 1716 amino acids.
7. The blood stage malaria vaccine as claimed in claim 2, wherein the PfRH5 encodes a protein having SEQ ID 11 comprises the amino acid region 28-526 of the 66 kDa native parasite protein having 526 amino acids.
8. The blood stage malaria vaccine as claimed in claim 2, wherein the PfAARP encodes a protein having SEQ ID 14 comprises the amino acid region 20-107 of the 24 kDa native parasite protein having 217 amino acids.
9. The blood stage malaria vaccine as claimed in claim 2, wherein the PfF2 encodes a protein having SEQ ID (17) comprises the amino acid region of the of 175 kDa native parasite protein having 1502 amino acids.
10. The blood stage malaria vaccine as claimed in claim 1, wherein the vaccine comprises a combination of two or more erythrocyte binding merozoite antigens are present.
11. The blood stage malaria vaccine as claimed in claim 1, wherein the vaccine comprises a combination of three erythrocyte binding merozoite antigens are present.
12. The blood stage malaria vaccine as claimed in claim 1, wherein the vaccine comprises a combination selected from PfAARP + PfRH5 + PfF2, PfAARP + PfRH5 + PfRHl, PfAARP + PfRH5 + PfRH2, PfAARP + PfRH5 + PfRH4, PfAARP + PfRH2 + PfF2, PfAARP + PfRH2 + PfRH4, PfAARP + PfRH2 + PfRHl, PfAARP + PfRHl + PfRH4, PfAARP + PfF2 + PfRHl, PfAARP + PfF2 + PfRH4, PfRH5 + PfRH2 +
PfF2, PfRH5 + PfRH2 + PfRHl, PfRH5 + PfRH2 + PfRH4, PfRH5 + PfF2 + PfRHl, PfRH5 + PfF2 + PfRH4, PfRH5 + PfF2 + PfRH2, PfRH5 + PfRHl + PfRH4, PfRHl + PfRH2 + PfRH4, PfF2 + PfRHl + PfRH4, PfF2 + PfRH2 + PfRH4.
13. The blood stage malaria vaccine as claimed in claim 1, wherein the vaccine comprises a combination selected from MSP(Fusion) + PfAARP + PfRH5, MSP(Fusion) +
PfAARP + PfRH2, MSP(Fusion) + PfAARP + PfRHl, MSP(Fusion) + PfAARP + PfRH4, MSP(Fusion) + PfAARP + PfF2, MSP(Fusion) + PfRH5 + PfRH2, MSP(Fusion) + PfRH5 + PfRHl, MSP(Fusion) + PfRH5 + PfRH4, MSP(Fusion) + PfRH5 + PfF2, MSP(Fusion) + PfRH2 + PfRHl, MSP(Fusion) + PfRH2 + PfRH4, MSP(Fusion) + PfRH2 + PfF2, MSP(Fusion) + PfRHl + PfRH4, MSP(Fusion) +
PfRHl + PfF2, MSP(Fusion) + PfRH4 + PfF2.
14. The blood stage malaria vaccine as claimed in claim 1, wherein the antigens produce a potent invasion inhibition at an IgG content of 1.5-14 mg/ml.
15. The blood stage malaria vaccine as claimed in any preceding claims, wherein the antigens block the sialic acid independent as well as sialic acid dependent binding of the parasite P. falciparum to the erythrocyte.
16. A composition that blocks the sialic acid independent as well as sialic acid dependent receptors on the surface of Plasmodium falciparum comprising a combination of two or more erythrocyte binding merozoite antigens along with pharmaceutically excipients and/or adjuvants
17. The composition as claimed in claim 17, wherein the merozoite antigens are selected from the group consisting of PfF2, PfRHl, PfRH2, PfRH4, PfRH5, TRAMP, AARP and PfMSP-fusion.
18. The composition as claimed in claim 18, wherein the composition comprises three merozoite antigens.
19. The composition as claimed in claim 17, wherein the combination of the merozoite antigens is selected from PfAARP + PfRH5 + PfF2, PfAAR + PfRH5 + PfRHl, PfAARP + PfRH5 + PfRH2, PfAARP + PfRH5 + PfRH4, PfAARP + PfRH2 + PfF2, PfAARP + Pf H2 + PfRH4, PfAARP + PfRH2 + PfRHl, PfAARP + PfRHl + PfRH4, PfAARP + PfF2 + PfRHl, PfAARP + PfF2 + PfRH4, PfRH5 + PfRH2 + PfF2, PfRH5 + PfRH2 + PfRHl, PfRH5 + PfRH2 + PfRH4, PfRH5 + PfF2 + PfRHl, PfRH5 + PfF2 + PfRH4, PfRH5 + PfF2 + PfRH2, PfRH5 + PfRHl + PfRH4, PfRHl + PfRH2 + PfRH4, PfF2 + PfRH 1 + PfRH4, PfF2 + PfRH2 + PfRH4.
20. The composition as claimed in claim 17, wherein the combination of the merozoite antigens is selected from MSP(Fusion) + PfAARP + PfRH5, MSP(Fusion) + PfAARP + PfRH2, MSP(Fusion) + PfAARP + PfRHl, MSP(Fusion) + PfAARP + PfRH4, MSP(Fusion) + PfAARP + PfF2, MSP(Fusion) + PfRH5 + PfRH2, MSP(Fusion) + PfRH5 + PfRHl, MSP(Fusion) + PfRH5 + PfRH4, MSP(Fusion) + PfRH5 + PfF2, MSP(Fusion) + PfRH2 + PfRHl, MSP(Fusion) + PfRH2 + PfRH4, MSP(Fusion) + PfRH2 + PfF2, MSP(Fusion) + PfRHl + PfRH4, MSP(Fusion) + PfRHl + PfF2, MSP(Fusion) + PfRH4 + PfF2.
21. A recombinant protein PfRH5 having SEQ ID No. 11.
22. The recombinant protein PfRH5 as claimed in claim 22, wherein the protein is expressed in E.Coli.
23. A method of preparation of recombinant protein PfRH5, wherein said method comprises:
a. inserting a full length DNA sequence of SEQ ID 10 in E. coli expression vector, pET-24b;
b. transforming E. coli BL21 (DE3) cells with the vector of step a;
c. culturing the transformed cells of step b in super broth at 37°C;
d. inducing the cultured cells with ImM IPTG so that OD60o is around 0.8-0.9; e. harvesting the cells as pellets by centrifugation at 3000g; and
f. obtaining the protein as inclusion bodies by cell lysis of the pellets .
24. The method of preparation of recombinant protein PfRH5 as claimed in claim 24, wherein in step a the DNA sequence is inserted downstream of the T7 promoter.
25. The method of preparation of recombinant protein PfRH5 as claimed in claim 24, wherein the expression vector has 6-histidine (6-His) tag at C-terminal end.
26. The method of preparation of recombinant protein PfRH5 as claimed in claim 24, wherein the method also comprises purification of rPfRH5 protein by Ni-NTA (nitrilotriacetic acid) affinity chromatography of the inclusion bodies.
27. The method of preparation of recombinant protein PfRH5 as claimed in claim 25, wherein the purified rPfRH5 protein is refolded by rapid dilution method in MES based buffer.
28. The method of preparation of recombinant protein PfRH5 as claimed in claim 26, wherein the refolded protein is purified by dialysis with SP- sepharosecation exchange column.
29. The recombinant protein PfRH5 as claimed in claim 22 exhibits specific erythrocyte binding activity consistent with that of the native Pf H5 parasite protein.
30. Use of merozoite antigens for the blocking the sialic acid independent and sialic acid dependent receptors on the surface of P. falciparum wherein the merozoite antigens comprises a combination of two or more antigens selected from the group consisting of PfF2, PfRHl , PfRH2, PfRH4, PfRH5, TRAMP, AARP and PfMSP-fusion.
31. Use of merozoite antigens for the blocking the sialic acid independent and sialic acid dependent receptors on the surface of P. falciparum wherein the merozoite antigens comprises a combination of three antigens.
32. A method of treatment and / or prevention of a pathological condition wherein the method comprises injecting a composition or a vaccine comprising a combination of two or more merozoite antigens selected the group consisting of PfF2, PfRHl, PfRH2, PfRH4, PfRH5, TRAMP, AARP and PfMSP-fusion.
33. A method of treatment and / or prevention of a pathological condition as claimed in claim 32, wherein the antigens block the sialic acid independent as well as sialic acid dependent binding of the parasite P. falciparum to the erythrocyte
34. A method of treatment and / or prevention of a pathological condition as claimed in claim 33, wherein the antigens produce a potent invasion inhibition at an IgG content of 1.5-14 mg/ml.
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