The present invention relates to a vaccine for therapeutic or prophylactic treatment of a pathological condition. In particular, the present invention relates to vaccine for therapeutic or prophylactic treatment of malaria.

Malaria is widely spread in tropical and subtropical regions. Malaria is caused by infection with malaria parasites mediated by anopheles. Of four kinds of human malaria, falciparum and vivax malaria account for the majority of them. Both cause symptoms, such as fever and anemia. Falciparum malaria causes death if accompanied by serious complications. After World War II, the number of deaths caused by malaria was reduced by measures against mediating mosquitoes using insecticides such as DDT and the appearance of a specific medicine, chloroquine. However, as chloroquine-resistant Plasmodium falciparum and insecticide-resistant mosquitoes subsequently emerged, the number of patients increased again. Currently, about 300 million people are affected by falciparum malaria, causing estimated deaths of more than 860,000 every year. Thus, malaria vaccines have attracted attention as new specific medicines.

However, malaria parasites express vastly different genes depending on the developmental stages of their complicated life cycles. Hence, three types of malaria vaccines have been investigated: (1) vaccines to prevent the infection targeting to sporozoites and liver-stage parasites, (2) vaccines to prevent the developing the disease targeting to erythrocyte-stage parasites and (3) vaccines to prevent the spreading of parasites in the mosquito gut. However, none has been put to practical use. Thus, the development of malaria vaccines is awaited.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Any document referred to herein is hereby incorporated by reference in its entirety. The fate of the malaria infection depends on the efficiency with which the invasive forms released from the mammalian host cell, merozoites, attach to and invade erythrocytes. Red blood cell selection is not random, as the tropism towards erythrocytes of different ages varies with the Plasmodium species. Furthermore, merozoite invasion occurs within minutes, and while free in the blood stream, these forms are particularly vulnerable to immune attack. Notwithstanding such a brief exposure, the molecules implicated in red blood cell selection and invasion are attractive targets for vaccine development. This prompted sustained efforts to unravel this complex sequence of events and to identify the parasite and host proteins that mediate them.

Knowledge of erythrocyte invasion has been mainly accrued for P. falciparum, a parasite that can invade all erythrocyte subsets, through detailed and elegant investigations made possible by the ability to culture this species in vitro and to generate genetic variants. This led to the discovery of multiple parasite-ligand/host-receptor combinations mediating distinct invasion pathways, with the most recent PfRh5/basigin providing a particularly promising target for vaccine development{Crosnier, 2011 #10;Douglas, 2014 #50}. In contrast, invasion by P. vivax, a species long known to have a strict predilection towards reticulocytes{Hegner, 1938 #18;Mons, 1988 #51;Malleret, 2015 #3}, cannot be adequately continuously maintained in vitro and has consequently been far less studied. To date the cognate reticulocyte-specific receptors of the putative P. vivax ligands are yet to be identified (Fig. la). One clue was provided by a single study conducted using one P. vivax strain (Belem, from South America) that suggested that this receptor was unaffected by trypsin treatment{Barnwell, 1989 #12}; an observation that we have extended of late to multiple P. vivax isolates from Thailand{Malleret, 2017 #68}.

We have recently shown that immature reticulocytes expressing CD71 (transferrin receptor 1) a marker that is lost from the surface membrane during reticulocyte maturation to normocyte{Frazier, 1982 #67}, are preferentially targeted by the P. vivax merozoite{Malleret, 2015 #3}. We adopted a strategy of a differential proteomic screen of CD71+ versus CD71- erythrocyte ghost membranes to determine dynamic changes on surface protein composition and to identify abundant reticulocyte proteins. The criteria to select candidate reticulocyte- specific receptors were: a) expression and presence on the immature reticulocytes but not on the normocytes, and b) insensitivity to trypsin treatment.

The proteomic screen identified a number of proteins with reduced expression on CD71- erythrocytes (Fig. lb and Fig. 4){Chu, 2018 #79} of which a subset was resistant to trypsin treatment (Fig. lb, Fig. 3 and Table 1).

Table 1. Differential proteomic screen of CD71+ versus CD71- erythrocyte ghost membranes. The above table depicts an average log2 (fold-change) of CD71+ cells compared to CD71- cells for the membrane fraction for the depicted antigens. Dashes indicate that the proteins were not identified in the proteomics dataset which could be a limitation for detecting certain peptides that are highly glycosylated.

Two surface proteins displayed a significant fold-change reduction in their expression levels between CD71+ and CD71- erythrocytes, namely CD71 (ca. 6-fold) and CD98 (ca. 4-fold), while all others showed less than a 2-fold change (Fig. lb). Of these two proteins only CD98 proved resistant to trypsin treatment (Fig. 5). CD98 (also known as 4F2hc) is the heavy chain component of the Large neutral Amino acid Transporter (LAT) complex{Boado, 1999 #31;Segawa, 1999 #30} that is exposed to outside of the reticulocyte surface membrane (Fig. lc){Fort, 2007 #15}. The diminishment of CD98 expression during erythrocyte maturation from CD71+ to CD71- erythrocytes was confirmed by careful immunophenotyping (Fig. Id), immunofluorescence (Fig. le), and Western blotting (Fig. If) analyses of in vitro matured CD71+ reticulocytes.

In order to demonstrate the involvement of CD98 in the invasion of reticulocytes we conducted invasion inhibition assays using different clinical P. vivax isolates{Russell, 2011 #6}. Polyclonal and monoclonal anti-CD98 antibodies significantly abrogated the invasion of P. vivax merozoites into CD71+ reticulocytes, with levels of inhibition (ca. 70%) close to those observed (98%) with antibodies directed against DARC (Fig. 2a, b). Antibodies directed against two highly expressed trypsin-resistant erythrocyte surface proteins, CD240DCE (Rh) and CD147 (basigin), a known receptor of P. falciparum merozoite invasion{Crosnier, 2011 #10}, had little or no effect on invasion efficiency (Fig. 2a). While the anti-CD147 antibody had a strong and specific inhibitory effect on P. falciparum invasion on CD71+ or CD71- erythrocytes, anti-CD98 antibodies did not affect P. falciparum invasion on CD71+ reticulocytes. Our attempts to confirm the central role for CD98 through genetic silencing experiments failed to yield viable reticulocytes, most probably because disruption of this amino acid transporter is essential for erythropoietic development. It is interesting to note that CD98 could not be detected at the surface of the P. vivax rings stage collected from patients, suggesting that CD98 exposure to cell surface is lost early after invasion of reticulocytes by P. vivax (Fig. 2c, d). In order to identify the corresponding parasite ligand that binds CD98, we generated a small pDisplay surface-expression library of selected P. vivax genes potentially implicated in merozoite invasion (Fig. 6 and Table 2). The P. vivax antigen library, cloned in the pDisplay vector, consisted of 28 different constructs encoding for 8 different P. vivax proteins that were successfully transfected into HEK293 cells for cell surface expression. Binding assays using these cells with CD71+ versus CD71- erythrocytes identified fragments encoded by two genes (PvRBP2a and PvRBP2b) that mediated strong binding to CD71+ reticulocytes (Fig. 3a, b and Table 2). However when this assay was conducted in presence anti-CD98 antibodies, there was no effect PvRBP2b binding to on reticulocytes (Extended Fig. 3c). The highest level of binding, more than two-fold than that measured for the other constructs, was observed with the HEK cells displaying PvRBP2a ₂₃ - ₇ 67 or PvRBP2ai ₆ o-ii ₃ 5. HEK cells transfected with constructs corresponding to other regions of PvRBP2a (Fig. 9) showed significantly weaker binding (Fig. 3a, Fig. 6b and Ta ble 2). The HEK cells transfected with the PvRBP2a ₂₃ -767 construct bound reticulocytes and the binding attributed to CD98 was specific to the reticulocytes as it was much reduced by the presence of anti-CD98 antibodies (Fig. 3c). When this similar binding assay was conducted with normocytes the presence of anti-CD98 antibodies did not inhibit the binding (Fig. 6d). These observations also indicate that the PvRBP2a protein can bind additional proteins on the surface of normocytes. I nteraction between CD98 and PvRBP2a was further demonstrated in a competitive binding assay using soluble crosslinked CD98 dimers as a competitor (Fig. 7), since CD98 forms dimers in its native form at the surface of human cells{Fort, 2007 #15}. Table 2. List of Plasmodium vivax genes used for reticulocyte binding assay. Twenty-eight gene fragments corresponding to 8 different P. vivax genes were amplified and cloned into the pDisplay vector.

Range of binding reticulocytes (mean of duplicates) corresponding to the following symbols: (-) 0 cell; (+/-) 1-60 cells; (+) 61-200 cells; (++) 201-260 cells; (++) > 261 cells.

As expected binding of cells transfected with the PvRBP2a ₂₃ -767 construct to reticulocytes was abolished by the presence of two different clones of anti-PvRBP2a mouse monoclonal antibodies (Fig. 3d and Fig. 8). Importantly these antibodies also inhibited the invasion of reticulocytes by P. vivax isolates (Fig. 3e). The levels of inhibition observed is likely to depend on affinity of the different anti-PvRBP2a antibodies to the native protein, genetic polymorphism of PvRBP2a or the presence of alternative receptors like CD71{Gruszczyk, 2018 #80}.

The interaction was assessed biochemically by isothermal titration calorimetry. We found that monomeric CD98 interacts with PvRBP2a with a KD value of 3.4 μΜ (Fig. 3f), similar to Duffy-binding protein (PvDBP) first binding event{Batchelor, 2014 #81}. Based on the available crystal structures{Gruszczyk, 2016 #37}, we expect a higher affinity between CD98 and PvRBP2a multimers due to avidity effects ( Fig. 10).

The distinct tropism for different subsets of red blood cells by Plasmodium parasites infecting humans as well as other mammalian hosts has exercised the interest of malariologists for more than eighty years. This was not for mere scientific curiosity, as the erythrocyte niche favoured by the parasite clearly has a major influence on the parasitological course and the clinical outcome of the infection. Restriction to reticulocytes could be considered as a natural means to moderate the parasite burden and the consequent pathology. I ndeed in humans clinical severity is less frequent in vivax than in falciparum malaria where the parasite invades all erythrocyte subsets. The data presented here provides evidence for a central role of CD98 as a host receptor in reticulocyte selection and further identifies PvRBP2a as the corresponding parasite ligand. PvRBP2a is a member of a multigene family of 11 members{Carlton, 2008 #58}, some of which have also been implicated in reticulocyte invasion by P. i /Vox{Galinski, 1992 #19;Gruszczyk, 2016 #37}. It now becomes important to determine whether the PvRBP2a/CD98 interaction is essential to reticulocyte recognition for all P. vivax populations. Indeed, the occurrence of alternative invasion pathways, known for P. falciparum, has recently been raised for P. vivax when infections by this parasite once considered strictly Duffy-dependent were recorded in Duffy-negative individuals in Africa and South America{Ryan, 2006 #62;Menard, 2010 #25;Cavasini, 2007 #61}. The identification of reticulocyte-specific receptor/ligand pair could help in the development of continuous in vitro culture of P. vivax, a tool whose lack severely hampers research on this parasite. Ultimately, it provides the community with a novel vaccine candidate that could contribute to the control and eventual elimination of this globally important pathogen. In an aspect of the present invention, there is provided an agent having a specificity for a PvRBP expressed on the surface of a Plasmodium merozoite or a human CD98 and/or CD147, wherein the agent blocks or reduces binding of the PvRBP with the human CD98 and/or CD147.

Plasmodium vivax, a pathogen of global importance, has long been known to have a strict preference to invade immature red blood cells. However, the molecular basis for this preference is unknown. Using a differential proteomic strategy based on a previous demonstration of selective invasion of CD71+ but not CD71- reticulocytes, and associated to a set of biological and biochemical assays, we identified CD98 as a reticulocyte-specific receptor and PvRBP2a as its cognate parasite ligand. Advantageously, because it has now been found that PvRBP2a/CD98 interaction is essential for reticulocyte recognition by all P. vivax parasite populations, the present invention provides a potential vaccine candidate against vivax malaria. We have also already started to generate crystal structures of the PvRBP2a/CD98 complex. This structural data will help us to design anti-CD147/CD98 bi- specific antibodies by phage display with the antibody platform of Singapore Immunology Network. The structural data will give us also the opportunity to do some epitope mapping to design the blocking peptides for the interactions between PvRBP2a and CD98. This knowledge and tools have a strong therapeutic potential and will surely be part of the armamentarium needed for P. vivax eradication.

Such an agent is useful in the therapeutic or prophylactic treatment of a P. vivax infection in a subject by blocking the binding or interaction between human CD98 (and/or CD147) and PvRBP of P. vivax.

By "agent", it is meant to include any agent, molecule, antibody, fragment of an antibody, nucleic acid molecule, oligonucleotide, peptide, polypeptide or the like that has a binding affinity to the targets described in the present invention. The agent may comprise polypeptides or antibodies, or antigen-binding fragments thereof. In various embodiments, the antibody is a monoclonal antibody. Further, the monoclonal antibody is humanised, human or chimeric antibody. In various aspects, the agent has a specificity for PvRBP2a ₂ 3-n35 of the Plasmodium merozoite. Preferably, the agent has a specificity for PvRBP2a ₂ 3-767 of the Plasmodium merozoite. The Plasmodium merozoite may be a Plasmodium vivax merozoite.

In another aspect of the present invention, there is provided nucleic acid molecule encoding the agent according to an embodiment of the present invention.

In yet another aspect of the present invention, there is provided a vector comprising a nucleic molecule according to an embodiment of the present invention. In yet another aspect of the present invention, there is provided host cell comprising a nucleic acid molecule according to an embodiment of the present invention or a vector according to an embodiment of the present invention, where the nucleic acid molecule or vector encodes the agent according to an embodiment of the present invention. In yet another aspect of the present invention, there is provided method of producing an antibody according to an embodiment of the present invention, wherein the method comprises culturing a host cell in vitro to produce the antibody.

In yet another aspect of the present invention, there is provided hybridoma which produces an antibody according to an embodiment of the present invention

In an aspect of the present invention, the agent is useful in medicine. In particular, the agent may be used in a method for the therapeutic or prophylactic treatment of malaria. As such, the invention may include a composition comprising an agent according to an aspect of the present invention and at least one pharmaceutically acceptable diluent or carrier. It includes the use of the agent in the manufacture of a medicament for the therapeutic or prophylactic treatment of malaria. The compositions, methods, and agents of the present invention includes a method of administering the agent as a vaccine. In yet another aspect of the present invention, there is provided method for the therapeutic or prophylactic treatment of malaria in a subject, the method comprising administering to the subject the agent according to an embodiment of the present invention.

In yet another aspect of the present invention, there is provided of the vaccine comprising the agent, and such use of it in the manufacture of a medicament for the treatment of malaria.

In yet another aspect of the present invention, there is provided an agent having a specificity for a human CD98 and CD147, wherein the agent blocks or reduces binding of a Plasmodium vivax merozoite or a Plasmodium falciparum merozoite with the human CD98 and CD147. In various embodiments, the agent is a bi-specific antibody.

In yet another aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of malaria in a subject, the method comprising administering to the subject an agent having a specificity for a human CD98 and CD147. It also includes the use of such an agent in the manufacture of a medicament for the therapeutic or prophylactic treatment of malaria. Preferably, the agent is an antibody. In a preferred embodiment, the agent targets the CD98 and CD147 to prevent their binding with P. vivax's PvRBP.

As such, the inventors have now discovered that CD98 is a specific receptor for the invasion of Plasmodium vivax merozoites in the immature red blood cells (reticulocytes). This protein is not involved in Plasmodium falciparum invasion. The other well-known Plasmodium vivax receptor is DARC (Duffy antigen/chemokine receptor) but this protein is expressed on the mature red blood cells, so it does not drive the reticulocyte tropism of Plasmodium vivax. Therefore, blocking CD98 with a specific antibody results in inhibition of Plasmodium invasion. In addition, blocking PvRBP of the Plasmodium vivax may also prevent it from binding with CD98 which, in turn, inhibits Plasmodium invasion. Still further, a CD98/CD147 bi-specific antibody which blocks Plasmodium vivox and erythrocyte interaction may also result in Plasmodium invasion.

I n order that the present invention may be fully understood and readily put into practical effect, there shall now be described by way of non-limitative examples only preferred embodiments of the present invention, the description being with reference to the accompanying illustrative figures.

I n the Figures:

Figure 1. CD98, a trypsin-resistant protein, is expressed specifically on CD71+ reticulocytes a, Schematic diagram depicting the presence of an unknown vivax receptor involved in reticulocyte tropism (Reticulocyte specific receptor or RSR). b, Relative fold changes of proteins expressed at the surface of CD71+ reticulocyte quantified by mass-spectrometry. The "R" in blue denotes trypsin resistance (see also Fig. 4). c, CD98 (4F2hc) protein structure with a schematic representation of the light subunit in the LAT complex{Fort, 2007 #15}. d, Flow cytometry profile of CD98 expression on CD71+ or CD71- CD235a+ erythrocytes for three different human cord blood samples compared to secondary antibody only (gray), e, CD98 and Demantin (Band 4.9) expression in CD71- and CD71+ erythrocytes, as recorded through immunofluorescence microscopy. The scale represents 2 μιη. f, Detection of CD98 by Western blot of ghost membranes derived from CD71-, and CD71+ erythrocytes matured ex vivo for 20 and 40 h.

Figure 2. CD98 is essential for P. vivax invasion, a, P. vivax invasion inhibition assay in presence of anti-CD147, anti-CD240DCE, anti-CD98 (polyclonal), anti-CD98 (monoclonal) or anti-DARC F(ab ^' )2 antibodies (final concentration of 25 μg/ml). Inhibition values are represented as mean ± SD, Kruskal-Wallis test (p<0.0001) followed by Dunn's post-test, ***p < 0.001 and **p < 0.01. b, Representative micrographs of Giemsa thin film smea rs of cultures treated with or without anti-CD98 or anti-DARC antibodies. Red asterisks indicate ring stage invasions and the scale represents 10 μιη. c, Flow cytometry histogram of CD98 expression (right) at the surface of P. vivax rings, early infected reticulocyte forms, gated on Hoechst- positive cells (left). The infected cells positive for Hoechst (a DNA stain) are in blue and the uninfected red blood cells (uRBC), which do not contain DNA, are in red. d, Comparison of delta of geometric mean fluorescence intensity (MFI) between immature reticulocytes from cord blood and P. vivax rings from an infected patient, *p < 0.05, unpaired Student t-test. Figure 3. P. vivax Reticulocyte binding protein (PvRBP2a) is the CD98 ligand. a, Binding of reticulocytes or normocytes to HEK cells transfected with different gene or gene fragment from P. vivax. HEK cells expressing a fragment of Duffy binding protein II (DBP), known to contained an erythrocyte binding domain{Chitnis, 1994 #45}, different fragment of PvRPB2a (PvRBP2a _23-767 , PvRBP2ai ₆ o-ii35, PvRBP2a _448-1090 , PvRBP2a _448-1314 and PvRBP2a _765-1314 ), or a fragment of Merozoite Surface Protein 7 (MSP73-359) were assessed for binding. Specific binding numbers to transfected HEK were determined after subtraction of the number of reticulocytes or normocytes binding to non-transfected HEK cells (left). Screening was done twice with monoplicates (left panel). I n an additional experiment, binding of reticulocytes to HEK transfected with the PvRBP2a ₂ 3-767 fragment was done in octuplicate and was shown to follow a normal distribution as determine by the D'Agostino's K-squared test, and differed significantly from binding to non-transfected HEK cells, ****p < 0.0001, Student t-test (right panel), b, Representative images of binding of reticulocytes (CD71+) or normocytes (CD71-) loaded with the fluorescent dye CFSE to HEK cells expressing the Duffy binding protein I I (DBPII) fragment or PvRBP2a ₂ 3-767 in the presence or absence of anti-CD98 antibodies. The scale represents 50 μιη. c, Binding assays of reticulocytes to HEK cells expressing PvRBP2a ₂ 3- 767 in the presence or absence of isotype control or anti-CD98 antibodies (binding of normocytes to HEK cells expressing PvRBP2a ₂ 3-767 was specific since addition of antibodies against CD98 did not abrogate binding to normocytes (Fig. 6d)), * p < 0.05, unpaired t-test. d, Binding assays with reticulocytes in the presence of PvRBP2a ₂ 3-767 fragment and antibodies against RBP2a (1C3 and 3A11). Values are expressed as mean ± SD, ****p < 0.0001, ANOVA followed Dunnett post hoc test, e, Inhibition of P. vivax invasion using two different mouse monoclonal anti-PvRBP2a (1C3 and 3A11) antibodies and one anti-DARC antibody with four different isolates tested in parallel. The inhibition was normalized to invasion efficiencies obtained without antibodies. Values are expressed as mean ± SD, * p < 0.05; ** p < 0.01; ****p < 0.0001. Repeated measures one-way ANOVA test with Greenhouse correction followed by Tukey's post-test, f, CD98-PvRBP2a microcalorimetry measurements. The top panels show heat differences upon injection of PvRBP2a and lower panels show integrated heats of injection and the best fit (solid line) using Origin ^® . Fitting values are n= 0.980 sites, K _a = 2.88e5 M ¹ , 0H=-3411cal/mol and 0S= 13.5 cal/mol/deg and K _D = 1/K _a .

Figure 4. Proteomic analysis of CD71+ and CD71- erythrocytes, a, demonstrates the 14 exclusive unique peptides covering 27% of the protein 4F2 cell surface heavy chain antigen (SLC3A2) (heavy chain of the CD98 dimer) which are highlighted in yellow and the trypsin cleavage sites along with fixed modifications are highlighted in green, b, Example of a fragmentation pattern of the triply charged peptide ADLLLSTQPGREEGSPLELER (residues 503 to 535) from the same protein. The bar graph on the left describes mean log2 normalized intensities for iTRAQ reporter ions where iTRAQ-116 and iTRAQ-117 represent the membrane fractions of CD71- and CD71+ cells, c, Log2 fold changes for every peptide used for the aggregate quantification of the protein 4F2 where quant 11,3, and 7 represent biological replicates of CD71- fractions of the cord blood and quant 12, 4, and 8 represent biological replicates of CD71+ fractions of the cord blood.

Figure 5. Reticulocyte phenotyping. Trypsin resistance profile of different markers expressed at the surface of cord blood reticulocytes. The black and red histograms represent the level of expression before and after trypsin treatment respectively. The trypsin sensitive proteins are annotated in red and the resistant ones in black. The isotype antibody used as control is represented in grey.

Figure 6. Binding assay of erythrocytes to HEK cells expressing P. vivax genes, a, Schematic description of the development of the library and its use in the erythrocyte binding assay: (1) cloning of P. vivax genes in the pDisplay plasmid, (2) transfection of HEK cells, (3) expression of the protein containing MYC and HA tags at the su rface of HEK cells and (5) binding assay with erythrocytes, b, Schematic representation of full-length PvRBP2a and of its recombinant protein fragments used in this study (right). Signal peptide (SP), transmembrane helix (TM) and nucleotide-binding domain (green) are indicated, c, Binding assays of reticulocyte to HEK cells expressing PvRBP2b _{481- 1530} in the presence or absence of anti-CD98 antibodies. No significant differences were observed, d, Binding assays of normocyte to HEK cells expressing PvRBP2a ₂ 3-767 in the presence or absence of anti-CD98 antibodies. No significant differences were observed. Figure 7. Competitive binding assay to reticulocytes using soluble CD98 proteins.

Reticulocyte binding assay to PvRBP2a ₂₃ -767 transfected HEK cells was performed in the presence of 36.4 Eg/ml of BSA, or different concentrations (5, 10 or 25 of CD98 protein

dimers).

Figure 8. Antigen specificity of anti-PvRBP2a antibodies. Mouse 1C3 and 3A11 mAbs recognize specifically the PvRBP2a fragment, PvRBP2a ₂₃ -767, expressed by HEK cells when tested by flow cytometry. As positive control, rabbit anti-myc antibodies were used. Secondary anti-mouse IgG antibodies coupled with e660 or mouse anti-rabbit immunoglobulin coupled with Alexa 674 were used as negative control.

Figure 9. Structure of PvRPB2a. a, Ribbon representation of PvRPB2a. colors according to the sequence position, alpha helices are numbered according to their position, disulfide bonds are shown as balls (yellow) and involved cysteine are numbered. Protein surface is represented in light gray, b, Surface representation of PvRBP2a. Colors according to the electrostatic potential. Images were obtained using PvRBP2a structure (PDB 4Z8N) using PyMol (Schroedinger). Electrostatic map calculated with the approach APBS/PDB2PQR{Unni, 2011 #76}.

Figure 10. Structural model of potential interaction between human CD98hc homodimer and Plasmodium vivax RBP2a (PvRPB2a). The homodimer of PvRBP2a (158-455) and CD98/4F2hc (ED) are represented in green/yellow and cyan/purple respectively. On each dimer the negative charges (red) and positive charges (blue) are also represented.

Plasmodium vivax is a major species of parasitic protozoa that causes malaria in humans. More than a third of the world's population is exposed to vivax malaria particularly in Asia{Organization, 2015 #70}. This pathogen of global health importance has long been known to have a strict preference to invade immature red blood cells named reticulocytes{Hegner, 1938 #18;Mons, 1988 #51}. The molecular basis for this preference is unknown. Our perception of red blood cell invasion by P. vivax has been singularly defined by the demonstration forty years ago of a strict dependence on the presence of a red blood cell surface antigen, the Duffy antigen/receptor for chemokine (DARC) {Miller, 1976 #13}. This phenomenon prompted the search for and the eventual discovery of Duffy binding proteins in P. wVox{Wertheimer, 1989 #53;Fang, 1991 #52}. However, DARC is clearly not the receptor that defines the selectivity to reticulocytes since this protein is also present on RBCs of all ages{Malleret, 2013 #9}. Previous investigations had also led to the identification of reticulocyte-binding proteins (PvRBP) in P. vivax{Galinski, 1992 #19}, and subsequent genome sequencing subsequently revealed that these were encoded by a multigene family{Carlton, 2008 #58}, suggesting that alternate invasion pathways might also be a characteristic of P. vivax invasion. Here we identified CD98 as a reticulocyte-specific receptor and PvRBP2a as its cognate parasite ligand. We characterized the strict expression level of CD98 at the surface of immature reticulocytes (CD71+) and we found an unambiguous interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results demonstrate for the first time a host membrane protein that is directly associated to the P. vivax reticulocyte tropism. This significantly reinforces the potential of RBP2a as a vaccine candidate against vivax malaria. Knowledge on the role of CD98 might help accelerate the development of continuous in vitro culture protocols for P. vivax.

The present invention relates to a vaccine for therapeutic or prophylactic treatment of a pathological condition, the vaccine comprising a agent with specificity for a cell surface molecule of a reticulocyte or a reticulocyte binding protein expressed on the surface of the merozoite pathogen, wherein the agent blocks or reduces binding of the reticulocyte binding protein of the pathogen and the cell surface molecule.

I n particular, the invention specifically uses such antibodies for therapeutic uses. The antibodies preferably specifically bind to CD98 and/or CD147 and PvRBP2a of Plasmodium vivax, that is they bind to CD98/CD147/PvRBP2a but they do not bind, or bind at a lower affinity, to other molecules. The term CD98/CD147 as used herein refers to human CD98. (CD98 sequence: https://www-ncbi-nlm-nih-gov.ejproxy.a-star.edu.sg/gene/6520 and CD147 sequence: https://www-ncbi-nlm-nih-gov.ejproxy.a-star.edu.sg/gene/682) .

An antibody of the present invention may have some binding affinity for CD98/CD147 from other mammals, for example primate or murine CD98/CD147. The antibodies preferably bind to human CD98/CD147 when localised on the surface of a cell. An antibody of the invention has the ability to bind to CD98/CD147 in its native state and in particular to CD98/CD147 localised on the surface of a cell. Preferably, an antibody of the invention will bind specifically to CD98/CD147. That is, an antibody of the invention will preferably bind to CD98/CD147 with greater binding affinity than that at which it binds to another molecule.

By "localised on the surface of a cell" it is meant that CD98/CD147 is associated with the cell such that one or more region of CD98/CD147 is present on the outer face of the cell surface. For example, CD98/CD147 may be inserted into the cell plasma membrane (i.e. orientated as a transmembrane protein) with one or more regions presented on the extracellular surface. This may occur in the course of expression of CD98/CD147 by the cell. Thus, in one embodiment, "localised on the surface of a cell" may mean "expressed on the surface of a cell." Alternatively, CD98/CD147 may be outside the cell with covalent and/or ionic interactions localising it to a specific region or regions of the cell surface. In particular, in the present invention, CD98/CD147 is localised on the cell surface of a reticulocyte.

The terms "binding activity" and "binding affinity" are intended to refer to the tendency of an antibody molecule to bind or not to bind to a target. Binding affinity may be quantified by determining the dissociation constant (Kd) for an antibody and its target. Similarly, the specificity of binding of an antibody to its target may be defined in terms of the comparative dissociation constants (Kd) of the antibody for its target as compared to the dissociation constant with respect to the antibody and another, non-target molecule.

Typically, the Kd for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than Kd with respect to the other, non-target molecule such as unrelated material or accompanying material in the environment. More preferably, the Kd will be 50- fold less, even more preferably 100-fold less, and yet more preferably 200-fold less. The value of this dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984). For example, the Kd may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993).

A preferred method for the evaluation of binding affinity for CD98/CD147 is by ELISA. Preferably, an antibody of the invention has an affinity for CD98/CD147 (measured as an EC50) of 2500 ng/ml or lower, 1500 ng/ml or lower, 1000 ng/ml or lower, 600 ng/ml or lower, 350 ng/ml or lower, 50 ng/ml or lower, 40 ng/ml or lower, 30 ng/ml or lower, 20 ng/ml or lower, or 10 ng/ml or lower. The EC50 will typically be higher than 1 ng/ml and thus the EC50 may be between 1 ng/ml and any of the upper limits specified in the preceding sentence. Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, Western blots, RIAs, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of the antibody also can be assessed by standard assays known in the a rt, such as by surface plasmon resonance (e.g. Biacore™ system) analysis. This form of analysis is also described in the Examples. The affinity constant (KD) for binding to CD98/CD147 for an antibody of the invention is preferably in the range 1- 10 nM. The association rate (ka) is preferably in the range 0.4-3.4 x 10 ⁶ 1/M. The dissociation rate (kd) is preferably in the range the range 1-10 x 10 " ³ 1/s. These values may typically be determined by surface plamson resonance. A competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another, known ligand of that target, such as another antibody. The concentration at which 50% inhibition occurs is known as the Ki. Under ideal conditions, the Ki is equivalent to Kd. The Ki value will never be less than the Kd, so measurement of Ki can conveniently be substituted to provide an upper limit for Kd.

An antibody of the invention is preferably capable of binding to its target with an affi nity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than its affinity for binding to another non-target molecule. An antibody of the present invention blocks or reduces binding of the reticulocyte binding protein of the pathogen (PvRBP2a of P. Vivax) and the cell surface molecule (CD 98 and/or CD147). These characteristics may be assessed by any suitable method, such as the methods described herein including in the Examples.

An antibody of the invention may have the ability to cross-compete with another antibody of the invention for binding to CD98/CD147/PVRBP or another appropriate target as described herein. For example, an antibody of the invention may cross-compete with one or more of the antibodies described herein. Such cross-competing antibodies can be identified based on their ability to cross-compete with a known antibody of the invention in standard binding assays. For example, BIAcore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition. Such cross-competition may suggest that the two antibodies bind to the same or similar epitopes.

An antibody of the invention may therefore be identified by a method that comprises a binding assay which assesses whether or not a test antibody is able to cross-compete with a known antibody of the invention for a binding site on the target molecule. Methods for carrying out competitive binding assays are well known in the art. For example they may involve contacting together a known antibody of the invention and a target molecule under conditions under which the antibody can bind to the target molecule. The antibody/target complex may then be contacted with a test antibody and the extent to which the test antibody is able to displace the antibody of the invention from antibody/target complexes may be assessed. An alternative method may involve contacting a test antibody with a target molecule under conditions that allow for antibody binding, then adding an antibody of the invention that is capable of binding that target molecule and assessing the extent to which the antibody of the invention is able to displace the test antibody from antibody/target complexes.

The ability of a test antibody to inhibit the binding of an antibody of the invention to the target demonstrates that the test compound can compete with an antibody of the invention for binding to the target and thus that the test antibody binds to the same epitope or region on the CD98/CD147/PVRBP protein as the known antibody of the invention. A test antibody that is identified as cross-competing with a known antibody of the invention in such a method is also a potential antibody according to the present invention. The fact that the test antibody can bind CD98/CD147/PVRBP in the same region as a known antibody of the invention and cross-compete with the known antibody of the invention suggests that the test antibody may act as a ligand at the same binding site as the known antibody and that the test antibody may therefore mimic the action of the known antibody.

The known antibody of the invention may be an antibody as described herein, such as one of the CD98/CD147/PVRBP antibodies as described herein or any variant or fragment thereof as described herein that retains the ability to bind to CD98/CD147/PVRBP. An antibody of the invention may bind to the same epitope as one or more of the antibodies as described herein or any variant or fragment thereof as described herein that retains the ability to bind to CD98/CD147/PVRBP.

Specific binding may be assessed with reference to binding of the antibody to a molecule that is not the target. This comparison may be made by comparing the ability of an antibody to bind to the target and to another molecule. This comparison may be made as described above in an assessment of Kd or Ki. The other molecule used in such a comparison may be any molecule that is not the target molecule.

Preferably the other molecule is not identical to the target molecule. Preferably the target molecule is not a fragment of the target molecule.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. An antibody of the invention may be a monoclonal antibody or a polyclonal antibody. In one embodiment, an antibody of the invention is a monoclonal antibody. Polyclonal antibodies are antibodies that are derived from different B cell lines. A polyclonal antibody may comprise a mixture of different immunoglobulin molecules that are directed against a specific antigen. The polyclonal antibody may comprise a mixture of different immunoglobulin molecules that bind to one or more different epitopes within an antigen molecule. Polyclonal antibodies may be produced by routine methods such as immunisation with the antigen of interest. For example a mouse capable of expressing human antibody sequences may be immunised with human CD98/CD147/PVRBP. Blood may be subsequently removed and the Ig fraction purified.

Monoclonal antibodies are immunoglobulin molecules that are identical to each other and have a single binding specificity and affinity for a particular epitope. Monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example those disclosed in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Hurrell (CRC Press, 1982).

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, such as CD98/CD147/PVRBP. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a F(ab') ₂ fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

An antibody of the invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody of interest, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA seq uences.

An antibody of the invention may be a human antibody. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody. Antibodies of the invention can be tested for binding to the target protein by, for example, standard ELISA or Western blotting. An ELISA assay can also be used to screen for hybridomas that show positive reactivity with the target protein. The binding specificity of an antibody may also be determined by monitoring binding of the antibody to cells expressing the target protein, for example by flow cytometry.

The specificity of an antibody of the invention for target protein may be further studied by determining whether or not the antibody binds to other proteins. For example, where it is desired to produce an antibody that specifically binds CD98/CD147/PVRBP or a particular part, e.g. epitope, of CD98/CD147/PVRBP, the specificity of the antibody may be assessed by determining whether or not the antibody also binds to other molecules or modified forms of CD98/CD147/PVRBP that lack the part of interest. Once a suitable antibody has been identified and selected, the amino acid sequence of the antibody may be identified by methods known in the art. The genes encoding the antibody can be cloned using degenerate primers. The antibody may be recombinantly produced by routine methods. A "polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term "polypeptide" thus includes short peptide sequences and also longer polypeptides and proteins. As used herein, the term "amino acid" refers to either natural and/or unnatural or synthetic ami no acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

The present inventors have identified antibodies as described in the examples. The present invention encompasses these antibodies and variants and fragments thereof which retain one or more activities of these antibodies. The activities of these antibodies include the ability to bind to CD98/CD147/PVRBP, and the ability to bind to human CD98/CD147/PVRBP when expressed on the surface of a cell. A suitable fragment or variant of this antibody will retain the ability to bind to CD98/CD147/PVRBP. It will preferably retain the ability to specifically bind to CD98/CD147/PVRBP. It will preferably retain the ability to specifically bind to the same epitope or region of the CD98/CD147/PVRBP molecule as the antibody from which it is derived. It will also retain one or more additional functions of the antibody from which it is derived, such as the ability to:

Bind to any one of CD 98 or CD 147, or in combi nation thereof, CD 98 and CD 147 be cell surface molecules expressed in a reticulocyte;

Bind to PvRBP of a merozoite, in particular that of a P. vivax;

The binding blocks or reduces binging of the PvRBP to the CD98 and/or CD147.

Polypeptide or antibody "fragments" according to the invention may be made by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and/or C terminal in this way. Fragments may also be generated by one or more internal deletions.

An antibody of the invention may be, or may comprise, a fragment of the antibodies or a variant thereof. The antibody of the invention may be or may comprise an antigen binding portion of these antibodies or a variant thereof as discussed further above. For example, the antibody of the invention may be a Fab fragment of one of these antibodies or a variant thereof or may be a single chain antibody derived from one of these antibodies or a variant thereof.

The present invention includes derivatives or variants to the amino acid sequences of the antibodies. Preferred "derivatives" or "variants" include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected. Derivatives and variants as described above may be prepared during synthesis of the antibody or by post- production modification, or when the antibody is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.

Preferably variant antibodies according to the invention have an amino acid sequence which has more than 60%, or more than 70%, e.g. 75 or 80%>, preferably more than 85%, e.g. more than 90 or 95% amino acid identity to the VL or VH domain, or a fragment thereof, of an antibody disclosed herein. This level of amino acid identity may be seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on,

Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized. The invention also relates to polynucleotides that encode the antibodies of the invention. Thus, a polynucleotide of the invention may encode any antibody as described herein. The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the invention may be provided in isolated or purified form. A nucleic acid sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

A suitable polynucleotide sequence may alternatively be a variant of one of these specific polynucleotide sequences. For example, a variant may be a substitution, deletion or addition variant of any of the above nucleic acid sequences. A variant polynucleotide may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and/or deletions from the sequences given in the sequence listing.

Suitable variants may be at least 70% homologous to a polynucleotide of any one of nucleic acid sequences of the present invention, preferably at least 80 or 90% and more preferably at least 95%, 97% or 99% homologous thereto. Preferably homology and identity at these levels is present at least with respect to the coding regions of the polynucleotides. Methods of measuring homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of nucleic acid identity. Such homology may exist over a region of at least 15, preferably at least 30, for instance at least 40, 60, 100, 200 or more contiguous nucleotides. Such homology may exist over the entire length of the unmodified polynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (e.g. used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can also be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. The homologue may differ from a sequence in the relevant polynucleotide by less than 3, 5, 10, 15, 20 or more mutations (each of which may be a substitution, deletion or insertion). These mutations may be measured over a region of at least 30, for instance at least 40, 60 or 100 or more contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specific sequences given in the sequence listing by virtue of the redundancy in the genetic code. The DNA code has 4 primary nucleic acid residues (A, T, C and G) and uses these to "spell" three letter codons which represent the amino acids the proteins encoded in an organism's genes. The linear sequence of codons along the DNA molecule is translated into the linear sequence of amino acids in the protein(s) encoded by those genes. The code is highly degenerate, with 61 codons coding for the 20 natural amino acids and 3 codons representing "stop" signals. Thus, most amino acids are coded for by more than one codon - in fact several are coded for by four or more different codons. A variant polynucleotide of the invention may therefore encode the same polypeptide sequence as another polynucleotide of the invention, but may have a different nucleic acid sequence due to the use of different codons to encode the same amino acids. Polynucleotide "fragments" according to the invention may be made by truncation, e.g. by removal of one or more nucleotides from one or both ends of a polynucleotide. Up to 10, up to 20, up to 30, up to 40, up to 50, up to 75, up to 100, up to 200 or more amino acids may be removed from the 3' and/or 5' end of the polynucleotide in this way. Fragments may also be generated by one or more internal deletions. Preferably such fragments are between 30 and 300 residues in length, e.g. 30 to 300, 30 to 200, 30 to 100, 100 to 200 or 200 to 300 residues. Alternatively, fragments of the invention may be longer sequences, for example comprising at least 50%, at least 60%, at least 70%>, at least 80% or at least 90% of a full length polynucleotide of the invention. An antibody of the invention may thus be produced from or delivered in the form of a polynucleotide which encodes, and is capable of expressing, it. Where the antibody comprises two or more chains, a polynucleotide of the invention may encode one or more antibody chains. For example, a polynucleotide of the invention may encode an antibody light chain, an antibody heavy chain or both. Two polynucleotides may be provided, one of which encodes an antibody light chain and the other of which encodes the corresponding antibody heavy chain. Such a polynucleotide or pair of polynucleotides may be expressed together such that an antibody of the invention is generated. Polynucleotides of the invention can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

The nucleic acid molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the antibody of the invention in vivo. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

The present invention thus includes expression vectors that comprise such polynucleotide sequences. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a peptide of the invention.

Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al.

The invention also includes cells that have been modified to express an antibody of the invention. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors or expression cassettes encoding for an antibody of the invention include mammalian HEK293T, CHO, HeLa, NSO and COS cells. Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide.

Such cell lines of the invention may be cultured using routine methods to produce an antibody of the invention, or may be used therapeutically or prophylactically to deliver antibodies of the invention to a subject. Alternatively, polynucleotides, expression cassettes or vectors of the invention may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject. In another aspect, the present invention provides compositions and formulations comprising molecules of the invention, such as the antibodies, polynucleotides, vectors and cells described herein. For example, the invention provides a pharmaceutical composition comprising one or more molecules of the invention, such as one or more antibodies of the invention, formulated together with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for parenteral, e.g. intravenous, intramuscular or subcutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the antibody may be coated in a material to protect the antibody from the action of acids and other natural conditions that may inactivate or denature the antibody. Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

An antibody or fragment of the present invention, or a composition comprising said antibody or fragment, may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies or compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection. Alternatively, an antibody or composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

Sterile injectable solutions can be prepared by incorporating the active agent (e.g. antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active agent plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Pharmaceutical compositions of the invention may comprise additional active ingredients as well as an antibody of the invention. As mentioned above, compositions of the invention may comprise one or more antibodies of the invention. They may also comprise additional therapeutic or prophylactic agents.

Local administration is preferred, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intra cavity infusion, intravesicle administration, and inhalation. However, the antibody or composition may also be administered systemically.

A suitable dosage of an antibody of the invention may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular antibody employed, the route of administration, the time of administration, the rate of excretion of the antibody, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A suitable dose of an antibody of the invention may be, for example, in the range of from about O.lg/kg to about lOOmg/kg body weight of the patient to be treated. For example, a suitable dosage may be from about 10 g/kg to about lOmg/kg body weight per day or from about 10 g/kg to about 5 mg/kg body weight per day. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Antibodies may be administered in a single dose or in multiple doses. The multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, antibodies can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the antibody in the patient and the duration of treatment that is desired. The dosage and frequency of administration can also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage may be administered, for example until the patient shows partial or complete amelioration of symptoms of disease.

Combined administration of two or more agents may be achieved in a number of different ways. In one embodiment, the antibody and the other agent may be administered together in a single composition. In another embodiment, the antibody and the other agent may be administered in separate compositions as part of a combined therapy. For example, the antibody may be administered before, after or concurrently with the other agent. The antibody of the invention may be administered in combination with or sequential ly to any other types of agents that may be suitable for treating or preventing malaria. Also within the scope of the present invention are kits comprising antibodies or other compositions of the invention and instructions for use. The kit may further contain one ore more additional reagents, such as an additional therapeutic or prophylactic agent as discussed above.

The antibodies in accordance with the present invention maybe used in therapy. In therapeutic applications, antibodies or compositions are administered to a subject already suffering from a disorder or condition, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom- free periods. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for a given purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. As used herein, the term "subject" includes any human.

In particular, antibodies to CD98/CD147/PvRBP may be useful in the treatment or prevention of malaria. Accordingly, the invention provides an antibody of the invention, or fragment thereof, for use in the treatment or prevention of malaria. The invention also provides a method of treating or preventing malaria comprising administering to an individual an antibody of the invention, or a fragment thereof. The invention also provides an antibody of the invention, or fragment thereof, for use in the manufacture of a medicament for the treatment or prevention of malaria.

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Example 1

Methods

CD98 cloning and expression in SF9 cells The coding sequence of the soluble domain of CD98 (W117-A529) was cloned using the primers A 5'-TATCCACCTTTACTGTTAGGCCGCGTAGG-3' and B 5'- TACTTCCAATCCATGTGGTGGCACACGGGC-3' and the amplified fragment was purified before insertion into the pFasBac-LIC-BseR1 (Addgene, USA) plasmid using the Gibson Assembly ^® Cloning Kit (NEB, USA). E. coli ToplO competent cells were transformed with the ligated plasmid (pFasBac-CD98) and spread on LB plates supplemented with ampicillin. E. coli DHlOBac competent cells were transformed with the purified pFastbac-CD98 plasmid. Extraction of the bacmid and virus packaging was done according to Bac-to-Bac ^® (Invitrogen, USA). For expression Sf-9 cells were infected at a MOI of 1 for 72 hours at 27°C under agitation. Cells were harvested at 3000g for 15min and cell pellet is stored at -20^C.

CD98 purification and dimerization

Cells were resuspended in 20mM Tris pH 7.5, 500mM NaCI, lOmM Imidazole and lysed by sonication. After centrifugation at 20,000 g for 45 minutes, the filtered supernatant is injected on a nickel affinity column (HiTrap 1 ml, GE Healthcare, USA). After a washing step of 6% of 20 mM Tris-HCI pH 7.5, 500 mM NaCI and 500 mM imidazole (Buffer B), the protein is eluted with 100% of Buffer B and injected on a gel filtration Superdex 75 26/60 (GE Hea lthcare, USA) equilibrated in PBS. The protein fractions are pooled, concentrated to 19 mg per ml and stored at -80°C. Protein at a concentration of 333 μΜ was supplemented with 0.02% of glutaraldehyde for 2 hours at 4°C. After quenching the reaction with addition of an equal volume of 1M Tris pH 7.5 the mixture is injected on a S200 10/300 gel filtration column. The fractions containing the dimer of CD98 were assessed by SDS-PAGE pooled and concentrated (e.g. to 8mg/ml) prior to storage at -80°C. Reticulocyte enrichment

Enrichment of the CD71+ reticulocytes was performed using the MACS system (Miltenyi, Singapore). One to two ml of blood at 50% haematocrit in PBS were passed through an LS column, the purity of the positive and negative fractions was monitored by flow cytometry using Thiazole orange, which stains RNA (the only nucleic acid species present in reticulocytes). Purity of CD71+ cell was > 80%.

Sample preparation and quantitative mass spectrometry Post MACs enrichment, samples were prepared and analyzed on a mass spectrometer as described in Chu et al. {Chu, 2018 #79}. Briefly, both reticulocytes and normocytes upon isolation were lysed using 0.02% saponin in phosphate buffered saline (PBS) with protease inhibitors. Following this, the membrane and soluble fractions were separated by differential centrifugation and precipitated with ice-cold acetone. For the membrane fraction, the sample was reduced using 5 mM Tris 2-carboxy-ethyl phosphine hydrochloride (TCEP) for 3 h at 30°C and then alkylated in the dark using 55 mM iodoacetamide (IAA) for lh at room temperature followed by in-solution trypsin digestion at 37°C for 16 h. These samples were desalted and then labelled with isobaric tags. We labelled CD71- membrane sample with 116 and CD71+ membrane sample with 117 isobaric tags respectively. These samples were further fractionated using an X-Bridge C18 column (Waters; 4.6 x 250 mm, 5-μιη, 300A). Fractions obtained from this LC were analysed using an Ultimate 3000 RSLC nano-HPLC system (Dionex, Amsterdam, NL) coupled to a QExactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific Inc., Bremen, Germany).

Proteomic Analysis Database searching

All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.4.1). Mascot was set up to search the con_uni_human_20150401_20150623 database (unknown version, 180822 entries) assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine and iTRAQ4plex of lysine and the n-terminus were specified in Mascot as fixed modifications. Deamidated of asparagine and glutamine, oxidation of methionine and iTRAQ4plex of tyrosine were specified in Mascot as variable modifications.

Criteria for protein identification

Scaffold (version Scaffold_4.4.3, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm{Keller, 2002 #47} with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm{Nesvizhskii, 2003 #48}.

Flow cytometry phenotyping of cord blood samples and HEK cells expressing RBP2a23 767 Three Percoll-enriched reticulocyte samples from human cord blood were treated or not with Trypsin (Sigma) 1 mg/ml{Russell, 2011 #6}. Five hundred nanoliters of packed cord blood (47.4% ± 14.5% CD71+, n= 3) were stained with different mouse anti-human markers antibodies followed by a F(ab')2 anti-mouse Immunoglobulin antibody coupled with e660 (eBioscience){Malleret, 2013 #9}. The antibodies used in this study are described in Table 3. Fifty thousand events were acquired on the LSR II flow cytometer (BD Biosciences) for each samples and the data were analysed using FlowJo software (Three Star).

Table 3. List of the antibodies used for the reticulocyte phenotyping by flow cytometry.

HEK293 cells expressing RBP2a ₂₃ -767 were stained with two mouse anti-PvRBP2a mAbs (1C3 and 3A11) at 25 μg/ ml followed by a (Fab')2 anti-mouse Immunoglobulin antibody coupled with e660 (eBioscience) or with anti-myc (Miltenyi Biotec) antibodies followed by a (Fab')2 anti-rabbit Immunoglobulin antibody coupled with Alexa647 (Invitrogen).

Profiling CD98 expression on red blood cells by Western Blotting Immature reticulocytes (CD71+) were purified from cord blood using magnetic beads conjugated to anti-CD71 antibody using the MACS purification system (Miltenyi Biotec). Purified CD71+ cells were allowed to mature ex vivo in RPMI medium supplemented with 5% Albumax at 37°C. Aliquots of 3 μί packed cells were collected at different time points, 0, 20 and 40 hours. Cells were incubated with 40 volumes of 5 mM Sodium phosphate, 10 minutes on ice with occasional gentle mixing, inducing membrane breaks and release of the cell cytoplasmic contents. The lysate was then centrifuged at 16,000xg/ lOmin/ 4°C to sediment the red blood cell ghosts that were again washed twice in ice-cold 5mM Sodium phosphate. Purified ghost membranes were next treated with 0.25% Triton X100/PBS. Membrane extract corresponding to 10 μg total protein (estimated using the BCA assay) obtained at different time point during reticulocyte maturation, together with a normocyte membrane sample prepared alongside, were extracted in reduced Laemmli buffer, incubated at 37°C for 10 min, then resolved on a 10% SDS-PAGE and transferred to PVDF membrane. Samples were probed using anti-CD98 antibody (E-5: sc-376815, Santa Cruz Biotechnology) at a 1:500 dilution in PBS followed by a mouse secondary antibody conjugated to HRP and visualized through ECL detection (Thermo Fisher Scientific). Antibody against Band-3 anion exchanger was used for loading control.

Immunofluorescence assay

Red blood cells (CD71+ and CD71-) were fixed in 4% paraformaldehyde (Sigma Aldrich) and 0.0075% glutaraldehyde (Sigma Aldrich) in PBS for 30 min at RT. Subsequently, cells were washed in IX PBS, quenched in 0.125 M Glycine/PBS for 15 min RT, washed again and permeabilized in 0.1% Triton X-100/PBS for 10 min at RT. After blocking for lh in blocking solution (3% BSA (w/v) in PBS), samples were incubated with rabbit anti-CD98 antibody (EPR3548 clone, Abeam ), or rabbit anti-Dematin (Band 4.9) antibody (Thermo Fisher Scientific) both at a 1:100 dilution, for lh, followed by Alexa Fluor 546-conjugated anti-rabbit secondary antibodies (1:200, Invitrogen). Images were acquired with a Zeiss LSM 700 microscope (Carl Zeiss) using 63x/1.4 Oil DIC objective lens. Images were processed using LSM software Zen 2009 (Carl Zeiss).

P. vivax parasites collection and cryo-preservation P. wVox-infected blood samples were collected from malaria patients receiving treatment at clinics run by the Shoklo Malaria Research Unit located at the North Western border of Thailand. The project was explained to all the patients before they provided informed consent prior to collection of blood by venepuncture. Five ml of whole blood were collected in lithium heparin collection tubes. These samples were cryopreserved in Glycerolyte 57 (Baxter) after leukocyte depletion using cellulose columns (Sigma cat #C6288){Sriprawat, 2009 #1}. After thawing, the parasites present in the packed cells (1.5 ml per isolate) were cultured to the schizont stage in 12 ml of McCoy 5A medium supplemented with 2.4 g/l D-glucose, and 20% heat-inactivated human AB serum, in 5% 0 ₂ at 37.5°C{Malleret, 2015 #3}.

P. vivax invasion assay

Invasion assays using freshly isolated P. vivax field samples were performed as previously described{Russell, 2011 #6}. In some experiments, anti-human CD147 (BD Bioscience, clone HIM6), CD240DCE (AbD Serotec, clone BRIC 69), polyclonal CD98 (TransGenic Inc.), monoclonal CD98 (BD Bioscience, clone UM7F8), anti-DARC (Fy6){Wasniowska, 2002 #75} F(ab')2 antibodies were added. The antibodies were prepared using the Pierce Fab Micro Preparation Kit and Resin Kit (ThermoScientific Pierce) as described previously{Lee, 2014 #5}. We chose to use F(ab')2 antibodies to avoid steric hindrance or agglutination of erythrocytes. All antibodies were diluted in PBS and were azide- and glycerol-free. Concentrated mature schizont preparations were mixed with the CD71+ reticulocyte enriched fraction pre- incubated for 10 minutes with each antibody (final concentration of 25 μg/ml){Russell, 2011 #6}. After 24 hours of culture at 37°C, blood thin smears were made and stained with Giemsa (Sigma-Aldrich). One to five thousand erythrocytes were counted per slide and the lowest parasitemia was 0.3% for the negative control (no mAb) condition used to nornalize the invasion assays.

Invasion assay was performed with three different Plasmodium falciparum lines (deriving from two field isolates) and assessed by flow cytometry as described (30). Briefly, CD71- and CD71+ erythrocytes were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) and incubated with or without anti-human CD147 or CD98 antibody at a final concentration of 25 Eg/ml. The proportion of invasion was defined by double positive events for CFSE and Hoechst (Sigma). Cell acquisition was performed using a FACS LSR II (Becton Dickinson).

The anti-human CD147 (BD Bioscience, clone HIM6) aborgated the invasion of 3 P. falciparum samples (one field isolate and 2 clones) with an efficiency of 87.6% ±2.4% and 86.0% ±5.4% in CD71- and CD17+ reticulocytes respectively.

Plasmodium vivax antigen library

The P. vivax antigen library was designed and established as previously described for a P. falciparum library{Peng, 2016 #64}. Briefly, 28 gene fragments corresponding to 8 different for P. vivax genes were amplified using PCR and either P. vivax genomic DNA or RNA as template, and cloned into the pDisplay vector (Invitrogen) (Table 2). Cloning of PvRBP2a,b,c into expression vector codon-optimized DNA of PvRBP2a,b,c was purchased from Genscript The resultant plasmids were then transfected into HEK293 cells using lipofectamine 2000 (Invitrogen) for surface expression of the P. vivax antigens. The P. vivax antigens were tagged with a hemagglutinin (HA) short sequence at the N-terminal and a myc short sequence at the C-terminal part of the protein. These tags allow the assessment of transfection efficacy and expression level of the P. vivax antigen using an anti-HA (Sigma) or anti-myc (Miltenyi Biotec) antibodies. Only HEK cells expressing P. vivax genes with transfection efficiency >10% (in some cases, more than 15%) were used.

Erythrocyte binding assay using the P. vivax antigen library

Adherent HEK293 cells transfected with P. vivax antigens were incubated with CD71+ or CD71- erythrocytes loaded with carboxy-fluorescein diacetate succinimidyl ester (CFSE) for 1 h at 37°C under agitation. After five washing steps using PBS, cell binding was recorded using confocal microscope. One thousand HEK cells were counted at 40x magnification and each CFSE stained erythrocyte in contact with HEK cells were counted as a binding cell using the software IMARIS software (Bitplane, South Windsor, CT). For each experiment the number of erythrocytes binding untransfected HEK cells were subtracted.

Reticulocyte binding inhibition and the competition assay were conducted by incubating the PvRBP2a-transfected HEK cells before adding reticulocytes with an isotype control anti- TNP (BD Bioscience, clone Alll-3) and two different mouse anti-PvRBP2a antibodies (clonelC3 and 3A11, described below) or mouse anti-CD98 mAb (BD Bioscience, clone UM7F8) and mouse anti-human CD147 (BD Bioscience, clone HIM6) at 25 μg/mΙ of PBS, or with soluble CD98 dimer protein (at 3 different concentration 5, 10 or 25 μg/mΙ of PBS). BSA (Sigma) at a concentration of 36.4 μg/mΙ (Sigma) (equimolar concentration of 25 μg/mΙ of CD98 dimer) was used as control.

Anti-PvRBP2a mouse monoclonal antibodies production

Anti-PvRBP2a mAbs were produced at the Monoclonal Antibody Facility at the Walter and Eliza Hall Institute. BALB/c and C57BL/6 mice received three immunizations of recombinant PvRBP2ai6o-ii35 purified as described{Gruszczyk, 2016 #37}. At day 0, complete Freund adjuvant was mixed with the antigen into an emulsion and injected intraperitoneally. At days 30 and 60, the antigen was mixed with incomplete Freund adjuvant, and the resulting emulsion injected intraperitoneally. Serums were taken at day 72 and ELISA were next performed using the same recombinant protein. The mouse with the best response received a final injection of antigen in saline, and splenocytes were harvested three days later. Spleen cells were fused with SP2/0 myeloma cells to generate hybridomas. Hybridomas were grown in hypoxanthine-aminopterine thymidine growth medium. ELISA was used to select hybridomas producing antibodies specific to PvRBP2a. Hybridomas were cloned by limiting dilution in multi-well plates aiming for one cell or less per well. The sub-cloning supernatants were screened by ELISA. Two or more rounds of limiting dilution cloning were generally required before the hybridomas were deemed monoclonal. The antibodies were purified from monoclonal hybridoma supernatants with protein A sepharose. Isothermal titration microcalorimetry measurements

Isothermal titration microcalorimetry experiments were performed with a PEAQ-ITC isothermal titration calorimeter from Malvern. The experiments were carried out at 25°C. Protein concentration in the microcalorimeter cell (0.2 mL) is 35 μΜ of monomeric CD98. Nineteen injections of 2 μΙ of pvRbp2a at a concentration of 350 μΜ were performed at intervals of 120 s while stirring at 600 rpm. The experimental data was fitted to theoretical titration curves with software integrated to ORIGIN ^® . This software uses the relationship between the heat generated by each injection and ΔΗ (enthalpy change in Kcal.Mol ^-1 ), K _a (the association binding constant in M-l), n (the number of binding sites), total protein concentration and free and total ligand concentrations {Wiseman, 1989 #72}.

Structural Modeling

We used YASARA software{Krieger, 2009 #73} to dock the crystal structure of human CD98hc (PDB:2DH3){Fort, 2007 #15} to the crystal structure of the erythrocyte-binding domain from Plasmodium vivax reticulocyte-binding protein 2a PvRBP2a (PDB:4Z8N{Gruszczyk, 2016 #37} using a semi-automated approach. Since it is possible but not yet confirmed that interaction could happen as dimers, we searched for an interaction model that is compatible with both monomer as well as dimer interaction modes. Starting with the orientation of the CD98 homodimer membrane interacting side facing downwards we placed the RBP2 on top while optimizing charge complementarity at the negatively charged surface region that may function as a putative erythrocyte-binding site{Gruszczyk, 2016 #37}. Following this targeted docking step for a starting conformation based on constraints suggested from literature, we ran short simulated annealing Molecular Dynamics simulations using the Energy Minimization function in YASARA to automatically optimize the predicted interface {Krieger, 2009 #73}.

Statistical analysis

A non-parametric Kruskal Wallis test followed by Dunn's post -test were used (Fig. 2a) for the invasion inhibition assays, as the percentage of infected reticulocytes are not normally distributed {Russell, 2011 #6}. For the difference of geometric mean (log normalized data) fluorescence intensity (MFI), an unpaired Student t-test was used (Fig. 2d) A D'Agostino's K- squared test was used to determine the normal distribution of reticulocyte binding assay (Fig. 3a) and an unpaired Student t-test for the comparison between transfected and non- transfected HEK cells assuming unequal variances of the data (Fig. 3a, c). A one way ANOVA with Geisser-Greenhouse correction (variance not equal) followed by Tukey post-test was used for the comparison of reticulocyte binding to HEK transfected cells with isotype control or anti-PvRBP2a antibodies (Fig. 3d). For All statistical analysis used Graph Pad Prism (7.0). Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.