Patent Publication Number: US-2022233667-A1

Title: Treatment and prevention of malaria

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the National Stage of International Application No. PCT/GB2019/052885, filed Oct. 10, 2019, which claims priority to GB 1816542.3, filed Oct. 10, 2018 and GB 1907609.0, filed May 29, 2019, which are entirely incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to antigens, antibodies and vaccines for treatment or prevention of malaria. 
     BACKGROUND OF THE INVENTION 
     Malaria places the gravest public-health burden of all parasitic diseases, leading to ˜215 million human clinical cases and ˜440,000 deaths annually, with the majority of deaths in children. The infection of red blood cells (RBCs) by the blood-stage form of the  Plasmodium  parasite is responsible for the clinical manifestations of malaria. Examples of  Plasmodium  parasite include the species  P. falciparum, P. vivax, P. ovale  and  P. malariae . The most virulent parasite species,  P. falciparum , is endemic in large parts of sub-Saharan Africa and Latin America. It causes the majority of malaria deaths. It can infect RBCs of all ages and is not limited to immature RBCs.  P. falciparum , is therefore of particular interest and is a major target for vaccine development, as it would be highly desirable to develop a vaccine. 
     The most advanced malarial vaccine aiming for licensure is RTS,S/AS01 (Mosquirix™), which is based on the RTS,S protein. The RTS,S protein acts by blocking infection of  P. falciparum  in the liver. However, this vaccine has achieved only partial efficacy (˜30-50% in phase II/III clinical trials). There is therefore an urgent need for a vaccine which can protect against the disease-causing blood-stage  Plasmodium  parasite, potentially by emulating natural immunity or by other immune mechanisms. 
     Previous studies have investigated the potential for antigens to induce antibodies which are effective against blood-stage malaria parasites in vitro, using the standard growth inhibitory activity (GIA) assay. One such antigen is apical membrane antigen 1 (PfAMA1). 
     GIA assay investigations into other protein families involved in blood-stage  Plasmodium  parasite invasion of RBCs have found them to be ineffective or less effective than PfAMA1. 
     PfAMA1 has therefore been a major focus of research on countering blood-stage malarial parasites, with ongoing clinical trials. However, antibodies against PfAMA1 appear only to be effective at an extremely high concentration. In addition, PfAMA1 induces strain-specific antibodies which are not effective against genetically diverse strains of the  Plasmodium  parasite (A. L. Goodman, S. J. Draper,  Ann. Trop. Med. Parasitol.  104, 189 (2010)). In addition, vaccine development has been hampered by the requirement for potentially reactogenic chemical adjuvants in addition to the antigen to induce sufficient antibody responses in human subjects. 
     Research has also been ongoing to identify other candidate malarial antigens for vaccines. In particular, the present inventors have previously identified Reticulocyte-binding protein Homologue 5 (PfRH5) as a potential antigen candidate for malarial vaccines (WO 2012/114125). 
     The Reticulocyte binding Homologue (PfRH) family comprises six members (PfRH1, PfRH2a, PfRH2b, PfRH3, PfRH4 and PfRH5), each of which is involved in the binding of the  Plasmodium  parasite to RBCs, with the possible exception of PfRH3 which may be a non-expressed pseudogene. The PfRH family has been identified as adhesins on the surface of the merozoite form of the  Plasmodium  parasite, which bind to receptors on the surface of the erythrocyte and hence permit invasion of RBCs by the parasite in its blood-stage. The PfRH5 antigen has an approximate molecular weight of 63 KDa. In vitro cleaved fragments of approximately 45 KDa and 28 KDa have been reported. 
     The present inventors have previously demonstrated that PfRH5 induces antibodies which are highly effective in the GIA assay against the blood-stage  Plasmodium falciparum  parasite and which neutralise parasites more effectively than PfAMA1 and remain effective at lower concentrations of immunoglobulin. In addition, PfRH5 induces antibodies which are effective against genetically diverse strains of the  Plasmodium  parasite. Therefore, PfRH5 is a promising candidate antigen for a malarial vaccine. 
     Earlier work by the present inventors has improved upon the full-length PfRH5 as a vaccine candidate by the development of rationally designed PfRH5 fragments, which contain regions or amino acid residues from within PfRH5 that give rise to protective antibodies, whilst excluding other regions of the full-length PfRH5 sequence which may be associated with unwanted side effects. In addition, rationally designed PfRH5 antigens have been developed with improved expression profiles and thermal stability without compromising immunological efficacy. 
     However, there is an ongoing need for the development of antibodies against PfRH5 with improved activity, and to develop rationally designed antigens capable of stimulating the production of such antibodies. In particular, there is a need for improved antibodies that are effective at low concentrations, and that can be used in concert to bolster the immune defence without unwanted interference or competition between said antibodies, and for improved antigens that will induce such antibodies. At the same time, it is necessary for said antibodies to be effective against genetically diverse strains of the  Plasmodium  parasite, with a view to future elimination campaigns, where they would be used alongside conventional antimalarial drugs. 
     The present invention addresses one or more of the above needs by providing combinations of non-neutralising antibodies for PfRH5 and neutralising antibodies for PfRH5 and other merozoite antigens, which together provide a synergistic effect. The invention also provides antigens which induce such antibodies, vectors encoding the antibodies and antigens, and antibody-like molecules including aptamers and peptides raised against the same antigen, together with the use thereof (either alone or in combination) in the prevention or treatment of malaria. 
     SUMMARY OF THE INVENTION 
     The present inventors have for the first time now shown that a class of antibodies which bind to PfRH5 and which in and of themselves have no neutralising activity in vitro but which significantly reduce the speed of red blood cell invasion by the merozoite form of the malarial parasite. Therefore, these non-neutralising antibodies can, when combined, potentiate all neutralising antibodies directed to PfRH5, as well as synergising with antibodies against other malaria invasion proteins. In particular, the present inventors have shown that non-neutralising antibodies which bind to a region near the N-terminus of the α-helical core are capable of potentiating the growth inhibitory activity of neutralising anti-PfRH5 antibodies and neutralising antibodies which bind to PfAMA1, PfCyRPA and PfRipr. 
     Accordingly, the present invention provides a non-neutralising antibody, or binding fragment thereof, that specifically binds an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO:1). Said non-neutralising antibody, or binding fragment thereof, of claim  1 , may comprise: (a) a heavy chain with a CDR1 sequence of SEQ ID NO: 11; a CDR2 sequence of SEQ ID NO: 12 and a CDR3 sequence of SEQ ID NO: 13, and a light chain with a CDR1 sequence of SEQ ID NO: 20; a CDR2 sequence of SEQ ID NO: 21 and a CDR3 sequence of SEQ ID NO: 22; (b) a heavy chain with a CDR1 sequence of SEQ ID NO: 14; a CDR2 sequence of SEQ ID NO: 14 and a CDR3 sequence of SEQ ID NO: 16, and a light chain with a CDR1 sequence of SEQ ID NO: 23; a CDR2 sequence of SEQ ID NO: 24 and a CDR3 sequence of SEQ ID NO: 25; or (c) a heavy chain with a CDR1 sequence of SEQ ID NO: 17; a CDR2 sequence of SEQ ID NO: 18 and a CDR3 sequence of SEQ ID NO: 19, and a light chain with a CDR1 sequence of SEQ ID NO: 26; a CDR2 sequence of SEQ ID NO: 21 and a CDR3 sequence of SEQ ID NO: 27. Preferably, said non-neutralising antibody, or binding fragment thereof comprises: (a) a heavy chain variable region sequence of SEQ ID NO: 47 and a light chain variable region sequence of SEQ ID NO: 48; (b) a heavy chain variable region sequence of SEQ ID NO: 49 and a light chain variable region sequence of SEQ ID NO: 50; or (c) a heavy chain variable region sequence of SEQ ID NO: 51 and a light chain variable region sequence of SEQ ID NO: 52. Said non-neutralising antibody, or binding fragment thereof may be: (a) a monoclonal or polyclonal antibody; or (b) an Fab, F(ab′)2, Fv, scFv, Fd or dAb. 
     The invention further provides a composition comprising: (a) one or more non-neutralising antibody, or binding fragment thereof as defined herein; and (b) one or more neutralising antibody, or binding fragment thereof, that specifically binds a  Plasmodium  merozoite antigen. The  Plasmodium  merozoite antigen may be selected from: (a) PfRH5; (b) a non-PfRH5 antigen within the PfRH5 invasion complex; and/or (c) a target closely linked to the PfRH5-basigin interaction. Typically said  Plasmodium  merozoite antigen is selected from PfRH5, PfRipr, PfCyRPA, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181 and PfAARP. The  Plasmodium  merozoite antigen may be PfRH5 and the one or more neutralising antibody, or binding fragment thereof, specifically binds an epitope within SEQ ID NO: 3 (RH5ΔN) or SEQ ID NO: 7 (RH5ΔNL); and preferably the one or more neutralising antibody, or binding fragment thereof, specifically binds an epitope corresponding to: (a) residues Lys196 to Ser197; Asn347 to Asn352; Arg357 to His365; and Lys443 to Arg448 of PfRH5 (SEQ ID NO: 1); (b) residues Gly201 to Lys219; and Lys327 to Tyr335 of PfRH5 (SEQ ID NO: 1); (c) residues Gly201 to Lys219 and Lys327 to Gln342 of PfRH5 (SEQ ID NO: 1); or (d) residues Lys196, Ser197, Tyr346 to Asn354 and Lys443 to Lys452PfRH5 (SEQ ID NO: 1); preferably the epitope of (a) or (b). In some preferred embodiments, the one or more neutralising antibody, or binding fragment thereof; comprises: (a) a heavy chain with a CDR1 sequence of SEQ ID NO: 54; a CDR2 sequence of SEQ ID NO: 55 and a CDR3 sequence of SEQ ID NO: 56, and a light chain with a CDR1 sequence of SEQ ID NO: 60; a CDR2 sequence of SEQ ID NO: 61 and a CDR3 sequence of SEQ ID NO: 62; or (b) a heavy chain with a CDR1 sequence of SEQ ID NO: 57; a CDR2 sequence of SEQ ID NO: 58 and a CDR3 sequence of SEQ ID NO: 59, and a light chain with a CDR1 sequence of SEQ ID NO: 63; a CDR2 sequence of SEQ ID NO: 64 and a CDR3 sequence of SEQ ID NO: 65; wherein optionally the one or more neutralising antibody, or binding fragment thereof; comprises: (i) a heavy chain variable region sequence of SEQ ID NO: 81 and a light chain variable region sequence of SEQ ID NO: 82; or (ii) a heavy chain variable region sequence of SEQ ID NO: 83 and a light chain variable region sequence of SEQ ID NO: 84. In the compositions of the invention each of the one or more non-neutralising antibodies and the one or more neutralising antibodies, or binding fragment thereof, is independently selected from: (a) a monoclonal or polyclonal antibody; or (b) an Fab, F(ab′)2, Fv, scFv, Fd or dAb. 
     The invention also provides a neutralising antibody, or binding fragment thereof, which comprises: (a) a heavy chain with a CDR1 sequence of SEQ ID NO: 54; a CDR2 sequence of SEQ ID NO: 55 and a CDR3 sequence of SEQ ID NO: 56, and a light chain with a CDR1 sequence of SEQ ID NO: 60; a CDR2 sequence of SEQ ID NO: 61 and a CDR3 sequence of SEQ ID NO: 62; or (b) a heavy chain with a CDR1 sequence of SEQ ID NO: 57; a CDR2 sequence of SEQ ID NO: 58 and a CDR3 sequence of SEQ ID NO: 59, and a light chain with a CDR1 sequence of SEQ ID NO: 63; a CDR2 sequence of SEQ ID NO: 64 and a CDR3 sequence of SEQ ID NO: 65; wherein optionally the one or more neutralising antibody, or binding fragment thereof; comprises: (i) a heavy chain variable region sequence of SEQ ID NO: 81 and a light chain variable region sequence of SEQ ID NO: 82. 
     The invention further provides a bispecific dual variable domain molecule comprising a non-neutralising antibody of the invention, or binding fragment thereof and a neutralising antibody of the invention, or binding fragment. 
     The invention also provides a composition comprising: (a) an oligonucleotide aptamer that specifically binds an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1); and (b) an oligonucleotide aptamer that specifically binds to a  Plasmodium  merozoite antigen of the invention. 
     The invention also provides a viral vector, RNA vaccine or DNA plasmid that expresses one or more non-neutralising antibody of the invention, or binding fragment thereof; or one or more neutralising antibody of the invention. 
     The invention also provides a viral vector, RNA vaccine or DNA plasmid that expresses: (a) one or more non-neutralising antibody of the invention, or binding fragment thereof; and (b) one or more neutralising antibody of the invention, or binding fragment thereof; wherein the one or more non-neutralising and neutralising antibodies or binding-fragments thereof are expressed by: (i) the same viral vector, RNA vaccine or DNA plasmid; or (ii) by two separate viral vectors, RNA vaccines or DNA plasmids; or (iii) a viral vector, RNA vaccine or DNA plasmid that expresses a bispecific dual variable domain molecule of the invention. 
     The invention also provides a vector that: (a) expresses or displays an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1); or a  Plasmodium  merozoite antigen as defined herein; or (b) induces a non-neutralising antibody or binding fragment thereof of the invention, or a neutralising antibody of the invention; wherein optionally said vaccine is selected from a viral vector, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine. 
     The invention further provides a vaccine that: (a) expresses or displays (i) an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1) and (ii) a  Plasmodium  merozoite antigen of the invention; or (b) induces (i) a non-neutralising antibody of the invention or binding fragment thereof, and (ii) a neutralising antibody, or binding fragment thereof, that specifically binds a  Plasmodium  merozoite antigen as defined herein, preferably a neutralising antibody of the invention; and optionally (c) (i) expresses or displays one or more additional antigen selected from PfRipr, PfCyRPA, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181 and PfAARP, or a fragment thereof, or (ii) induces an antibody that specifically binds to said one or more additional antigen; wherein optionally: (i) said vaccine is selected from a viral vector, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine; and/or (ii) wherein the epitope of (a)(i), the  Plasmodium  merozoite antigen of (a)(ii) and optionally the one or more additional antigen of (c)(i) are expressed or displayed by; or the non-neutralising antibody or binding fragment thereof of (b)(i), the neutralising antibody of (b)(ii) and optionally the one or more additional antigen of (c)(ii) are induced by: (i) the same vaccine, preferably the same viral vector, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine, even more preferably as a fusion protein; or (ii) by separate vaccines, preferably separate viral vectors, RNA vaccines, DNA plasmids, virus-like particles or proteins. 
     In some embodiments: (a) the one or more non-neutralising antibody, or binding fragment thereof, and the one or more neutralising antibody, or binding fragment thereof, or the bispecific dual variable domain molecule, or epitope each further comprise a signal peptide; wherein optionally the signal peptides direct secretion from human cells, and preferably said signal peptide is a mammalian signal peptide from tissue plasminogen activator; and/or (b) the viral vector, RNA vaccine, DNA plasmid or vaccine is capable of expression in a mammalian cell; and/or (c) the DNA plasmid is capable of expression in a heterologous protein expression system; and optionally the viral vector is a human or simian adenovirus, an adeno-associated virus (AAV), or a pox virus, preferably an AdHu5, ChAd63, ChAdOX1, ChAdOX2 or modified vaccinia Ankara (MVA) vector. 
     The invention also provides a vaccine composition comprising: (i) a composition of the invention; (ii) a non-neutralising antibody of the invention, or binding fragment thereof and optionally a neutralising antibody, or binding fragment thereof as described herein; (iii) a bispecific dual variable domain molecule of the invention; or (iv) a vaccine, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine of the invention. 
     The invention further provides a vaccine composition comprising: (i) a composition of the invention; (ii) a non-neutralising antibody of the invention, or binding fragment thereof and optionally a neutralising antibody as described herein, or binding fragment thereof; (iii) a bispecific dual variable domain molecule of the invention; or (iv) a vaccine, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine of the invention, for use in a method of treating and/or preventing malaria. 
     The invention also provides the use of a vaccine composition comprising: (i) a composition of the invention; (ii) a non-neutralising antibody of the invention, or binding fragment thereof and optionally a neutralising antibody as described herein, or binding fragment thereof; (iii) a bispecific dual variable domain molecule of the invention; or (iv) a vaccine, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine of the invention in the manufacture of a medicament for the prevention and/or treatment of malaria. 
     The invention also provides the vaccine composition for use as described herein, or the use of a vaccine composition as described herein, wherein: (a) the non-neutralising antibody is provided in the form of (i) an antibody as defined herein; (ii) a viral vector, RNA vaccine or DNA plasmid as described herein; or (iii) a vaccine of the invention; and/or (b) the neutralising antibody is provided by (i) an antibody as described herein; (ii) a viral vector, RNA vaccine or DNA plasmid which expresses said neutralising antibody, said viral vector, RNA vaccine or DNA plasmid optionally as defined herein; (iii) a malarial vaccine comprising a  Plasmodium  merozoite antigen as defined herein; or (iv) a malarial vaccine comprising a vector expressing said  Plasmodium  merozoite antigen, or which induces said neutralising antibody, wherein optionally said vaccine is a vaccine as described herein. 
     The invention also provides a composition of the invention; a non-neutralising antibody of the invention, or binding fragment and optionally a neutralising antibody, or binding fragment thereof as described herein; a bispecific dual variable domain molecule of the invention; or a vaccine, RNA vaccine, DNA plasmid, virus-like particle or a protein-based vaccine of the invention, or a vaccine composition of the invention for use in immunising a subject, which has a growth inhibitory activity (GIA) of at least 50% against the blood-stage  Plasmodium  parasite; wherein optionally: (a) the growth inhibitory activity (GIA) of at least 50% is against a plurality of genetic strains of the blood-stage  Plasmodium  parasite; and/or (b) the  Plasmodium  parasite is  Plasmodium falciparum.    
     The invention also provides a host cell containing one or more recombinant expression vector which encodes for: (a)(i) one or more non-neutralising antibody, or binding fragment thereof, that specifically binds an epitope within residues corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1); and (ii) one or more neutralising antibody, or binding fragment thereof, that specifically binds a  Plasmodium  merozoite antigen as described herein; or (b)(i) an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1); and (ii) one or more  Plasmodium  merozoite antigen as described herein. 
    
    
     
       DESCRIPTION OF FIGURES 
         FIG. 1 : Description and binding characteristics of anti-PfRH5 mAbs. (A) Genetic lineage of variable regions from PfRH5-specific mAbs and their donor origin. The percentage of nucleotide substitutions relative to germline is shown. (B) Iso-affinity plot showing the kinetic rate constants of binding for all 21 mAbs to PfRH5FL as determined by SPR. Diagonal dotted lines represent equal affinity at equilibrium. (C) Real-time analysis by Bio-Layer Interferometry (BLI) of mAb binding to PfRH5FL variants with the five most common naturally-occurring amino acid substitutions. Bars represent fold change compared to wild type PfRH5FL (3D7 sequence) binding. 
         FIG. 2 : Growth inhibitory properties of human PfRH5-specific mAbs. (A) In vitro GIA of each mAb tested at 3 mg/mL against 3D7 clone  P. falciparum . Data points are coded to reflect potency (“GIA-high” (≥75%) as squares, “GIA-low” (75%&gt;GIA&gt;25%) as triangles and “GIA-negative” (≤25%) as circles). (B) In vitro GIA dilution series against the 3D7 reference clone. EC50 values were determined by interpolation after fitting the data to a four-parameter dose-response curve. (C) In vitro GIA dilution series of GIA-high mAbs against heterologous parasite laboratory lines and isolates. (D) Table of amino acids at the five most common polymorphic sites of PfRH5 for the seven parasite lines used in the GIA assays. Deviations from the 3D7 reference sequence are in bold. All GIA data points are the mean of duplicate wells. 
         FIG. 3 : Anti-PfRH5ΔNL IgG recapitulates the growth inhibitory properties of anti-PfRH5FL IgG. (A) Binding of anti-PfRH5 mAbs to PfRH5ΔNL by ELISA. Bars show the mean and standard deviation of 4 replicate wells. (B) GIA of purified total IgG from seven different PfRH5FL-vaccinated human volunteers alone or in the presence of 0.5 μM of PfRH5FL or PfRH5ΔNL protein (30 μg/mL and 20 μg/mL, respectively). Bars represent the mean of duplicate wells. (C) GIA of purified total IgG from rabbits immunized with PfRH5FL or PfRH5ΔNL (n=6 rabbits per group). The concentration of PfRH5FL-specific polyclonal IgG in each test sample is plotted on the x-axis and was measured by ELISA using a readout with a μg/mL conversion factor determined by calibration-free concentration analysis. (D) GIA EC 50  comparison between both groups. EC 50  values were determined by interpolation after fitting the data from panel C to a four-parameter dose-response curve. Black horizontal bars represent the mean and error bars are standard deviation. All GIAs were carried out using 3D7 clone  P. falciparum . (E) Intravital luminescence signal of humanized mice infected with transgenic  P. falciparum  (NF54-luciferase) following passive transfer of 15 mg of pre-vaccination or PfRH5ΔNL-vaccinated rabbit IgG at d6 post-infection. Starting group sizes were n=2 for the PBS group and n=4 for the two IgG passive transfer groups. Mice which died before the experiment endpoint (d13) were: one at d7 and another at d13 in the PBS group, one at d9 and another at d12 in the pre-vaccination IgG group and one at d6, one at d10 and another at d12 in the vaccinated IgG group. Individual data points are plotted with a connecting line representative of the mean. (F) Concentration time-course of PfRH5-specific rabbit IgG in the passive transfer experiment shown in panel E, as determined by ELISA binding to PfRH5FL. Data points are the mean, error bars are the standard deviation. 
         FIG. 4 : Epitope-binning groups mAbs by functional phenotype and the correlation between nAb kinetic binding parameters and GIA. (A) Epitope bins determined by a matrix of sequential PfRH5FL-binding assays for different mAbs as measured by BLI. Re=red bin, Bl=blue bin; Ol=olive bin; Pu=purple bin; Ye=yellow bin; Gr=green bin; and Or=orange bin. (B) Potency of anti-PfRH5 mAb invasion inhibition grouped by epitope bin. EC 30  values were interpolated from data in  FIG. 2B . (C) The effect of mAbs on the binding of PfRH5FL to its receptor basigin and to its binding partners PfCyRPA and PfP113 (PfP113Nt used here), as determined by SPR assays. Binding of PfRH5FL to PfCyRPA, PfP113Nt and basigin in the absence of mAb is shown by the black bars in each assay as a control (far left). The colours used in B and C correspond to those of the epitope bins in panel A. (D) The binding parameters of nAbs (Kon, Koff and KD) were correlated with growth inhibition using the GIA EC 30  as a measure of potency. Only GIA-high nAbs were included in this analysis because of their overt ability to bind merozoite-bound PfRH5, as evidenced by their growth inhibitory properties. EC 30  values were calculated from data shown in  FIG. 2B . The reported P-value is two-tailed. 
         FIG. 5 : Identification of the R5.004 and R5.016 mAb epitopes. (A) Crystal structure of the complex of PfRH5ΔNL bound to R5.004 and R5.016 Fab fragments. Insets show close-up views of the epitopes. (B) The top PfRH5 peptides identified as protected by HDX-MS upon binding to R5.004 mAb (blue) and R5.016 mAb (red), respectively, are highlighted on the structure of PfRH5ΔNL. The positions of the Fab fragments in the crystal structure are overlaid as faded cartoons. (C) Overlay of PfRH5ΔNL:BSG co-complex from PDB entry 4U0Q (BSG coloured in teal) with R5.004 Fab or R5.016 Fab structures at their respective binding sites in the PfRH5ΔNL:R5.004:R5.016 co-complex. Fab fragments are color-coded according to their respective epitope bin colour as defined in  FIG. 4A . 
         FIG. 6 : Non-neutralizing mAb R5.011 potentiates the effect of anti-PfRH5 nAbs against 3D7 clone  P. falciparum . (A) GIA of nAbs R5.008, R5.016 and R5.018 alone (grey data points) or in the presence of an equimolar concentration of a mAb which blocks binding to PfRH5FL in vitro (black data points). R5.008 was used at 300 μg/mL, R5.016 was used at 150 μg/mL and R5.018 was used at 400 μg/mL. (B) GIA of human anti-PfRH5 mAbs at 150 μg/mL alone (grey data points) or in the presence of 150 μg/mL R5.011 (black data points). (C) GIA of total human IgG from five PfRH5FL-vaccinated volunteers (1017, 1020, 2205, 2207 and 2210) at a concentration of 5 mg/mL alone (grey data points) or in the presence of 300 μg/mL of R5.011 or R5.003 (black data points). (D) GIA of a dilution series of mAbs R5.004, R5.016 and R5.011 alone (triangle, triangle and circle respectively) as well as a dilution series of the R5.004 and R5.016 nAbs in the presence of an excess of R5.011 (500 μg/mL) (square) to determine the maximal effect of R5.011. “Concentration of test mAb” refers to the concentration of nAb in mAb combinations. (E) GIA of total IgG from rabbits immunized with PfMSP1, PfRH4, PfCyRPA, PfRipr or PfAMA1 (left most data point for each immunisation antigen as indicated) with the addition of 300 μg/mL of R5.011 or R5.009 (middle and right data points for each antigen as indicated). The concentrations of vaccinated IgG used were 10 mg/mL for PfRH4 and PfRipr, 5 mg/mL for PfMSP1 and PfCyRPA and 3.25 mg/mL for PfAMA1, to achieve approximately 30-60% GIA in the absence of mAb. All data points are the mean of 3 replicate wells, error bars are standard deviation. Parasites used were 3D7 clone  P. falciparum . (F) GIAs of nAbs R5.004 and R5.016 alone or with R5.011 as various mAb+Fab fragment combinations. Neither bivalency nor the Fc is necessary for the potentiating effect of R5.011. X-axis concentration values are plotted as the concentration of nAb binding site. mAb-mAb combinations are equimolar and mAb-Fab combinations are equimolar in terms of binding sites. (G) mAb R5.011 is titrated in increasing concentrations into three fixed concentrations of nAb R5.004 or R5.016 to determine the concentration at which R5.011 enhancement reaches a maximum. (H) GIA of titration curves of R5.016+R5.011 combinations in different molar ratios. (I) GIA of the most potent single human anti-PfRH5 mAb (R5.016), the most potent combination of two human anti-PfRH5 mAbs (R5.016+R5.011) and of a 1611 bispecific DVD-Ig™. The cartoon below is a schematic of the 1611 DVD-Ig™ comprising R5.011 and R5.016 variable regions. All data points are the mean of 3 replicate wells, error bars are standard deviation. Parasites used were 3D7 clone  P. falciparum.    
         FIG. 7 : Potentiating mAb binding site analysis: PfRH5ΔNL in complex with R5.011 and R5.016 Fab fragments. (A) Crystal structure of a complex of PfRH5ΔNL bound to Fab fragments from R5.011 and R5.016. The top left inset box shows a close-up of the R5.011 epitope. The PfRH5 peptide most protected upon R5.011 mAb binding as determined by HDX-MS (green) is highlighted on a close-up of the R5.011 binding site in the bottom left inset box. The position of the R5.011 Fab fragment in the crystal structure is overlaid as a faded cartoon. (B) Overlay of R5.004, R5.011 and R5.016 Fab fragment structures on PfRH5ΔNL. All Fabs are color-coded according to their respective epitope bin colour as defined in  FIG. 4A . (C) Structural alignment of PfRH5 from the PfRH5ΔNL:R5.004:R5.016 co-complex (in beige), the PfRH5ΔNL:R5.011:R5.016 co-complex (in magenta) and the PfRH5 from PDB entry 4WAT (in light purple), to highlight differences in conformation of the N-terminus. (D) Structural alignment of R5.011 Fab fragment unbound (white) and bound to PfRH5ΔNL (green). Light chain and heavy chain CDR loops are annotated. 
         FIG. 8 : Non-neutralizing mAb R5.011 potentiates the effect of anti-PfRipr nAbs against 3D7 clone  P. falciparum . (A) GIA of total IgG from PfRipr-vaccinated rabbits alone (open circles, grey) or in the presence of 1 mg/mL of R5.011 mAb (crossed squares, black). The x-axis shows the concentration of animal-derived IgG only and does not include the R5.011 concentration. Each panel represents the GIA of one rabbit&#39;s IgG. All data points show the mean+standard deviation of three replicate wells. (B) GIA of total IgG from a PfRH4-vaccinated rabbit (left), a PfCyRPA-vaccinated rat (centre), and a PfAMA1-vaccinated rabbit (right) in the presence of an excess of R5.011 mAb (1.5 mg/mL, all square data points) showing the maximal extent of R5.011-mediated synergy. The x-axis shows the concentration of animal-derived IgG only and does not include the R5.011 concentration. 
         FIG. 9 : Influence of R5.011 on parasite invasion time as determined by live-cell microscopy. (A) Total invasion time required for RBC invasion in the presence of R5.011 or an irrelevant isotype-matched antibody control (α-EBOV). (B) Invasion time required in early invasion (pre-penetration) and late invasion (penetration). Data are presented as box-and-whiskers plots showing the interquartile range and total range overlaid on individual data points. Solid black lines show the mean for each group. 3D7 clone  P. falciparum  parasites were used. Both mAbs were used at a concentration of 500 μg/mL and 21 and 22 invasion events were recorded for α-EBOV and R5.011, respectively. **** signifies P&lt;0.0001 and n.s.=non-significant. 
         FIG. 10 : Additional information from live-cell microscopy experiments. (A) Mean and median early (pre-penetration), late (penetration) and total invasion times of merozoites incubated with 500 μg/mL of R5.011 or α-EBOV. (B) Total invasion times of merozoites in the presence of neutralizing mAb R5.016, synergistic mAb combination R5.011+R5.016, non-neutralizing, non-potentiating mAb R5.009 or α-EBOV mAb at the concentrations indicated. Data are representative of n=9 invasions for R5.009, R5.016 10 μg/mL and R5.016+R5.011, n=12 for R5.016 500 μg/mL, n=21 for α-EBOV and are presented as box-and-whiskers plots showing the interquartile range and total range overlaid on individual data points. Solid black lines show the mean for each group. 3D7 clone  P. falciparum  parasites were used. n.s=non-significant. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. 
     PfRH5 Antibodies 
     The following definitions are generally applicable to all antibodies of the invention, including the non-neutralising antibodies (non-nAb) and neutralising antibodies (nAb) discussed below. 
     The term “antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody entities are known in the art, non-limiting embodiments of which are discussed below. 
     In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. 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). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies may be polyclonal (pAb) or monoclonal (mAb). Typically the antibodies of the invention are mAbs. 
     Antibodies of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass and may be from any species (e.g., mouse, human, chicken, rat, rabbit, sheep, shark and camelid). 
     The term “antigen-binding fragment” of an antibody (or simply “binding fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by one or more fragments of a full-length antibody. Single chain antibodies are also encompassed. Such antigen-binding fragments may also be bispecific, dual specific, or multi-specific, specifically binding to two or more different antigens. Thus, examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include Fab, Fv, scFv, dAb, Fd, Fab′ or F(ab′)2, tandem scFv and diabodies. 
     Also encompassed are antibody constructs, defined as a polypeptide comprising one or more the antigen binding fragment of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. 
     An antibody of the invention may be a “human antibody”; defined as an antibody having variable and constant regions derived from human germline immunoglobulin sequences, but which 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), for example in the CDRs and in particular CDR3. Recombinant human antibodies are also encompassed by the present invention. 
     An antibody of the invention may be a “chimeric antibody”; defined as an antibody which comprises heavy and light chain variable region sequences from one species and constant region sequences from another species. The present invention encompasses chimeric antibodies having, for example, murine heavy and light chain variable regions linked to human constant regions. 
     An antibody of the invention may be a “CDR-grafted antibody”; defined as an antibody which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3 or all three CDRs) has been replaced with human CDR sequences. 
     An antibody of the invention may be a “humanized antibody”; defined as an antibody which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. 
     The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). 
     Antibodies of the invention are not limited to a particular method of generation or production. Thus, the invention provides antibodies which have been manufactured from a hybridoma that secretes the antibody, as well as antibodies produced from a recombinantly produced cell that has been transformed or transfected with a nucleic acid or nucleic acids encoding the antibody. Such hybridomas, recombinantly produced cells, and nucleic acids form part of the invention. 
     An antibody, or antigen-binding fragment thereof, of the invention is selective or specific for PfRH5, or a particular epitope of PfRH5 as described herein. By specific, it will be understood that the antibody binds to the molecule of interest, in this case the PfRH5 fragment of the invention, with no significant cross-reactivity to any other molecule, particularly any other protein. For example, a binding compound or antibody of the invention that is specific for a particular PfRH5 epitope of the invention will show no significant cross-reactivity with other PfRH5 epitopes. As another example, a binding compound or antibody of the invention that is specific for PfRH5 will show no significant cross-reactivity with other malarial invasion proteins. Cross-reactivity may be assessed by any suitable method. Cross-reactivity of a binding compound (e.g. antibody) for a PfRH5 epitope with another PfRH5 epitope or non-PfRH5 protein may be considered significant if the binding compound (e.g. antibody) binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to the PfRH5 epitope. A binding compound (e.g. antibody) that is specific for the PfRH5 fragment may bind to another molecule such as PfAMA1 at less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that it binds to the PfRH5 epitope. Preferably, the binding compound (e.g. antibody) binds to the other molecule at less than 20%, less than 15%, less than 10% or less than 5%, less than 2% or less than 1% the strength that it binds to the PfRH5 epitope. Binding affinity may be quantified in any suitable way, e.g. by K D . 
     The binding affinity of a PfRH5 antibody (neutralising or non-neutralising) of the invention for PfRH5 may be quantified in terms of dissociation constant (K D ). K D  may be determined using any appropriate technique, but SPR is generally preferred in the context of the present invention. A PfRH5 antibody of the invention may bind to PfRH5 with a K D  of less than 1 μM, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 1 nM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 10 pM, less than 5 pM, or less. Typically a PfRH5 antibody of the invention binds to PfRH5 with a K D  of less 50 nM, less than 10 nM or less than 1 nM. 
     Further antibodies which bind to the epitopes/antigens of the invention may be generated by producing variants of the antibodies of the invention. Such variants may have CDRs sharing a high level of identity with the CDRs of an antibody of the invention, for example may have CDRs each of which independently may differ by one or two amino acids from the antibody of the invention from which the variant antibody is derived, and wherein the variant retains the binding and functional properties of the antibody of the invention. Additionally, such antibodies may have one or more variations (e.g. a conservative amino acid substitution) in the framework regions. By way of a non-limiting example, variants of antibody mAb11 or mAb16 may have CDRs that differ by 1 or 2 amino acid residues in any one or more of the CDRs of antibody mAb11 or mAb16. Conservative amino acid substitutions are particularly contemplated (see the disclosure herein in relation to PfRH5 antigen variants, which applies equally to variants of antibodies of the invention). The variations in the amino acid sequences of the antibodies of the invention should maintain at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and up to 99% sequence identity. Variants having at least 90% sequence identity with the antibodies of the invention, particularly the specific antibodies exemplified herein are specifically contemplated. Variation may or may not be limited to the framework regions and not present in the CDRs. 
     A particular exemplified antibody of the invention may be interchangeably referred to as mAbX (where X is an identification number) or as R5.X (where X is the same identification number). 
     DNA oligonucleotide aptamers, RNA oligonucleotide aptamers, and other engineered biopolymers against a PfRH5 antigen of the invention may also be able to replicate the activity of the antibodies and combinations thereof described here. As a vaccine, PfRH5 antigens of the invention are likely amenable to expression by recombinant viral vectored vaccines, as well as nucleic acid-based vaccines such as RNA or DNA; and recombinant protein or virus-like particles (VLPs) expressed in mammalian expression systems or insect cell systems. It is also possible to express the PfRH5 antigens of the invention as proteins or VLPs in bacteria or yeast, as well as plant/algae systems. 
     Non-Neutralising Antibodies 
     The present inventors have identified a novel epitope on PfRH5 which elicits antibodies which themselves possess no neutralising activity. However, the inventors have surprisingly shown that non-neutralising antibodies which bind to this epitope on PfRH5 potentiate the activity of all neutralising antibodies directed to PfRH5. In addition, these non-neutralising antibodies (non-nAb) also synergise with antibodies against other malarial invasion proteins. 
     The present inventors have previously solved the crystal structure of PfRH5 (see WO2016/016651, which is incorporated by reference in its entirety, particularly Example 1 and  FIGS. 1 and 2A and 2B  thereof). The present inventors have now demonstrated in the Examples herein, the non-neutralising antibodies of the present invention bind to an epitope in the vicinity of the interface between the disordered N-terminus and the rigid α-helical core of the full-length PfRH5 protein. Thus, this epitope is situated at the N-terminus of both the PfRH5ΔN fragment (which lacks the signal peptide (corresponding to residues 1 to 23 of SEQ ID NO: 1 or 2) and the flexible/disordered N-terminal region (corresponding to residues 24 to 139 or 24 to 159 (preferably 24 to 139) of SEQ ID NO: 1 or 2) of PfRH5), as exemplified by SEQ ID NOs: 3 to 6; and the PfRH5ΔNL fragment (which lacks both the signal peptide and flexible/disordered N-terminal region identified above and the flexible loop region corresponding to amino acid residues 248 to 296 of SEQ ID NO: 1 or 2), as exemplified by SEQ ID NOs: 7 to 10. Accordingly, the invention provides a novel epitope defined by amino acids Ser153 to Ile163 and Aps305 to Lys319 of PfRH5. In particular, the invention provides an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of SEQ ID NO: 1, or the corresponding amino acid residues of another PfRH5 sequence (SEQ ID NO: 2) or fragment (such as any one of SEQ ID NOs: 3 to 10). More particularly, the epitope comprises or consists of amino acid residues corresponding to Ser153 to Asn156; Asn159; Ile161; Ile163; Asp305; Asn308; Lys312; Lys316 and Lys 319 of PfRH5 as defined above. With regards to PfRH5 fragments, the numbering of the amino acids of the epitope may differ from the residues indicated above (as these are defined by reference to the full-length sequence of PfRH5, typically SEQ ID NO: 1 or 2). However, the corresponding residues within a PfRH5 fragment may be readily identified using only routine skill and methods (e.g. sequence alignment). Indeed, by way of example, these epitopes are identified in the fragments of SEQ ID NOs: 3 to 10 in the sequence information section herein. More particularly, the epitope comprises or consists of amino acid residues corresponding to Ser153 to Asn156; Asn159; Ile161; Ile163; Asp305; Asn308; Lys312; Lys316 and Lys319 of PfRH5 or fragments thereof as defined above. Typically the epitopes described above give rise to (induce) non-neutralising antibodies of the invention, such as those exemplified herein. 
     A non-neutralising antibody or binding fragment thereof of the invention may comprise three heavy chain CDRs having sequences: (a) a CDR1 sequence of SEQ ID NO: 11; a CDR2 sequence of SEQ ID NO: 12 and a CDR3 sequence of SEQ ID NO: 13; (b) a CDR1 sequence of SEQ ID NO: 14; a CDR2 sequence of SEQ ID NO: 15 and a CDR3 sequence of SEQ ID NO: 16; or (c) a CDR1 sequence of SEQ ID NO: 17; a CDR2 sequence of SEQ ID NO: 18 and a CDR3 sequence of SEQ ID NO: 19. 
     A non-neutralising antibody or binding fragment thereof of the invention may comprise three light chain CDRs having sequences: (a) a CDR1 sequence of SEQ ID NO: 20; a CDR2 sequence of SEQ ID NO: 21 and a CDR3 sequence of SEQ ID NO: 22; a CDR1 sequence of SEQ ID NO: 23; a CDR2 sequence of SEQ ID NO: 24 and a CDR3 sequence of SEQ ID NO: 25; or (c) a CDR1 sequence of SEQ ID NO: 26; a CDR2 sequence of SEQ ID NO: 21 and a CDR3 sequence of SEQ ID NO: 27. 
     In a preferred embodiment, a non-neutralising antibody or binding fragment thereof of the invention may comprise: (a) a heavy chain with a CDR1 sequence of SEQ ID NO: 11; a CDR2 sequence of SEQ ID NO: 12 and a CDR3 sequence of SEQ ID NO: 13, and a light chain with a CDR1 sequence of SEQ ID NO: 20; a CDR2 sequence of SEQ ID NO: 21 and a CDR3 sequence of SEQ ID NO: 22; (b) a heavy chain with a CDR1 sequence of SEQ ID NO: 14; a CDR2 sequence of SEQ ID NO: 15 and a CDR3 sequence of SEQ ID NO: 16, and a light chain with a CDR1 sequence of SEQ ID NO: 23; a CDR2 sequence of SEQ ID NO: 24 and a CDR3 sequence of SEQ ID NO: 25; or (c) a heavy chain with a CDR1 sequence of SEQ ID NO: 17; a CDR2 sequence of SEQ ID NO: 18 and a CDR3 sequence of SEQ ID NO: 19, and a light chain with a CDR1 sequence of SEQ ID NO: 26; a CDR2 sequence of SEQ ID NO: 21 and a CDR3 sequence of SEQ ID NO: 27. In a more preferred embodiment, a non-neutralising antibody or binding fragment thereof of the present invention comprises the CDRs of mAb11 or the CDRs of mAb14. 
     A non-neutralising antibody or binding fragment thereof of the invention may comprise four heavy chain framework regions (FRs) having sequences: (a) a FR1 sequence of SEQ ID NO: 28; a FR2 sequence of SEQ ID NO: 29; a FR3 sequence of SEQ ID NO: 30; and a FR4 sequence of SEQ ID NO: 31; (b) a FR1 sequence of SEQ ID NO: 32; a FR2 sequence of SEQ ID NO: 33; a FR3 sequence of SEQ ID NO: 34; and a FR4 sequence of SEQ ID NO: 31; or (c) a FR1 sequence of SEQ ID NO: 35; a FR2 sequence of SEQ ID NO: 36; a FR3 sequence of SEQ ID NO: 37; and a FR4 sequence of SEQ ID NO: 38. 
     A non-neutralising antibody or binding fragment thereof of the invention may comprise four light chain framework regions (FRs) having sequences: (a) a FR1 sequence of SEQ ID NO: 39; a FR2 sequence of SEQ ID NO: 40; a FR3 sequence of SEQ ID NO: 41; and a FR4 sequence of SEQ ID NO: 42; (b) a FR1 sequence of SEQ ID NO: 43; a FR2 sequence of SEQ ID NO: 44; a FR3 sequence of SEQ ID NO: 41; and a FR4 sequence of SEQ ID NO: 42; or (c) a FR1 sequence of SEQ ID NO: 45; a FR2 sequence of SEQ ID NO: 40; a FR3 sequence of SEQ ID NO: 46; and a FR4 sequence of SEQ ID NO: 42. 
     Preferably a non-neutralising antibody of the invention has the heavy and light chain CDRs of the mAb11 and the corresponding heavy and light chain FRs of the mAb11 antibody; the heavy and light chain CDRs of the mAb14 and the corresponding heavy and light chain FRs of the mAb14 antibody; or the heavy and light chain CDRs of the mAb10 and the corresponding heavy and light chain FRs of the mAb10 antibody; as identified above. In a preferred embodiment, a non-neutralising antibody of the invention, or binding fragment thereof, comprises: (a) a heavy chain variable region sequence of SEQ ID NO: 47 and/or a light chain variable region sequence of SEQ ID NO: 48; (b) a heavy chain variable region sequence of SEQ ID NO: 49 and/or a light chain variable region sequence of SEQ ID NO: 50; or (c) a heavy chain variable region sequence of SEQ ID NO: 51 and/or a light chain variable region sequence of SEQ ID NO: 52. Particularly preferred are antibodies with both the VH and VL sequences listed above. In a more preferred embodiment, a non-neutralising antibody or binding fragment thereof of the present invention comprises the VH and VL sequences of mAb11 or mAb14. 
     The term “non-neutralising antibody” is defined herein to mean an antibody which by itself (in the absence of any other PfRH5 antibody or other antibody against a malarial invasion protein) lacks the ability, or has negligible ability, to inhibit the growth of  Plasmodium falciparum  parasites, and more preferably across a plurality of strains of blood-stage  P. falciparum  parasites. 
     This activity or lack thereof may be quantified using any appropriate technique and measured in any appropriate units. For example, the activity of PfRH5 antibodies of the invention may be given in terms of their growth inhibitory activity (GIA), half maximal effective concentration (EC 50 ), antibody titre stimulated (in terms of antibody units, AU) and/or EC 50  in terms of AU. The latter of these gives an indication of the quality of the response elicited by the PfRH5 antibody of the invention. Any appropriate technique may be used to determine the GIA, EC 50 , AU or EC 50 /AU. Typically the activity of polyclonal antibodies is quantified using EC 50 /AU, and the activity of monoclonal antibodies is quantified using EC 50 . Exemplary techniques are described in the examples and conventional techniques are known in the art. 
     Typically non-neutralising antibodies of the invention have a GIA of less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less against  Plasmodium  parasites, down to zero GIA against  Plasmodium  parasites. 
     The growth inhibitory activity (GIA) may be measured at any appropriate concentration of the antibodies, for example the GIA may be measured at 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2 mg/ml or 3 mg/ml of purified IgG antibody. 
     As described herein, without being bound by theory, the evidence suggests that non-neutralising antibodies of the invention slow the invasion of host red blood cells by the merozoite form of the  Plasmodium  parasite (referred to interchangeably herein as increasing invasion time), giving longer for neutralising antibodies to bind to their epitope on the merozoite. A non-neutralising antibody of the invention may increase invasion time by at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times or more, preferably at least 2 times and more preferably at least 3 times, compared with the time taken for red blood cell invasion by merozoites (typically a merozoite from the same malarial strain) in the absence of said non-neutralising antibody. By way of non-limiting example, a non-neutralising antibody of the invention may increase invasion time by at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds or more compared with the time taken for red blood cell invasion by merozoites (typically a merozoite from the same malarial strain) in the absence of said non-neutralising antibody. Typically, a non-neutralising antibody of the invention increases the phase of invasion immediately preceding merozoite penetration of a red blood cell (see schematic in  FIG. 9  B). Any disclosure herein in relation to non-neutralising antibodies increasing invasion time may also more specifically relate to increasing the time for the phase of invasion immediately preceding merozoite penetration of a red blood cell. 
     Neutralising Antibodies 
     The present inventors have further identified three novel epitopes on PfRH5 which elicits antibodies which possess significant neutralising activity. The inventors have also shown the effects of these neutralising antibodies (nAb) are potentiated when used in combination with the non-neutralising antibodies of the invention. 
     All the novel neutralising antibodies identified by the present inventors bind to the PfRH5ΔNL fragment. As indicated above, this fragment lacks both the signal peptide (corresponding to residues 1 to 23 of SEQ ID NO: 1 or 2); the flexible/disordered N-terminal region (corresponding to residues 24 to 139 or 24 to 159 (preferably 24 to 139) of SEQ ID NO: 1 or 2) of PfRH5) and the flexible loop region corresponding to amino acid residues 248 to 296 of SEQ ID NO: 1 or 2). The PfRH5ΔNL fragment is exemplified by SEQ ID NOs: 7 to 10. 
     Notably, the PfRH5ΔNL fragment lacks resides K33 to K51 of the full-length PfRH5 protein, which have previously been reported as the minimal PfP113 binding region, and which was thought to be a potential site for antibody-mediated neutralisation 
     In particular, the inventors identified three epitope bins which contain potent neutralising antibodies. The binding sites of mAb16 and mAb4 as defined herein both have significant overlap with the basigin binding site of PfRH5 (as defined in WO2016/016651, particularly in Example 1 which is incorporated by reference in its entirety, particularly Example 1 and  FIGS. 3A and 3D  thereof). mAb4 binds PfRH5 towards the tip of the “kite-like” structure, contacting the N-terminus of helix 4 and each of the three loops that link the converging helices at this apex of PfRH5. These interactions are mediated by five of the CDR loops of the antibody, with only light chain CDR2 (L2) not participating. mAb16 binds predominantly to the N-terminus of helix 2 of PfRH5. The major contact is mediated by the heavy chain CDR3 (H3) loop, which lies along the groove between helices 2 and 3. Additional interactions are mediated by the heavy chain CDR1 (H1) and CDR2 (H2) and L2 loops. 
     Accordingly, the invention provides a novel epitope within RH5ΔN (as exemplified by SEQ ID NOs: 3 to 6) or RH5ΔNL as exemplified by SEQ ID NOs: 7 to 10). In particular, the invention provides novel epitopes corresponding to (a) (i) residues Lys196 to Ser197; Asn347 to Asn352; Arg357 to His365; and Lys443 to Arg448 of PfRH5 (SEQ ID NO: 1); or (ii) residues Gly201 to Lys219; and Lys327 to Tyr335 of PfRH5 (SEQ ID NO: 1); (b) residues Gly201 to Lys219 and Lys327 to Gln342 of PfRH5 (SEQ ID NO: 1); or (c) residues Lys196, Ser197; Tyr346 to Asn354 and Lys443 to Lys452 of PfRH5 (SEQ ID NO: 1). In a preferred embodiment, the invention provides novel epitopes corresponding to (i) residues Lys196 to Ser197; Asn347 to Asn352; Arg357 to His365; and Lys443 to Arg448 of PfRH5 (SEQ ID NO: 1); or (ii) residues Gly201 to Lys219; and Lys327 to Tyr335 of PfRH5 (SEQ ID NO: 1). Alternatively, said epitopes may be defined in terms of the corresponding amino acid residues of another PfRH5 sequence (SEQ ID NO: 2) or fragment (such as any one of SEQ ID NOs: 3 to 10). With regards to PfRH5 fragments, the numbering of the amino acids of the epitope may differ from the residues indicated above (as these are defined by reference to the full-length sequence of PfRH5, typically SEQ ID NO: 1 or 2). However, the corresponding residues within a PfRH5 fragment may be readily identified using only routine skill and methods (e.g. sequence alignment). Indeed, by way of example, these epitopes are identified in the fragments of SEQ ID NOs: 3 to 10 in the sequence information section herein. More particularly, the epitope comprises or consists of amino acid residues corresponding to (a) Lys196; Ser197; Asn347; Asn352; Arg357; Asp361; Glu362; His365; Lys443; Trp447 and Arg448; or (b) Gly201; Lys202; Ile204; Asp207 to Phe209; Lys211; Lys212; Glu215; Lys219; Lys327, Asp331 and Tyr335 of PfRH5 or fragments thereof as defined above. Typically the epitopes described above give rise to (induce) neutralising antibodies of the invention, such as those exemplified herein. 
     A neutralising antibody or binding fragment thereof of the invention may comprise three heavy chain CDRs having sequences: (a) a CDR1 sequence of SEQ ID NO: 54; a CDR2 sequence of SEQ ID NO: 55 and a CDR3 sequence of SEQ ID NO: 56; or (b) a CDR1 sequence of SEQ ID NO: 57; a CDR2 sequence of SEQ ID NO: 58 and a CDR3 sequence of SEQ ID NO: 59. 
     A neutralising antibody or binding fragment thereof of the invention may comprise three light chain CDRs having sequences: (a) a CDR1 sequence of SEQ ID NO: 60; a CDR2 sequence of SEQ ID NO: 61 and a CDR3 sequence of SEQ ID NO: 62; or (b) a CDR1 sequence of SEQ ID NO: 63; a CDR2 sequence of SEQ ID NO: 64 and a CDR3 sequence of SEQ ID NO: 65. 
     In a preferred embodiment, a neutralising antibody or binding fragment thereof of the invention may comprise: (a) a heavy chain with a CDR1 sequence of SEQ ID NO: 54; a CDR2 sequence of SEQ ID NO: 55 and a CDR3 sequence of SEQ ID NO: 56, and a light chain with a CDR1 sequence of SEQ ID NO: 60; a CDR2 sequence of SEQ ID NO: 61 and a CDR3 sequence of SEQ ID NO: 62; or (b) a heavy chain with a CDR1 sequence of SEQ ID NO: 57; a CDR2 sequence of SEQ ID NO: 58 and a CDR3 sequence of SEQ ID NO: 59, and a light chain with a CDR1 sequence of SEQ ID NO: 63; a CDR2 sequence of SEQ ID NO: 64 and a CDR3 sequence of SEQ ID NO: 65. 
     A neutralising antibody or binding fragment thereof of the invention may comprise four heavy chain framework regions (FRs) having sequences: (a) a FR1 sequence of SEQ ID NO: 66; a FR2 sequence of SEQ ID NO: 67; a FR3 sequence of SEQ ID NO: 68; and a FR4 sequence of SEQ ID NO: 69; or (b) a FR1 sequence of SEQ ID NO: 70; a FR2 sequence of SEQ ID NO: 67; a FR3 sequence of SEQ ID NO: 71; and a FR4 sequence of SEQ ID NO: 72. 
     A neutralising antibody or binding fragment thereof of the invention may comprise four light chain framework regions (FRs) having sequences: (a) a FR1 sequence of SEQ ID NO: 73; a FR2 sequence of SEQ ID NO: 74; a FR3 sequence of SEQ ID NO: 75; and a FR4 sequence of SEQ ID NO: 76; or (b) a FR1 sequence of SEQ ID NO: 77; a FR2 sequence of SEQ ID NO: 78; a FR3 sequence of SEQ ID NO: 79; and a FR4 sequence of SEQ ID NO: 80. 
     Typically a neutralising antibody of the invention has both the heavy and light chain CDRs of the same antibody of the invention (e.g. the heavy and light chain CDRs of mAb16) and the heavy and light chain FRs from this same antibody (e.g. the heavy and light chain FRs of mAb16). Preferably a neutralising antibody of the invention comprises the heavy and light chain CDRs of the mAb16 antibody and the corresponding heavy and light chain FRs of the mAb16 antibody; or the heavy and light chain CDRs of the mAb4 and the corresponding heavy and light chain FRs of the mAb4 antibody; as identified above. 
     In a preferred embodiment, a neutralising antibody of the invention, or binding fragment thereof, comprises: (a) a heavy chain variable region sequence of SEQ ID NO: 81 and/or a light chain variable region sequence of SEQ ID NO: 82; or (b) a heavy chain variable region sequence of SEQ ID NO: 83 and/or a light chain variable region sequence of SEQ ID NO: 84.Particularly preferred are neutralising antibodies with both the VH and VL sequences listed above. In a more preferred embodiment, a neutralising antibody or binding fragment thereof of the present invention comprises the VH and VL sequences of mAb16 or mAb4. 
     The term “neutralising antibody” is defined herein to mean an antibody which by itself (in the absence of any other PfRH5 antibody or other antibody against a malarial invasion protein) has the ability to inhibit the growth of  Plasmodium falciparum  parasites, and more preferably across a plurality of strains of blood-stage  P. falciparum  parasites. 
     This activity may be quantified using any appropriate technique and measured in any appropriate units as discussed above in relation to non-neutralising antibodies. This disclosure applies equally to neutralising antibodies of the invention (and to other binding compounds as described herein). 
     Typically, the neutralising PfRH5 antibodies of the invention have a GIA of at least at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more against  Plasmodium  parasites. In a preferred embodiment, the PfRH5 fragments of the invention induce antibodies that have a growth inhibitory activity (GIA) of at least 70%, at least 75%, at least 80%, at least 90% or more against  Plasmodium  parasites. 
     The growth inhibitory activity (GIA) may be measured at any appropriate concentration of the antibodies (again, as described in relation to non-neutralising antibodies and which equally applies to neutralising antibodies). For example, the neutralising antibodies of the invention may give a GIA of at least 40%, at least 50% and preferably at least 70% or more against the blood-stage  Plasmodium  parasite, at an IgG concentration of 1 mg/ml IgG, of the purified neutralising antibody. 
     Preferably the neutralising PfRH5 antibodies of the invention exert similarly high levels of GIA against both the vaccine-homologous clone, 3D7, and against a vaccine-heterologous strain, FVO. A neutralising PfRH5 antibody of the invention has an EC 50  which is at least comparable to total IgG against full length PfRH5, and preferably significantly lower than the EC 50  against full-length PfRH5 of the EC 50  of known anti-PfRH5 antibodies. A neutralising antibody of the invention preferably has an EC 50  significantly lower than that of the anti-PfAMA1 BG98 standard (Faber, B. W., et al., Infection and immunity, 2013; incorporated herein by reference). Typically a neutralising PfRH5 antibody of the invention has an EC 50  value of less than 500 μg/ml, less than 400 μg/ml, less than 300 μg/ml, less than 200 μg/ml, less than 150 μg/ml, less than 100 μg/ml, less than 90 μg/ml, less than 80 μg/ml, less than 70 μg/ml, less than 60 μg/ml, less than 50 μg/ml, less than 40 μg/ml, less than 30 μg/ml, less than 25 μg/ml, less than 20 μg/ml, less than 15 μg/ml, less than 10 μg/ml, less than 9 μg/ml, less than 8 μg/ml, less than 7 μg/ml, less than 6 μg/ml, less than 5 μg/ml, less than 4 μg/ml, less than 3 μg/ml, less than 2 μg/ml, less than 1 μg/ml, or less. 
     Typically the neutralising antibodies of the invention result in a GIA of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more against the blood-stage  Plasmodium  parasite. In a preferred embodiment, the neutralising antibodies of the invention will raise antibodies that result in a GIA of at least 70% against the blood-stage  Plasmodium  parasite measured at any appropriate concentration as described herein. 
     Typically the PfRH5 neutralising antibodies of the invention are effective against genetically diverse strains of the  Plasmodium  parasite. This is likely to be of importance in achieving protection against the variety of strains circulating in the natural environment. Accordingly, in a preferred embodiment, the neutralising antibodies of the invention will result in a GIA of at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more against a plurality of genetic strains of the blood-stage  Plasmodium  parasite. In a preferred embodiment, the neutralising antibodies of the invention will result in a GIA of at least 70% or more against a plurality of genetic strains of the blood-stage  Plasmodium  parasite. 
     The PfRH5 fragment of the invention may have a comparable immunogenicity when compared with the full length PfRH5 antigen. 
     Antibody Combinations 
     As outlined above and as demonstrated in the Examples below, the present inventors have surprisingly shown that non-neutralising antibodies which bind to a particular epitope on PfRH5 potentiate the activity of all neutralising antibodies directed to PfRH5, and also synergise with antibodies against other malarial invasion proteins. 
     Accordingly, the present invention provides a composition comprising: (a) one or more non-neutralising antibody, or binding fragment thereof, that specifically binds a non-neutralising antibody epitope of the invention, particularly, an epitope corresponding to residues Ser153 to Ile163 and Asp305 to Lys319 of PfRH5 (SEQ ID NO: 1); and (b) one or more neutralising antibody, or binding fragment thereof, that specifically binds a  Plasmodium  merozoite antigen. 
     The one or more non-neutralising antibody may be any non-neutralising antibody as described herein. The disclosure of non-neutralising antibodies of the invention is generally applicable to all disclosure herein in relation to the compositions of the invention. 
     The one or more neutralising antibody may bind to any  Plasmodium  merozoite antigen, also referred to interchangably herein as a malarial invasion protein (or a fragment thereof), including malarial invasion proteins already known in the art. The one or more neutralising antibody typically binds to: (a) PfRH5; (b) a non-PfRH5  Plasmodium  merozoite antigen that is within the PfRH5 invasion complex; or (c) a  Plasmodium  merozoite antigen that is closely linked to PfRH5. Closely linked  Plasmodium  merozoite antigens may act either prior to PfRH5 engagement (upstream of PfRH5) or after PfRH5 engagement (downstream of PfRH5). 
     In particular, the one or more neutralising antibody may bind to a  Plasmodium  merozoite antigen selected from: (a) PfRH5; (b) a non-PfRH5 antigen within the PfRH5 invasion complex; and/or (c) a target upstream or downstream of the PfRH5-basigin interaction. 
     As used herein, a non-PfRH5 antigen within the PfRH5 invasion complex is any malarial protein which associates with PfRH5 to form a complex essential for the invasion of red blood cells by the  Plasmodium  merozoites. Accordingly, non-PfRH5 antigens within the PfRH5 invasion complex include PfRipr, PfCyRPA, PfP113, PfRhopH3 and PfRAP2. 
     Non-limiting examples of  Plasmodium  merozoite antigens that are closely linked to PfRH5 include PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181 and PfAARP, or a fragment thereof Antibodies to one or more of these antigens, or any combination thereof, may be used according to the invention. 
     In some preferred embodiments the one or more neutralising antibody is an anti-PfRH5 neutralising antibody as defined herein. The disclosure of neutralising PfRH5 antibodies of the invention is generally applicable to all disclosure herein in relation to the compositions of the invention. 
     Any combinations of neutralising antibodies of the invention (e.g. any two, three, four or more neutralising antibodies to one or more of the  Plasmodium  merozoite antigens described herein) may be used in combination with a non-neutralising antibody. By way of a non-limiting example, a non-neutralising antibody of the invention may be used in combination with a neutralising antibody (or binding fragment thereof) to PfRH5 together with a neutralising antibody (or binding fragment thereof) against a PfCyRPA antigen, a neutralising antibody (or binding fragment thereof) against a PfAMA1 antigen and/or a neutralising antibody (or binding fragment thereof) against a PfRipr antigen. Such a combination may be equally effective against both the vaccine-homologous 3D7 clone and the vaccine-heterologous FVO strain. One or more additional neutralising antibodies to other  Plasmodium  merozoite antigen(s) can be used in combination with the non-neutralising antibody (or binding fragment thereof), neutralising PfRH5 antibody and other merozoite antigen neutralising antibody combination. One exemplary preferred combination is a non-neutralising antibody of the invention together with a neutralising antibody (or binding fragment thereof) to PfRH5 and with a neutralising antibody (or binding fragment thereof) against a PfCyRPA antigen, a PfAMA1 antigen or a PfRipr antigen. 
     The combination of a non-neutralising antibody of the invention and a neutralising antibody of the invention may be in the form of two separate antibody molecules or binding fragments thereof. In which case, the antibodies may be used or administered (or in the case of a therapeutic indication) sequentially or simultaneously, with simultaneous use or administration being preferred. If the use/administration is sequential, preferably the non-neutralising antibody is used/administered first. 
     Alternatively, the non-neutralising antibody or binding fragment thereof and the neutralising antibody or binding fragment thereof may be in the form of a single molecule. Thus, the invention provides bispecific molecules comprising both a non-neutralising antibody or binding fragment thereof and a neutralising antibody or binding fragment thereof Such bispecific molecules include fusion proteins, trifunctional antibodies, F(ab′) 2 , bispecific T-cell engager molecules, dual-variable-domain immunoglobulins (DVD-Ig™, in which the antigen-binding variable domains of two monoclonal antibodies are combined via linkers (typically naturally occurring linkers) to create a tetravalent, dual-targeting single agent). Examples of bispecific antibody structures are well known in the art, as are methods for their production, including the so-called “Knobs-into-Holes” (KiH) approach, the controlled Fab arm exchange method (cFAE) and using scFvs. 
     As well as use or administration of the non-neutralising antibodies and neutralising antibodies of the invention in protein (i.e. antibody) form as discussed above, the non-neutralising and neutralising antibodies of the invention may be used or administered in any other appropriate form. 
     In some embodiments, one or more non-neutralising antibody and/or one or more neutralising antibody is generated in vivo. In vivo generation of said antibodies may be via the administration of one or more vector (e.g. a viral vector, RNA vaccine or DNA plasmid) which encodes and expresses said one or more non-neutralising antibody and/or one or more neutralising antibody. Alternatively, in vivo generation of said antibodies may be by the administration of a vector or vaccine which (i) delivers an antigen which will induce the immune system to make said one or more non-neutralising antibody and/or one or more neutralising antibody, or (ii) encodes or expresses an antigen which will then induce the immune system to make said one or more non-neutralising antibody and/or one or more neutralising antibody; or (iii) natural exposure to malaria (particularly in the case of said one or more neutralising antibody). 
     Any combination of delivery of said one or more non-neutralising antibody and/or one or more neutralising antibody in protein form or in vivo generation of said one or more non-neutralising antibody and/or one or more neutralising antibody may be used. 
     For example, in some embodiments non-neutralising antibodies are used or administered in protein form (i.e. as antibodies), and the neutralising antibodies are generated in vivo. 
     The in vivo generation of the neutralising antibodies may be via the use or administration of a malarial vaccine encoding a  Plasmodium  merozoite antigen as described herein to a subject to be treated, by natural exposure to malaria, or by the use or administration of a vector or plasmid encoding the neutralising antibody (as exemplified herein). 
     In embodiments where the in vivo generation of the neutralising antibodies is via the use or administration of a malarial vaccine to a subject to be treated, the non-neutralising antibody or binding fragment thereof may be described as being used in combination with the antibodies generated in vivo in response to the malarial vaccine. The malarial vaccine may comprise any  Plasmodium  merozoite antigen as described herein. All the disclosure herein in relation to  Plasmodium  merozoite antigens to which neutralising antibodies of the invention specifically binds also applies to the  Plasmodium  antigens themselves as included in a malarial vaccine for use in combination with a non-neutralising antibody (or binding fragment thereof) of the invention. Thus, the malaria vaccine may contain one or more of: (a) PfRH5; (b) a non-PfRH5  Plasmodium  merozoite antigen that is within the PfRH5 invasion complex; or (c) a  Plasmodium  merozoite antigen that is closely linked to PfRH5; or may contain a vector which encodes one or more of said antigens. Examples of such antigens are described herein. Typically the malarial vaccine comprises or encodes a PfRH5 antigen, preferably comprising or encoding an epitope for a neutralising antibody as described herein. Non-limiting examples of vaccine vectors are described herein. 
     The combination of a non-neutralising antibody of the invention and a malarial vaccine may be used or administered (or in the case of a therapeutic indication) sequentially or simultaneously. If the use/administration is sequential, preferably the malarial vaccine is used/administered first. 
     Alternatively, the neutralising antibodies may be generated in vivo naturally via exposure to malaria. 
     The non-neutralising antibodies may be used or administered as a vector encoding said non-neutralising antibody. In such embodiments, the neutralising antibodies may be generated in vivo as described above (e.g. induced by a vaccine, including protein, VLP or other vaccine, or expressed by an expression vector) or used or administered in protein (i.e. antibody) form. The antibodies (in whatever form) may be used or administered (or in the case of a therapeutic indication) sequentially or simultaneously. If the use/administration is sequential, preferably the non-neutralising antibody (in whatever form) is used/administered first. 
     Any combination of delivery method may be used for the non-neutralising antibodies and neutralising antibodies of the invention. In particular, the non-neutralising antibody may be delivered by a means selected from vaccine induction, in vivo vector generation or direct antibody protein delivery as described herein, or a combination thereof, and independently the neutralising antibody of may be also delivered by a means selected from vaccine induction, in vivo vector generation or direct antibody protein delivery as described herein, or a combination thereof. In some preferred embodiments: (i) the neutralising antibody is induced in vivo by a vaccine and the non-neutralising antibody is given directly as a protein antibody; (ii) both the neutralising and non-neutralising antibodies are given directly as protein antibodies; or (iii) both the neutralising and non-neutralising antibodies are induced by vaccines. 
     Regardless of how the non-neutralising antibody is delivered (e.g. as a protein antibody, expressed in vivo by a vector or plasmid of the invention or generated in vivo by the administration of a malarial vaccine (protein, virus-like particle or any other type of vaccine, examples of which are described herein)), or how the neutralising antibody is delivered (e.g. generated in vivo by administration of a malarial vaccine (protein, virus-like particle or any other type of vaccine, examples of which are described herein), by exposure to malaria, as a protein antibody or expressed in vivo by a vector or plasmid of the invention), a non-neutralising antibody of the invention can synergise the activity of the neutralising antibodies as described herein. 
     The non-neutralising antibodies of the invention are able to potentiate or synergise the activity of the neutralising antibodies. The terms potentiate and synergise (or variants thereof) are used interchangeably herein. 
     By potentiation, it is meant that the non-neutralising antibody increases the effect of the neutralising antibody beyond the effect obtained using the non-neutralising antibody alone. This can be quantified in any suitable way and measured using conventional means, as exemplified in the Examples herein. 
     Typically a non-neutralising antibody of the invention or binding fragment thereof reduces the amount or concentration of neutralising antibody required to obtain the desired inhibitory effect on the  Plasmodium  parasite. As a non-limiting example, a non-neutralising antibody or binding fragment thereof of the invention may decrease the EC 50  value of a neutralising antibody or binding fragment thereof by at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, at least ten-fold or more compared with the EC 50  value of the neutralising antibody or binding fragment thereof alone. In some preferred embodiments, the non-neutralising antibody or binding fragment thereof decreases the EC 80  value of the neutralising antibody or binding fragment thereof by at least five-fold, more preferably at least seven-fold, even more preferably at least nine-fold or more compared with the EC 80  value of the neutralising antibody or binding fragment thereof alone. 
     Typically the maximum synergistic effect of a non-neutralising antibody of the invention or binding fragment thereof is achieved using a concentration of greater than 1 μg/ml, greater than 10 μg/ml, greater than 50 μg/ml, greater than 100 μg/ml, greater than 150 μg/ml, greater than 200 μg/ml, greater than 250 μg/ml, greater than 300 μg/ml, or more of the non-neutralising antibody or binding fragment thereof. In some preferred embodiments, the maximum synergistic effect of a non-neutralising antibody of the invention or binding fragment thereof is achieved using a concentration of greater than 150 μg/ml, even more preferably greater than 200 μg/ml of the non-neutralising antibody or binding fragment thereof. 
     The ratio of a neutralising antibody or binding fragment thereof to non-neutralising antibody or binding fragment thereof (nAb:non-nAb) may be about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1 or about 4.5:1. Typically the nAb:non-nAb ratio is about 4:1. The lower the concentration of neutralising antibody or binding fragment thereof, the greater the effect of nAb-biased ratios. Thus, for concentrations of neutralising antibody (or binding fragment thereof) below 100 μg/ml, a nAb:non-nAb ratio of 4:1 is preferred. However, at neutralising antibody (or binding fragment thereof) concentrations above 100 μg/ml, the nAb:non-nAb has less effect, with nAb:non-nAb ratios in the range of 1:4 to 4:1 being roughly equivalent. 
     Other Binding Compounds 
     PfRH5 is a component of the mechanism by which the  Plasmodium  parasite invades RBCs. Compounds that specifically bind to PfRH5 inhibit this process and prevent the invasion of RBCs. 
     Accordingly, other binding compounds and combinations thereof may be used as alternatives to the non-neutralising antibody and neutralising antibody combinations described herein, provided that these combinations of other binding compounds include a non-neutralising binding compound to the epitope identified herein for the non-neutralising antibodies of the invention, and a neutralising binding compound to a  Plasmodium  merozoite antigen as defined herein, and that the non-neutralising binding compound synergises the activity of the neutralising binding compound as for the non-neutralising antibody/neutralising antibody combinations described herein. 
     Thus, the invention provides a composition comprising: (a) an oligonucleotide aptamer that specifically binds to the non-neutralising epitope of the invention, particularly, an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1); and (b) an oligonucleotide aptamer that specifically binds to a  Plasmodium  merozoite antigen as defined herein. 
     Any of the disclosure herein in relation to non-neutralising antibodies/neutralising antibodies and combinations thereof of the invention applies equally to other binding compounds and combinations thereof. 
     The non-neutralising and/or neutralising binding compounds of the invention may be oligonucleotide aptamers which bind to the respective epitopes/antigens as defined herein in relation to the corresponding non-neutralising and neutralising antibodies of the invention. The aptamer(s) may specifically bind to the respective epitopes/antigens as defined herein in relation to the corresponding non-neutralising and neutralising antibodies of the invention. Specificity is discussed herein in relation to the antibodies of the invention and applies equally to aptamers (or other binding compounds of the invention). 
     The activity of neutralising aptamers and the synergistic effect of non-neutralising aptamers may be assessed using a GIA assay. Such aptamers can be found by known methods (e.g. as set out in D. H. J. Bunka, P. G. Stockley,  Nature Reviews Microbiology  4, 588 (2006)). An aptamer of the invention may be optimised to render it suitable for therapeutic use, e.g. it may be conjugated to a monoclonal antibody to modify its pharmacokinetics (e.g. half-life and biodistribution) and/or recruit Fc-dependent immune functions. 
     Vectors and Plasmids 
     The present invention provides vectors that express a non-neutralising antibody of the invention (or other non-neutralising binding compound as defined herein) and a neutralising antibody of the invention (or other neutralising binding compound as defined herein). Typically the vector is present in the form of a vaccine formulation. 
     The one or more non-neutralising and neutralising antibodies or binding-fragments thereof (or other binding compounds as defined herein) may be expressed by: (i) the same vector; or (ii) by two (or more in the case of multiple antibodies/binding proteins) separate vectors; or (iii) a vector that expresses a bispecific molecule (such as a bispecific dual variable domain molecule as defined herein). The vector(s) may be present in the form of a vaccine formulation. 
     The invention also provides a vector that expresses the non-neutralising antibody epitope, a vector that expresses one or more neutralising antibody epitope and a vector that expresses the non-neutralising antibody epitope and one or more neutralising antibody epitope of the invention. The vector(s) may be present in the form of a vaccine formulation. 
     The invention provides a vector that expresses: (a) a non-neutralising antibody epitope of the invention, particularly, an epitope corresponding to residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1); and (b) a  Plasmodium  merozoite antigen as defined herein; and optionally (c) one or more additional antigen selected from PfRipr, PfCyRPA, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181 and PfAARP, or a fragment thereof; wherein the epitope of (a), the  Plasmodium  merozoite antigen of (b) and optionally the one or more additional antigen of (c) are expressed by: (i) the same vector; or (ii) by separate vectors. The vector(s) may be present in the form of a vaccine formulation. 
     The vector(s) may be a viral vector. Such a viral vector may be an adenovirus (of a human serotype such as AdHu5, a simian serotype such as ChAd63, ChAdOX1 or ChAdOX2, or another form), an adeno-associated virus (AAV), or poxvirus vector (such as a modified vaccinia Ankara (MVA)). ChAdOX1 and ChAdOX2 are disclosed in WO2012/172277 (herein incorporated by reference in its entirety). ChAdOX2 is a BAC-derived and E4 modified AdC68-based viral vector. Preferably said viral vector is an AAV vector. 
     Viral vectors are usually non-replicating or replication impaired vectors, which means that the viral vector cannot replicate to any significant extent in normal cells (e.g. normal human cells), as measured by conventional means—e.g. via measuring DNA synthesis and/or viral titre. Non-replicating or replication impaired vectors may have become so naturally (i.e. they have been isolated as such from nature) or artificially (e.g. by breeding in vitro or by genetic manipulation). There will generally be at least one cell-type in which the replication-impaired viral vector can be grown—for example, modified vaccinia Ankara (MVA) can be grown in CEF cells. In one embodiment, the vector is selected from a human or simian adenovirus or a poxvirus vector. 
     Typically, the viral vector is incapable of causing a significant infection in an animal subject, typically in a mammalian subject such as a human or other primate. 
     The vector(s) may be a DNA vector, such as a DNA plasmid. In one embodiment the DNA vector(s) is capable of expression in a mammalian cell expression system, such as an immunised cell. The vector may be suitable for expression in a bacterial and/or insect host cell or expression system, such as any of those exemplified herein. A non-limiting example of a suitable expression vector is a pET15b vector, which may be optionally modified to encode an N-terminal tag, such as a hexa-histidine tag and/or a protease cleavage site, such as a TEV protease cleavage site. 
     The vector(s) may be a RNA vector, such as a self-amplifying RNA vaccine (Geall, A. J. et al., Proc Natl Acad Sci USA 2012; 109(36) pp. 14604-9; incorporated herein by reference). 
     The present invention may be a phage vector, such as an AAV/phage hybrid vector as described in Hajitou et al., Cell 2006; 125(2) pp. 385-398; herein incorporated by reference. 
     Vectors of the present invention also include virus-like particles (VLP), soluble proteins and/or fusion proteins comprising non-neutralising antibodies and neutralising antibodies (or other binding compounds), as described herein, or PfRH5 epitopes or  Plasmodium  merozoite antigens as described herein. Typically these comprise or express PfRH5 epitopes or  Plasmodium  merozoite antigens as described herein. Methods for generating VLPs are known in the art (see, for example, Brune et al. Sci. Rep. (2016), 19(6):19234, which is incorporated by reference in its entirety) and can readily be applied to the present invention. References herein to vectors of the invention may apply equally to VLP, soluble proteins and/or fusion proteins of the invention. 
     The antibodies/binding compounds or epitopes/antigens of the invention may be expressed by in a single vector. Wherein the vector of the invention expresses epitopes/antigens of the invention, the expressed epitopes/antigens are capable of inducing both non-neutralising and neutralising antibodies (or other binding proteins) as described herein. Alternatively, the modified PfRH5 antigen and the one or more additional antigen or fragment thereof may be delivered using a mixture of vectors expressing the individual non-neutralising and neutralising antibodies or epitopes/antigens which induce said antibodies (Forbes, E. K., et al., J Immunol, 2011. 187(7): p. 3738-50; and Sheehy, S. H., et al., Mol Ther, 2012. 20(12): p. 2355-68; both of which are incorporated herein by reference). Where the non-neutralising and neutralising antibodies or epitopes/antigens which induce said antibodies are co-expressed, this may be in the form of a fusion protein (Porter, D. W., et al., Vaccine, 2011. 29(43): p. 7514-22; incorporated herein by reference), or the non-neutralising and neutralising antibodies or epitopes/antigens which induce said antibodies expressed as separate transcripts under the control of separate promoters (Bruder, J. T., et al., Vaccine, 2010. 28(18): p. 3201-10; and Tine, J. A., et al., Infect Immun, 1996. 64(9): p. 3833-44; both of which are incorporated herein by reference), or the non-neutralising and neutralising antibodies or epitopes/antigens which induce said antibodies translated as a single polypeptide which undergoes cleavage to yield two separate antigens (Ibrahimi, A., et al.,  Hum Gene Ther,  2009. 20(8): p. 845-60; incorporated herein by reference). 
     The antibodies/binding compounds or epitopes/antigens of the invention may include a leader sequence, for example to assist in recombinant production and/or secretion. Any suitable leader sequence may be used, including conventional leader sequences known in the art. Suitable leader sequences include Bip leader sequences, which are commonly used in the art to aid secretion from insect cells and human tissue plasminogen activator leader sequence (tPA), which is routinely used in viral and DNA based vaccines and for protein vaccines to aid secretion from mammalian cell expression platforms. By way of a non-limiting example, the antibodies/binding compounds or epitopes/antigens of the invention may include the secretory signal from bovine tissue plasminogen activator, or may include another signal to direct the subcellular trafficking of the antibodies/binding compounds or epitopes/antigens. Alternatively, the antibodies/binding compounds or epitopes/antigens of the invention may be the mature form in which the N-terminal signal peptide has been removed. 
     The antibodies/binding compounds or epitopes/antigens of the invention may additionally comprise an N- or C-terminal tag, for example to assist in recombinant production and/or purification. Any N- or C-terminal tag may be used, including conventional tags known in the art. Suitable tags sequences include C-terminal hexa-histidine tags and the “C-tag” (the four amino acids EPEA at the C-terminus), which are commonly used in the art to aid purification from heterologous expression systems, e.g. insect cells, mammalian cells, bacteria, or yeast. Other examples of suitable tags include GST and MBP tags, or any other conventional tag which may be used to facilitate increased expression. In other embodiments, the antibodies/binding compounds or epitopes/antigens of the invention are purified from heterologous expression systems without the need to use a purification tag. 
     PfRH5 Antigens 
     The present invention provides PfRH5 antigens that give rise to non-neutralising antibodies as defined herein. The invention also provides PfRH5 antigens which give rise to neutralising antibodies as defined herein. The invention further relates to the use of other  Plasmodium  merozoite antigens which give rise to neutralising antibodies that can be used according to the present invention. 
     In particular, the invention provides PfRH5 antigens which comprise an epitope within residues corresponding to amino acid residues Ser153 to Ile163 and Aps305 to Lys319 of PfRH5 (SEQ ID NO: 1). As described herein, said antigens give rise to antibodies which are themselves non-neutralising, but which potentiate the activity of neutralising PfRH5 antibodies, and which also synergise with the activity of neutralising antibodies directed to other  Plasmodium  merozoite antigens as defined herein. The structure of such a PfRH5 antigen of the invention is not limited, provided that the epitope is held in the correct confirmation to give rise to non-neutralising antibodies according to the invention. 
     Full-length PfRH5 is defined by SEQ ID NO: 1 or 2. The mature form of PfRH5 in which the N-terminal signal peptide has been removed may comprise or consist of amino acid residues 26 to 526 of SEQ ID NO: 1 or 2. Said fragments may comprise or consist of at least 21, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, 110, 120, 130 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520 or more consecutive amino acid residues in length. Such fragments may have a common antigenic cross-reactivity with said unmodified (full-length) PfRH5 antigen. Unmodified PfRH5 antigens are described in detail in WO2012/114125 (herein incorporated by reference in its entirety). 
     The inventors have previously described rationally designed fragments of PfRH5 lacking some of the internal disorganised regions. These fragments, including fragments lack the flexible N-terminal domain and/or a flexible central linker. Said flexible N-terminal region of PfRH5 typically comprises amino acid residues corresponding to amino acid residues 1 to 139 or 1 to 159 of SEQ ID NO: 1 or 2. Amino acid residues corresponding to amino acid residues 1 to 23 of SEQ ID NO: 1 or 2 are typically a signal peptide that is cleaved from the mature PfRH5 protein. As used herein, the term flexible N-terminal region may include or exclude the signal peptide. Thus, the term flexible N-terminal region may include the signal peptide and so refer to the amino acids corresponding to amino acid residues 1 to 139 or 1 to 159 of SEQ ID NO: 1 or 2. Alternatively, the term flexible N-terminal region may exclude the signal peptide and so refer to the amino acids corresponding to amino acid residues 24 to 139 or 24 to 159 of SEQ ID NO: 1 or 2. Said flexible disordered central linker region of PfRH5 typically corresponds to amino acid residues 248 to 296 of SEQ ID NO: 1 or 2. The terms “flexible disordered central linker region”, “flexible central linker region” and “flexible central linker” are used interchangeable herein. The flexible central linker of PfRH5 as defined herein may comprise or consist of one of the recited sequences or variants thereof. Such rationally designed PfRH5 fragments may be discontinuous (i.e. contain consecutive runs of amino acid sequences that would be separated by additional amino acids in the full-length wild-type PfRH5). These rationally designed fragments are described in detail in WO 2016/016651 (herein incorporated by reference in its entirety). 
     Optimised PfRH5 fragments, derived from either unmodified PfRH5 or rationally designed PfRH5 fragments have also previously been developed, as described in WO 2018/055331 (herein incorporated by reference in its entirety). Such optimised fragments are typically more stable than the corresponding unmodified PfRH5 antigens, and also have improved expression profiles. 
     The PfRH5 antigens of the invention which give rise to non-neutralising antibodies of the invention may be derived from unmodified PfRH5 fragments, from rationally designed PfRH5 fragments, or from optimised PfRH5 fragments, provided they retain the epitope corresponding to amino acid residues Ser153 to Ile163 and Asp305 to Lys319 of PfRH5 (SEQ ID NO: 1) in the correct confirmation to give rise to non-neutralising antibodies with the synergistic activity described herein. 
     The invention provides PfRH5 antigens which comprise an epitope within SEQ ID NO: 3 (RH5ΔN) or SEQ ID NO: 7 (RH5ΔNL). As described herein, said antigens give rise to antibodies which are themselves neutralising, and the activity of which is potentiated by the non-neutralising PfRH5 antibodies of the invention as described herein. Typically, said PfRH5 antigens which give rise to neutralising PfRH5 antibodies of the invention comprise an epitope having residues corresponding to (i) amino acid residues Lys196 to Ser197; Asn347 to Asn352; Arg357 to His365; and Lys443 to Arg448 of PfRH5 (SEQ ID NO: 1); (ii) residues Gly201 to Lys219; and Lys327 to Tyr335 of PfRH5 (SEQ ID NO: 1); residues Gly201 to Lys219 and Lys327 to Gln342 of PfRH5 (SEQ ID NO: 1); or residues Lys196, Ser197, Tyr346 to Asn354 and Lys443 to Lys452 of PfRH5 (SEQ ID NO: 1). The structure of such a PfRH5 antigen of the invention is not limited, provided that the epitope is held in the correct confirmation to give rise to neutralising antibodies according to the invention. 
     The PfRH5 antigens of the invention which give rise to neutralising anti-PfRH5 antibodies of the invention may be derived from unmodified PfRH5 fragments, from rationally designed PfRH5 fragments, or from optimised PfRH5 fragments (as described above in relation to antigens which give rise to non-neutralising antibodies), provided they retain the epitope within SEQ ID NO: 3 (RH5ΔN) or SEQ ID NO: 7 (RH5ΔNL), and more particularly an epitope corresponding to (i) amino acid residues amino acid residues Lys196 to Ser197; Asn347 to Asn352; Arg357 to His365; and Lys443 to Arg448 of PfRH5 (SEQ ID NO: 1); (ii) residues Gly201 to Lys219; and Lys327 to Tyr335 of PfRH5 (SEQ ID NO: 1); residues Gly201 to Lys219 and Lys327 to Gln342 of PfRH5 (SEQ ID NO: 1); or residues Lys196, Ser197, Tyr346 to Asn354 and Lys443 to Lys452 of PfRH5 (SEQ ID NO: 1) in the correct confirmation to give rise to neutralising antibodies with the activity described herein. 
     The PfRH5 antigens of the invention (either which give rise to non-neutralising antibodies or to neutralising antibodies) may have variant sequences compared with the PfRH5 sequence (be it a wild-type/unmodified PfRH5 sequence, a rationally designed fragment or optimised PfRH5 sequence) from which it is derived. Such variants may exhibit at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% at least 98%, at least 99% or more identity with the PfRH5 sequence from which they are derived. 
     Conventional methods for determining amino acid sequence identity are known in the art. The terms “sequence identity” and “sequence homology” are considered synonymous in this specification. 
     By way of example, a polypeptide of interest may comprise an amino acid sequence having at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity with the amino acid sequence of a reference polypeptide. 
     There are many established algorithms available to align two amino acid sequences. Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid sequences for comparison may be conducted, for example, by computer implemented algorithms (e.g. GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms. 
     The BLOSUM62 table shown below is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff &amp; Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are indicated by the standard one-letter codes. The percent identity is calculated as: 
     
       
         
           
             
               
                 ( 
                 
                   Total 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   number 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   of 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   identical 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   matches 
                 
                 ) 
               
               
                 
                   
                     
                       [ 
                       
                         length 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         of 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         the 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         longer 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         sequence 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         plus 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         the 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         number 
                       
                     
                   
                 
                 
                   
                     
                       of 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       gaps 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       Introduced 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       into 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       the 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       longer 
                     
                   
                 
                 
                   
                     
                       
                         sequence 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         in 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         order 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         to 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         align 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         the 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         two 
                         ⁢ 
                         
                             
                         
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                         sequences 
                       
                       ] 
                     
                   
                 
               
             
             × 
             100 
           
         
       
     
     
       
         
           
               
               
            
               
                   
                 BLOSUM62 table 
               
               
                   
                 A R N D C Q E G H I L K M F PST W Y V 
               
               
                   
                   
               
               
                   
                 A 4 
               
               
                   
                   
               
               
                   
                 R-1 5 
               
               
                   
                   
               
               
                   
                 N-2 0 6 
               
               
                   
                   
               
               
                   
                 D-2-2 1 6 
               
               
                   
                   
               
               
                   
                 C 0-3-3-3 9 
               
               
                   
                   
               
               
                   
                 Q-1 1 0 0-3 5 
               
               
                   
                   
               
               
                   
                 E-1 0 0 2-4 2 5 
               
               
                   
                   
               
               
                   
                 G 0-2 0-1-3-2-2 6 
               
               
                   
                   
               
               
                   
                 H-2 0 1-1-3 0 0-2 8 
               
               
                   
                   
               
               
                   
                 I-1-3-3-3-1-3-3-4-3 4 
               
               
                   
                   
               
               
                   
                 L-1-2-3-4-1-2-3-4-3 24 
               
               
                   
                   
               
               
                   
                 K-1 2 0-1-3 1 1-2-1-3-2 5 
               
               
                   
                   
               
               
                   
                 M-1-1-2-3-1 0-2-3-2 1 2-1 5 
               
               
                   
                   
               
               
                   
                 F-2-3-3-3-2-3-3-3-1 0 0-3 0 6 
               
               
                   
                   
               
               
                   
                 P-1-2-2-1-3-1-1-2-2-3-3-1-2-4 7 
               
               
                   
                   
               
               
                   
                 S 1-1 1 0-1 0 0 0-1-2-2 0-1-2-1 4 
               
               
                   
                   
               
               
                   
                 T 0-1 0-1-1-1-1-2-2-1-1-1-1-2-1 1 5 
               
               
                   
                   
               
               
                   
                 W-3-3-4-4-2-2-3-2-2-3-2-3-1 1-4-3-2 11 
               
               
                   
                   
               
               
                   
                 Y-2-2-2-3-2-1-2-3 2-1-1-2-1 3-3-2-2 2 7 
               
               
                   
                   
               
               
                   
                 V 0-3-3-3-1-2-2-3-3 3 1-2 1-1-2-2 0-3-1 4 
               
            
           
         
       
     
     In a homology comparison, the identity may exist over a region of the sequences that is at least 10 amino acid residues in length (e.g. at least 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 520 amino acid residues in length)—e.g. up to the entire length of the reference sequence. 
     Substantially homologous polypeptides have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 1 to about 30 amino acids (such as 1-10, or 1-5 amino acids); and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. 
     Typically a PfRH5 antigen of the invention (either which give rise to non-neutralising antibodies or to neutralising anti-PfRH5 antibodies) is at least 12, at least 13, at least ILL at least 15 at least 16 at least 17 at least 18 at least 19 at least 20 at least 21, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, 110, 120, 130 140, 150 or more consecutive amino acids in length. The PfRH5 antigens of the invention may be linear or branched, preferably linear. 
     The PfRH5 antigens of the invention may have substitutions at amino acid residues corresponding to amino acid residue 40 and/or amino acid residue 216 and/or amino acid residue 286 and/or amino acid residue 299 of SEQ ID NO: 1 or 2, wherein the amino acid T is replaced by an amino acid other than T. In one embodiment amino acid residues corresponding to amino acid residues 40, 216, 286 and/or 299 of SEQ ID NO: 1 or 2 are replaced with A. Typically, amino acid residues corresponding to amino acid residues 40, 216, 286 and 299 of SEQ ID NO: 1 or 2 are each replaced with A. 
     A PfRH5 antigen of the invention may be modified relative to the PfRH5 antigen from which it is derived, provided that: (i) in the case of a PfRH5 antigen that gives rise to a non-neutralising antibody of the invention, the epitope defined herein is retained and the antigen still gives rise to a non-neutralising antibody; or (ii) in the case of a PfRH5 antigen that gives rise to a neutralising antibody of the invention, the epitope defined herein is retained and the antigen still gives rise to a neutralising antibody. 
     An amino acid modification according to the invention may be a substitution, deletion, addition or other modification, including post-translational modification, unless the relevant disclosure explicitly says otherwise. Preferably said modifications are amino acid substitutions. In other words, the amino acid at a specified position within the PfRH5 antigen of the invention is substituted by a naturally occurring or non-naturally occurring amino acid that is different to the amino acid present at that position in the PfRH5 sequence from which the PfRH5 antigen of the invention is derived. Alternatively, the amino acid at a specified position within the PfRH5 antigen of the invention may be modified post-translationally. Post-translational modifications include glycosylations, acetylations, acylations, de-aminations, phosphorylisations, isoprenylisations, glycosyl phosphatidyl inositolisations and further modifications known to a person skilled in the art. 
     The modification of one or more amino acid position as described herein may be performed, for example, by specific mutagenesis, or any other method known in the art. 
     In embodiments in which one or more amino acid position is substituted relative to the corresponding PfRH5 antigen from which the antigen of the invention is derived, the substitution may be a conservative substitution or a non-conservative substitution. A conservative substitution is defined as substitution by an amino acid pertaining to the same physiochemical group to the amino acid present in the PfRH5 antigen from which the antigen of the invention is derived. A non-conservative amino acid substitution is defined as substitution by an amino acid pertaining to a different physiochemical group to the amino acid present in the PfRH5 antigen from which the antigen of the invention is derived. In more detail, amino acids are, in principle, divided into different physiochemical groups. Aspartate and glutamate belong to the negatively-charged amino acids. Histidine, arginine and lysine belong to the positively-charged amino acids. Asparagine, glutamine, serine, threonine, cysteine and tyrosine belong to the polar amino acids. Glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine and tryptophan belong to the non-polar amino acids. Aromatic side groups are to be found among the amino acids histidine, phenylalanine, tyrosine and tryptophan. Thus, as a non-limiting example, a conservative substation may involve the substitution of a non-polar amino acid by another non-polar amino acid, such as substituting leucine with isoleucine. As another non-limiting example, a non-conservative substitution may involve the substation of a non-polar amino acid (e.g. leucine) with a negatively-charged amino acid (e.g. aspartate), a positively-charged amino acid (e.g. arginine), or a polar amino acid (e.g. asparagine). 
     Expression Systems 
     A PfRH5 antibody, binding compound or antigen of the invention may be expressed using any suitable systems. 
     Generation of antibodies once an epitope has been identified can be carried out using known methods in the art. The antibodies per se can be generated using such methods as mouse hybridomas, phage display or ribosome display. Other methods known to the person skilled in the art may also be used. The resultant functional antibodies from a mouse immunisation or a phage display screen can then have their binding confirmed by epitope mapping. Accordingly, the invention provides a hybridoma expressing any antibody (non-neutralising or neutralising) of the invention. 
     For example, one could readily determine whether an antibody binds to the epitope of the invention via a reference antibody such as the mAb11 antibody of the invention (for a non-neutralising antibody), or the mAb16 or mAb4 antibodies (for a neutralising antibody). 
     Such methods are a matter of routine in the art. By way of a non-limiting example, to determine whether a given antibody binds to the same epitope as antibody mAb11, the antibody mAb11 can be bound to PfRH5 under saturating conditions. Subsequently, the antibody of interest can be assessed for binding to PfRH5. If this antibody is capable of binding to PfRH5 following saturation binding with antibody mAb11, it is clear that the two antibodies are binding to different epitopes. If the antibody is not capable of binding to PfRH5 following saturation binding with antibody mAb11, then the antibody may be binding to the epitope of the invention. The binding profile of the antibody of interest should also be taken into consideration: if it binds with similar affinity (K D ) and/or potency (GIA) to PfRH5 as antibody mAb11, then this makes it far more likely that it is binding to the epitope of the invention. Conversely, if the binding profiles are very different, this suggests that it is not binding to the epitope of the invention. Additional routine experimentation (such as peptide mutation and binding analyses) can be carried out to confirm whether the observed lack of binding in this situation is in fact due to binding the epitope of the invention or if some other phenomenon (such as steric hindrance) is responsible. Such experiments can be carried out using ELISA, RIA, Biacore, flow cytometry or other known antibody binding assays. 
     Antigens of the invention may be expressed using conventional systems. Such a system may be a prokaryotic or a eukaryotic system. Examples of such systems are well-known in the art. Non-limiting examples of suitable host systems include  Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris , non-lytic insect cell expression systems such as Schneider 2 (S2) and Schneider 3 (S3) cells from  Drosophila melanogaster  and Sf9 and Sf21 cells from  Spodoptera frugiperda , and mammalian expression systems such as CHO cells and human embryonic kidney (HEK/HEK293) cells. 
     Accordingly, the invention provides a host cell containing a recombinant expression vector which encodes for a PfRH5 antigen of the invention. In a preferred embodiment the host cell is an insect cell, preferably a  Drosophila melanogaster  cell, or a  Pichia  yeast cell, or an  Escherichia coli  cell. 
     The term antigen as used herein refers to any peptide-based sequence that can be recognised by the immune system and/or that stimulates a cell-mediated immune response and/or stimulates the generation of antibodies. The PfRH5 antigens of the invention may be present in the form of a vaccine composition or vaccine formulation. 
     As described herein, the PfRH5 antigens of the invention raise non-neutralising or neutralising antibodies as described herein. The neutralising antibodies raised by PfRH5 antigens of the invention inhibit the growth of malarial parasites, i.e.  Plasmodium  parasites, preferably across a plurality of strains of blood-stage  Plasmodium  parasites. In a more preferred embodiment, the PfRH5 antigens of the invention raise neutralising antibodies that inhibit the growth of  Plasmodium falciparum  parasites, and more preferably across a plurality of strains of blood-stage  P. falciparum  parasites. The effectiveness of the PfRH5 antigens of the invention may be quantified using any appropriate technique and measured in any appropriate units. For example, the effectiveness of the PfRH5 antigens of the invention may be given in terms of their growth inhibitory activity (GIA), half maximal effective concentration (EC 50 ), antibody titre stimulated (in terms of antibody units, AU) and/or EC 50  in terms of AU (described herein in relation to antibodies of the invention). The latter of these gives an indication of the quality of the antibody response stimulated by the PfRH5 antigen of the invention. Any appropriate technique may be used to determine the GIA, EC 50 , AU or EC 50 /AU. Exemplary techniques are described in the examples and conventional techniques are known in the art. The disclosure herein in relation to quantifying the effectiveness of the antibodies of the invention applies equally to antibodies raised by PfRH5 antigens or epitopes of the invention. 
     The antibodies/binding compounds or epitopes/antigens of the invention may be expressed by in a single vector. Wherein the vector of the invention expresses epitopes/antigens of the invention, the expressed epitopes/antigens are capable of inducing both non-neutralising and neutralising antibodies (or other binding proteins) as described herein. Alternatively the combination of the non-neutralising and neutralising antibodies of the invention, or the combination of the corresponding epitopes/antigens, can be effected by mixing two separate recombinant protein vaccines (Pichyangkul, S., et al., Vaccine, 2009. 28(2): p. 452-62; and Ellis, R. D., et al., PLoS One, 2012. 7(10): p. e46094; both of which are incorporated herein by reference), or by co-delivery using vaccine platforms such as particle-based protein vaccine delivery (Bachmann, M. F., et al., Nat Rev Immunol, 2010. 10(11): p. 787-96; incorporated herein by reference), or virus-like particles (VLP), or by fusing or conjugating the PfRH5 epitopes/antigens to a construct or constructs that allow for particle formation and/or enhanced immunogenicity (Spencer, A. J., et al., PLoS One, 2012. 7(3): p. e33555; and Wu, Y., et al., Proc Natl Acad Sci USA, 2006. 103(48): p. 18243-8; both of which are incorporated herein by reference). In one embodiment, the PfRH5 antigen/epitope that gives rise to a non-neutralising antibody and the PfRH5 antigen/epitope that gives rise to a neutralising antibody may be delivered as a fusion protein (Biswas, S., et al., PLoS One, 2011. 6(6): p. e20977; incorporated herein by reference). Additionally or alternatively, the PfRH5 antigen/epitope that gives rise to a non-neutralising antibody and the PfRH5 antigen/epitope that gives rise to a neutralising antibody (or the non-neutralising antibody and neutralising antibodies themselves) may be delivered using a mixture of viral vectors expressing the individual antigens/epitopes (or antibodies) (Forbes, E. K., et al., J Immunol, 2011. 187(7): p. 3738-50; and Sheehy, S. H., et al., Mol Ther, 2012. 20(12): p. 2355-68; both of which are incorporated herein by reference), or viral vectors co-expressing both the PfRH5 antigen/epitope that gives rise to a non-neutralising antibody and the PfRH5 antigen/epitope that gives rise to a neutralising antibody (or the antibodies themselves). Co-expression may be in the form of a fusion protein (Porter, D. W., et al., Vaccine, 2011. 29(43): p. 7514-22; incorporated herein by reference), or as separate transcripts under the control of separate promoters (Bruder, J. T., et al., Vaccine, 2010. 28(18): p. 3201-10; and Tine, J. A., et al., Infect Immun, 1996. 64(9): p. 3833-44; both of which are incorporated herein by reference), or as a single polypeptide which undergoes cleavage to yield two separate antigens/epitopes (or antibodies) (Ibrahimi, A., et al.,  Hum Gene Ther,  2009. 20(8): p. 845-60; incorporated herein by reference). 
     Therapeutic Indications 
     The present invention also provides a method of stimulating or inducing an immune response in a subject comprising administering to the subject a composition of the invention, one or more antibodies of the invention, one or more binding proteins of the invention, one or more vectors of the invention, or one or more epitopes/antigens of the invention (as described above). 
     Thus, the invention provides a vaccine composition comprising a composition of the invention; a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention. 
     In one embodiment, the method of stimulating or inducing an immune response in a subject comprises administering a composition of the invention (including a vaccine composition); a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (as described above) to a subject. In the context of the therapeutic uses and methods, a “subject” is any animal subject that would benefit from stimulation or induction of an immune response against a  Plasmodium  parasite. Typical animal subjects are mammals, such as primates, for example, humans. 
     Thus, the present invention provides a method for treating or preventing malaria. 
     The present invention also provides a composition of the invention (including a vaccine composition); a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention for use in the prevention or treatment of malaria. 
     The present invention also provides the use of a composition of the invention (including a vaccine composition); a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention in the manufacture of a medicament for the prevention and/or treatment of malaria. 
     The one or more epitopes/antigens of the invention may be in the form of a recombinant protein, a protein particle, a virus-like particle, a fusion protein, or a combination thereof as described herein. In addition, the one or more epitopes/antigens of the invention may be used in combination with one or more further antigens selected from the group consisting of PfRipr, PfCyRPA, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181 and PfAARP, or a fragment thereof; for use in prevention or treatment of malaria. 
     The present invention provides the use of a composition of the invention (including a vaccine composition); a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (as described above) for use either alone or in combination in the prevention or treatment of malaria. 
     In one embodiment, the method for treating or preventing malaria comprises administering a therapeutically effective amount of a composition of the invention (including a vaccine composition); a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (as described above), either alone or in combination, to a subject. 
     Vaccine compositions of the invention, whilst they may express or display an epitope as defined herein, or induce an antibody as defined herein, typically do not express or display the full-length PfRH5 protein. Instead, such vaccine compositions of the invention express or display rationally designed fragments of PfRH5 which comprise or consist of one or more epitope as defined herein, or otherwise induce an antibody as defined herein. Nor do vaccine compositions of the invention comprise or consist of the full-length PfRH5 protein, but rather such vaccines of the invention comprise or consist of rationally designed fragments of PfRH5 which comprise or consist of one or more epitope as defined herein, or otherwise induce an antibody as defined herein. 
     As used herein, the term “treatment” or “treating” embraces therapeutic or preventative/prophylactic measures, and includes post-infection therapy and amelioration of malaria. 
     As used herein, the term “preventing” includes preventing the initiation of malaria and/or reducing the severity or intensity of malaria. The term “preventing” includes inducing or providing protective immunity against malaria. Immunity to malaria may be quantified using any appropriate technique, examples of which are known in the art. 
     A composition of the invention (including a vaccine composition), a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (as described above) may be administered to a subject (typically a mammalian subject such as a human or other primate) already having malaria, a condition or symptoms associated with malaria, to treat or prevent malaria. For example, the subject may be suspected of having come in contact with  Plasmodium  parasite, or has had known contact with  Plasmodium  parasite, but is not yet showing symptoms of exposure. 
     When administered to a subject (e.g. a mammal such as a human or other primate) that already has malaria, or is showing symptoms associated with  Plasmodium  parasite infection, a composition of the invention (including a vaccine composition), a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (as described above) can cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/or prolong the survival of a subject beyond that expected in the absence of such treatment. 
     Alternatively, a composition of the invention (including a vaccine composition), a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (as described above) may be administered to a subject (e.g. a mammal such as a human or other primate) who ultimately may be infected with  Plasmodium  parasite, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of malaria, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment, or to help prevent that subject from transmitting malaria. 
     The treatments and preventative therapies of the present invention are applicable to a variety of different subjects of different ages. In the context of humans, the therapies are applicable to children (e.g. infants, children under 5 years old, older children or teenagers) and adults. In the context of other animal subjects (e.g. mammals such as primates), the therapies are applicable to immature subjects and mature/adult subjects. 
     The present invention provides vaccine compositions comprising any of the compositions of the invention; non-neutralising antibodies, or binding fragments thereof, of the invention and the neutralising antibodies, or binding fragments thereof, of the invention; the bispecific dual variable domain molecules of the invention; the viral vectors, RNA vaccines or DNA plasmids of the invention, or one or more epitopes/antigens of the invention (described herein). Said vaccine compositions may further comprise one or more additional malarial antigens as described herein, and/or any further components as described herein. 
     A vaccine composition of the invention may further comprise one or more additional neutralising antibody to, a bispecific dual variable domain molecule binding to; a viral vector, RNA vaccine or DNA plasmid expressing such an antibody or binding molecule, or one or more epitopes/antigens (or vectors expressing) one or more additional antigen selected from the group consisting of PfRipr, PfCyRPA, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181 and PfAARP, or a fragment thereof. These may be expressed in any suitable form. As a non-limiting example, expression may be as a virus like particle (VLP). Recombinant particulate vaccines are well known in the art. They may be, for example, either fusion proteins or proteins chemically conjugated to particles. Examples of fusion proteins are hepatitis B surface antigen fusions (e.g. as in the RTS,S malaria vaccine candidate), hepatitis B core antigen fusions, or Ty-virus like particles. Examples of chemical fusion particles are the Q-beta particles under development by the biotechnology company Cytos (Zurich, Switzerland) and as in Brune et al. Sci. Rep. (2016), 19(6):19234. 
     The present invention further provides a vaccine composition comprising a PfRH5 epitope of the invention (which gives rise to a non-neutralising but synergistic antibody) and a  Plasmodium  merozoite antigen of the invention (which gives rise to a neutralising antibody synergised by the non-neutralising antibody of the invention). Optionally said vaccine may further comprise one or more additional antigen or a fragment thereof to an additional malarial antigen as described herein (particularly PfRipr or PfCyRPA or a fragment thereof), where either or both the PfRH5 epitope, the  Plasmodium  merozoite antigen and/or the one or more additional antigen or fragment thereof may be expressed as a soluble recombinant protein. Recombinant protein-based vaccines are well known in the art. They may be, for example, monomeric soluble proteins or soluble fusion proteins. Such proteins are typically administered or formulated in a vaccine adjuvant. Examples of protein-based vaccines are diphtheria and tetanus toxoids, or soluble malaria protein antigens such as the PfAMA1 protein vaccine candidates developed for blood-stage malaria (Spring, M. D., et al., PLoS ONE, 2009. 4(4): p. e5254; incorporated herein by reference). 
     The compositions of the invention; non-neutralising antibodies, or binding fragments thereof, of the invention and the neutralising antibodies, or binding fragments thereof, of the invention; the bispecific dual variable domain molecules of the invention; the viral vectors, RNA vaccines or DNA plasmids of the invention, or one or more epitopes/antigens of the invention (described herein) may be combined to provide a single vaccine product (as described above), e.g. by mixing two separate vaccines, or by co-delivery using vaccine platforms such as particle-based protein vaccine delivery, or by using a mixture of viral vectors expressing the individual components, or viral vectors co-expressing both components. 
     As used, herein, a “vaccine” is a formulation that, when administered to an animal subject such as a mammal (e.g. a human or other primate) stimulates or provides a protective immune response against  Plasmodium  parasitic infection. The immune response may be a humoral and/or cell-mediated immune response. A vaccine of the invention can be used, for example, to protect a subject from the effects of  P. falciparum  infection (i.e. malaria). 
     The lack of polymorphism at the PfRH5 locus (five non-synonymous SNP across its entire length in circulating  P. falciparum  parasites) suggest either a lack of substantial immune pressure, or a high degree of functional constraint that prevents mutations from freely occurring. This property makes it highly likely that the vaccine compositions comprising any of the compositions of the invention; non-neutralising antibodies, or binding fragments thereof, of the invention and the neutralising antibodies, or binding fragments thereof, of the invention; the bispecific dual variable domain molecules of the invention; the viral vectors, RNA vaccines or DNA plasmids of the invention, or one or more epitopes/antigens of the invention (described herein) according to the present invention will have broadly neutralising activity. 
     Thus, the compositions of the invention; non-neutralising antibodies, or binding fragments thereof, of the invention and the neutralising antibodies, or binding fragments thereof, of the invention; the bispecific dual variable domain molecules of the invention; the viral vectors, RNA vaccines or DNA plasmids of the invention, or one or more epitopes/antigens of the invention (described herein) typically provide a highly effective cross-strain GIA against the  Plasmodium  parasite. Thus, in one embodiment, the invention provides protection (such as long term protection) against disease caused by  Plasmodium  parasites. Typically, a composition of the invention; a non-neutralising antibody, or binding fragment thereof, of the invention and a neutralising antibody, or binding fragment thereof, of the invention; a bispecific dual variable domain molecule of the invention; a viral vector, RNA vaccine or DNA plasmid of the invention, or one or more epitopes/antigens of the invention (described herein) provides an antibody response (e.g. a neutralising antibody response) to  Plasmodium  parasitic infection. The compositions of the invention; non-neutralising antibodies, or binding fragments thereof, of the invention and the neutralising antibodies, or binding fragments thereof, of the invention; the bispecific dual variable domain molecules of the invention; the viral vectors, RNA vaccines or DNA plasmids of the invention, or one or more epitopes/antigens of the invention as described herein may be used to confer pre-erythrocytic or transmission-blocking protection against  Plasmodium  parasites. 
     The treatment and/or prevention of malaria according to the present invention may further comprise boosting a subject. Such “boosting” may comprise the administration of a pox virus, such as MVA. 
     Pharmaceutical Compositions and Formulations 
     The term “vaccine” is herein used interchangeably with the terms “therapeutic/prophylactic composition”, “formulation” or “medicament”. 
     The vaccine of the invention (as defined above) can be combined or administered in addition to a pharmaceutically acceptable carrier. Alternatively or in addition the vaccine of the invention can further be combined with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and/or antimicrobial compound. 
     Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. 
     Administration of immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (e.g. vaccines) is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral injection, for example, a subcutaneous, intradermal or intramuscular injection. Formulations comprising neutralizing antibodies may be particularly suited to administration intravenously, intramuscularly, intradermally, or subcutaneously. 
     Accordingly, immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (e.g. vaccines) of the invention are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules. 
     The active immunogenic ingredients (such as the non-neutralising antibodies, or binding fragments thereof, of the invention and the neutralising antibodies, or binding fragments thereof, of the invention; the bispecific dual variable domain molecules of the invention; the viral vectors, RNA vaccines or DNA plasmids of the invention, or one or more epitopes/antigens of the invention) are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. 
     Generally, the carrier is a pharmaceutically-acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as BSA. In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage. 
     Examples of additional adjuvants which may be effective include but are not limited to: complete Freund&#39;s adjuvant (CFA), Incomplete Freund&#39;s adjuvant (IFA), Saponin, a purified extract fraction of Saponin such as Quil A, a derivative of Saponin such as QS-21, lipid particles based on Saponin such as ISCOM/ISCOMATRIX,  E. coli  heat labile toxin (LT) mutants such as LTK63 and/or LTK72, aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl oxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion, the MF59 formulation developed by Novartis, and the AS02, ASO1, AS03 and ASO4 adjuvant formulations developed by GSK Biologicals (Rixensart, Belgium). 
     Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5). 
     Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. 
     Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. 
     It is within the routine practice of a clinician to determine an effective amount of a vaccine composition of the invention. An effective amount is an amount sufficient to elicit a protective immune response against malaria. A clinician will also be able to determine appropriate dosage interval using routine skill. 
     Sequence Information 
     PfRH5 Antigens from which Antigens/Epitopes of the Invention May be Derived 
     Full Length PfRH5 Amino Acid Sequence (3D7) Including Signal Sequence: SEQ ID NO: 1 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   KKEIDNDKEN IKTNNAKDHS TYIKSYLNTN VNDGLKYLFI PSHNSFIKKY SVFNQINDGM 
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 241 
                 PYDINNK NDD SYRYDISEEI DDKSEETDDE TEEVEDSIQD TDSNHTPSNK KKNDLM NRTF 
               
               
                   
               
               
                 301 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 361 
                   DEYIH KLILS VKSKNLNKDL SDMTNILQQS ELLLTNLNKK MGSYIYIDTI KFIHKEMKHI 
               
               
                   
               
               
                 421 
                 FNRIEYHTKI INDKTKIIQD KI KLNIWR TF QKDELLKRIL DMSNEYSLFI TSDHLRQMLY 
               
               
                   
               
               
                 481 
                 NTFYSKEKHL NNIFHHLIYV LQMKFNDVPI KMEYFQTYKK NKPLTQ 
               
            
           
         
       
         
         
           
             Signal sequence (amino acids 1 to 23) is in bold italics, flexible N-terminal (amino acids 1 to 139) and flexible loop (amino acids 248 to 296) regions are underlined. 
             The (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     Full Length PfRH5 Amino Acid Sequence (7G8) Including Signal Sequence: SEQ ID NO: 2 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   KKEIDNDKEN IKTNNAKDHS TYIKSYLNTN VNDGLKYLFI PSHNSFIKKY SVFNQINDGM 
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 241 
                 PYDINNK NDD SYRYDISEEI DDKSEETDDE TEEVEDSIQD TDSNHTPSNK KKNDLMN RTF 
               
               
                   
               
               
                 301 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 361 
                   DEYIH KLILS VKSKNLNKDL SDMTNILQQS ELLLTNLNKK MGSYIYIDTI KFIHKEMKHI 
               
               
                   
               
               
                 421 
                 FNRIEYHTKI INDKTKIIQD KI KLNIWR TF QKDELLKRIL DMSNEYSLFI TSDHLRQMLY 
               
               
                   
               
               
                 481 
                 NTFYSKEKHL NNIFHHLIYV LQMKFNDVPI KMEYFQTYKK NKPLTQ 
               
            
           
         
       
         
         
           
             Signal sequence (amino acids 1 to 23) is in bold italics, flexible N-terminal (amino acids 1 to 139) and flexible loop (amino acids 248 to 296) regions are underlined. 
             The (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (3D7) Excluding Signal Sequence and Flexible N-Terminal Region (Amino Acids 1 to 139): SEQ ID NO: 3 (3D7 RH5ΔN) 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 241 
                 LSDMTNILQQ SELLLTNLNK KMGSYIYIDT IKFIHKEMKH IFNRIEYHTK IINDKTKIIQ 
               
               
                   
               
               
                 301 
                 DKI KLNIWR T FQKDELLKRI LDMSNEYSLF ITSDHLRQML YNTFYSKEKH LNNIFHHLIY 
               
               
                   
               
               
                 361 
                 VLQMKFNDVP IKMEYFQTYK KNKPLTQ 
               
            
           
         
       
         
         
           
             Flexible loop (corresponding to amino acids 248 to 296 of full length PfRH5 of SEQ ID NO: 1) region is underlined. 
             The (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (7G8) Excluding Signal Sequence and Flexible N-Terminal Region (Amino Acids 1 to 139): SEQ ID NO: 4 (7G8 RH5ΔN) 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 NHENDFNKIC MDMKNYGTNL FEQLSCY NNN FCN TNGI RYH YDEYIH KLIL SVKSKNLNKD 
               
               
                   
               
               
                 241 
                 LSDMTNILQQ SELLLTNLNK KMGSYIYIDT IKFIHKEMKH IFNRIEYHTK IINDKTKIIQ 
               
               
                   
               
               
                 301 
                 DKI KLNIWR T FQKDELLKRI LDMSNEYSLF ITSDHLRQML YNTFYSKEKH LNNIFHHLIY 
               
               
                   
               
               
                 361 
                 VLQMKFNDVP IKMEYFQTYK KNKPLTQ 
               
            
           
         
       
         
         
           
             Flexible loop (corresponding to amino acids 248 to 296 of full length PfRH5 of SEQ ID NO: 2) region is underlined. 
             The (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (3D7) Excluding Signal Sequence and Flexible N-Terminal Region (Amino Acids 1 to 159): SEQ ID NO: 5 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 VKSKCNDIKN DLIATIKKLE HPYDINNK ND DSYRYDISEE IDDKSEETDD ETEEVEDSIQ   
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 FEQLSCY NNN FCNTN GI RYH YDEYIH KLIL SVKSKNLNKD LSDMTNILQQ SELLLTNLNK 
               
               
                   
               
               
                 241 
                 KMGSYIYIDT IKFIHKEMKH IFNRIEYHTK IINDKTKIIQ DKI KLNIWR T FQKDELLKRI 
               
               
                   
               
               
                 301 
                 LDMSNEYSLF ITSDHLRQML YNTFYSKEKH LNNIFHHLIY VLQMKFNDVP IKMEYFQTYK 
               
               
                   
               
               
                 361 
                 KNKPLTQ 
               
            
           
         
       
         
         
           
             Flexible loop (corresponding to amino acids 248 to 296 of full length PfRH5 of SEQ ID NO: 1) region is underlined. 
             Partial (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (7G8) Excluding Signal Sequence and Flexible N-Terminal Region (Amino Acids 1 to 159): SEQ ID NO: 6 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 VKSKCNDIKN DLIATIKKLE HPYDINNK ND DSYRYDISEE IDDKSEETDD ETEEVEDSIQ   
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 FEQLSCY NNN FCN TNGI RYH YDEYIH KLIL SVKSKNLNKD LSDMTNILQQ SELLLTNLNK 
               
               
                   
               
               
                 241 
                 KMGSYIYIDT IKFIHKEMKH IFNRIEYHTK IINDKTKIIQ DKI KLNIWR T FQKDELLKRI 
               
               
                   
               
               
                 301 
                 LDMSNEYSLF ITSDHLRQML YNTFYSKEKH LNNIFHHLIY VLQMKFNDVP IKMEYFQTYK 
               
               
                   
               
               
                 361 
                 KNKPLTQ 
               
            
           
         
       
         
         
           
             Flexible loop (corresponding to amino acids 248 to 296 of full length PfRH5 of SEQ ID NO: 2) region is underlined. 
             Partial (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (3D7) Excluding Signal Sequence, Flexible N-Terminal (Amino Acids 1 to 139) and Flexible Loop (Amino Acids 248 to 296) Regions: SEQ ID NO: 7 (3D7 RH5ΔNL) 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 LSVKSKNLNK DLSDMTNILQ QSELLLTNLN KKMGSYIYID TIKFIHKEMK HIFNRIEYHT 
               
               
                   
               
               
                 241 
                 KIINDKTKII QDKI KLNIWR  TFQKDELLKR ILDMSNEYSL FITSDHLRQM LYNTFYSKEK 
               
               
                   
               
               
                 301 
                 HLNNIFHHLI YVLQMKFNDV PIKMEYFQTY KKNKPLTQ 
               
            
           
         
       
         
         
           
             The (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (7G8) Excluding Signal Sequence, Flexible N-Terminal (Amino Acids 1 to 139) and Flexible Loop (Amino Acids 248 to 296) Regions: SEQ ID NO: 8 (7G8 RH5ΔNL) 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 LSVKSKNLNK DLSDMTNILQ QSELLLTNLN KKMGSYIYID TIKFIHKEMK HIFNRIEYHT 
               
               
                   
               
               
                 241 
                 KIINDKTKII QDKI KLNIWR  TFQKDELLKR ILDMSNEYSL FITSDHLRQM LYNTFYSKEK 
               
               
                   
               
               
                 301 
                 HLNNIFHHLI YVLQMKFNDV PIKMEYFQTY KKNKPLTQ 
               
            
           
         
       
         
         
           
             The (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (3D7) Excluding Signal Sequence, Flexible N-Terminal (Amino Acids 1 to 159) and Flexible Loop (Amino Acids 248 to 296) Regions: SEQ ID NO: 9 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 QSELLLTNLN KKMGSYIYID TIKFIHKEMK HIFNRIEYHT KIINDKTKII QDKI KLNIWR   
               
               
                   
               
               
                 241 
                 TFQKDELLKR ILDMSNEYSL FITSDHLRQM LYNTFYSKEK HLNNIFHHLI YVLQMKFNDV 
               
               
                   
               
               
                 301 
                 PIKMEYFQTY KKNKPLTQ 
               
            
           
         
       
         
         
           
             Partial (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     PfRH5 Amino Acid Sequence (7G8) Excluding Signal Sequence, Flexible N-Terminal (Amino Acids 1 to 159) and Flexible Loop (Amino Acids 248 to 296) Regions: SEQ ID NO: 10 
       
     
       
         
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 61 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 121 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 181 
                 QSELLLTNLN KKMGSYIYID TIKFIHKEMK HIFNRIEYHT KIINDKTKII QDKI KLNIWR   
               
               
                   
               
               
                 241 
                 TFQKDELLKR ILDMSNEYSL FITSDHLRQM LYNTFYSKEK HLNNIFHHLI YVLQMKFNDV 
               
               
                   
               
               
                 301 
                 PIKMEYFQTY KKNKPLTQ 
               
            
           
         
       
         
         
           
             Partial (discontinuous) epitope for the non-neutralising antibodies of the invention is shown in broken underline. 
             The (discontinuous) epitope for the neutralising mAb4 antibody of the invention is shown in double underline. 
             The (discontinuous) epitope for the neutralising mAb16 antibody of the invention is shown in wavy underline. 
           
         
       
    
     Heavy Chain CDR Sequences for Non-Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 CDR1 
                 CDR2  
                 CDR3  
               
               
                 Antibody 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb11 
                 SYAMN 
                 WINTNTGNPTY 
                 ESPNYYDSSGYY 
               
               
                   
                 (11) 
                 AQGFTG (12) 
                 SGYYFDY (13) 
               
               
                   
               
               
                 mAb14 
                 DYAMH 
                 GISWNSASMDY 
                 DPAPPYCGGDCY 
               
               
                   
                 (14) 
                 ADSVKG (15) 
                 PSFDY (16) 
               
               
                   
               
               
                 mAb10 
                 SYWMS 
                 NIKQDGSEKYY 
                 GASGYYYYNYGM 
               
               
                   
                 (17) 
                 VDSVKG (18) 
                 DV (19) 
               
               
                   
               
            
           
         
       
     
     Light Chain CDR Sequences for Non-Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                 Antibody 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb11 
                 GGKNIGSKSVH 
                 DDSDRPS 
                 QVWDSSSDHRV 
               
               
                   
                 (20) 
                 (21) 
                 (22) 
               
               
                   
               
               
                 mAb14 
                 GNNIGSKSVH 
                 YDSDRPS 
                 QVWDSSRDHVV 
               
               
                   
                 (23) 
                 (24) 
                 (25) 
               
               
                   
               
               
                 mAb10 
                 GGNNIGIKSVH 
                 DDSDRPS 
                 QVWDSSSDHPGV 
               
               
                   
                 (26) 
                 (21) 
                 (27) 
               
               
                   
               
            
           
         
       
     
     Heavy Chain Framework Region Sequences Non-Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 FR1  
                 FR2  
                 FR3  
                 FR4  
               
               
                 Antibody 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb11 
                 EVQLVQSGSDLKKP 
                 WVRQAPGQGLEWVG 
                 RFVFSLDTSVSTAY 
                 WGQGTLVTVSS 
               
               
                   
                 GASVKVSCKASGYT 
                 (29) 
                 LQISSLKAEDTAVY 
                 (31) 
               
               
                   
                 FT (28) 
                   
                 YCAR (30) 
                   
               
               
                   
               
               
                 mAb14 
                 EVQLVESGGGLVQP 
                 WVRQAPGKGLEWVS 
                 RFTISRDNAKNSLY 
                 WGQGTLVTVSS 
               
               
                   
                 GRSLRLSCAASGFT 
                 (33) 
                 LQMNSLRAEDTALY 
                 (31) 
               
               
                   
                 FD (32) 
                   
                 YCAK (34) 
                   
               
               
                   
               
               
                 MAb10 
                 EVQLVESGGGLVQP 
                 WVRQAPGKGLEWVA 
                 RFTISGDNAKNSLY 
                 WGQGTRVTVSS 
               
               
                   
                 GGSLRLSCAASGFT 
                 (36) 
                 LQMNSLRAEDTAVY 
                 (38) 
               
               
                   
                 FS (35) 
                   
                 YCAR (37) 
               
               
                   
               
            
           
         
       
     
     Light Chain Framework Region Sequences Non-Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 FR1  
                 FR2  
                 FR3  
                 FR4  
               
               
                 Antibody 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb11 
                 SYELTQPPSVSVAPG 
                 WYQQKPGQAPVLVVY 
                 GIPERFSGSNSGNTA 
                 FGGGTKLTVLGQP 
               
               
                   
                 QTATITC (39) 
                 (40) 
                 TLTISRVEAGDEADY 
                 (42) 
               
               
                   
                   
                   
                 YC (41) 
                   
               
               
                   
               
               
                 mAb14 
                 SYELTQPPSMSVAPG 
                 WYQQKPGQAPVLVIY 
                 GIPERFSGSNSGNTA 
                 FGGGTKLTVLGQP 
               
               
                   
                 KTARITCG (43) 
                 (44) 
                 TLTISRVEAGDEADY 
                 (42) 
               
               
                   
                   
                   
                 YC (41) 
                   
               
               
                   
               
               
                 mAb10 
                 QPVLTQPPSVSVAPG 
                 WYQQKPGQAPVLVVY 
                 GIPERFSGSNSANTA 
                 FGGGTKLTVLGQP 
               
               
                   
                 QTARITC (45) 
                 (40) 
                 TLTISRVEAGDEADY 
                 (42) 
               
               
                   
                   
                   
                 FC (46) 
               
               
                   
               
            
           
         
       
     
     VH and VL Sequences for Non-Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
             
               
                   
               
               
                 Antibody 
                 VH (SEQ ID NO) 
                 VL (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb11 
                 EVQLVQSGSDLKKPGASVKVSCKASGYTFTS 
                 SYELTQPPSVSVAPGQTATITCGGKNIGSKS 
               
               
                   
                 YAMNWVRQAPGQGLEWVGWINTNTGNPTYAQ 
                 VHWYQQKPGQAPVLVVYDDSDRPSGIPERFS 
               
               
                   
                 GFTGRFVFSLDTSVSTAYLQISSLKAEDTAV 
                 GSNSGNTATLTISRVEAGDEADYYCQVWDSS 
               
               
                   
                 YYCARESPNYYDSSGYYSGYYFDYWGQGTLV 
                 SDHRVFGGGTKLTVLGQP (48) 
               
               
                   
                 TVSS (47) 
                   
               
               
                   
               
               
                 mAb14 
                 EVQLVESGGGLVQPGRSLRLSCAASGFTFDD 
                 SYELTQPPSMSVAPGKTARITCGGNNIGSKS 
               
               
                   
                 YAMHWVRQAPGKGLEWVSGISWNSASMDYAD 
                 VHWYQQKPGQAPVLVIYYDSDRPSGIPERFS 
               
               
                   
                 SVKGRFTISRDNAKNSLYLQMNSLRAEDTAL 
                 GSNSGNTATLTISRVEAGDEADYYCQVWDSS 
               
               
                   
                 YYCAKDPAPPYCGGDCYPSFDYWGQGTLVTV 
                 RDHVVFGGGTKLTVLGQP (50) 
               
               
                   
                 SS (49) 
                   
               
               
                   
               
               
                 MAbi0 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSS 
                 QPVLTQPPSVSVAPGQTARITCGGNNIGIKS 
               
               
                   
                 YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD 
                 VHWYQQKPGQAPVLVVYDDSDRPSGIPERFS 
               
               
                   
                 SVKGRFTISGDNAKNSLYLQMNSLRAEDTAV 
                 GSNSANTATLTISRVEAGDEADYFCQVWDSS 
               
               
                   
                 YYCARGASGYYYYNYGMDVWGQGTRVTVSS 
                 SDHPGVFGGGTKLTVLGQP (52) 
               
               
                   
                 (51) 
               
               
                   
               
            
           
         
       
     
     Epitope for the Non-Neutralising Antibodies of the Invention: SEQ ID NO: 53 
       
                            SNYNIANSID IDEYNTKKKK LIKCIK            
This is a non-linear epitope corresponding to residues Ser153 to Ile163 and Asp305 to Lys319 of SEQ ID NO: 1 or 2
 
     Heavy Chain CDR Sequences for Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 CDR1  
                 CDR2  
                 CDR3  
               
               
                 Antibody 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb16 
                 SYGIS (54) 
                 WISGYDGNTNY 
                 DGPQVGDFDWQ 
               
               
                   
                   
                 AQKLQG (55) 
                 VYYYYGMDV (56) 
               
               
                   
               
               
                 mAb4 
                 NYAIN (57) 
                 GIIPIFATTNY 
                 DKHSWSYAFDI 
               
               
                   
                   
                 AQKFQG (58) 
                 (59) 
               
               
                   
               
            
           
         
       
     
     Light Chain CDR Sequences for Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                 Antibody 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
                 (SEQ ID NO) 
               
               
                   
               
             
            
               
                 mAb16 
                 RASQSINTWLA 
                 KASSLES 
                 QQYNSYLYT 
               
               
                   
                 (60) 
                 (61) 
                 (62) 
               
               
                   
               
               
                 mAb4 
                 SGSSSNIGSNTVN 
                 SNNQRPS 
                 AAWDDSLNGWV 
               
               
                   
                 (63) 
                 (64) 
                 (65) 
               
               
                   
               
            
           
         
       
     
     Heavy Chain Framework Region Sequences Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 FR1 
                 FR2 
                 FR3 
                 FR4 
               
               
                   
                 (SEQ ID 
                 (SEQ ID 
                 (SEQ ID 
                 (SEQ ID 
               
               
                 Antibody 
                 NO) 
                 NO) 
                 NO) 
                 NO) 
               
               
                   
               
             
            
               
                 mAb16 
                 AIRMTQSP 
                 WYQQKPGK 
                 GVPSRFSG 
                 FGQGTKV 
               
               
                   
                 STLSASVG 
                 APNLLIS 
                 SGSGTEF 
                 EIRRTV 
               
               
                   
                 DRVTITC 
                 (74) 
                 TLTISSL 
                 (76) 
               
               
                   
                 (73) 
                   
                 QPDDFAT 
                   
               
               
                   
                   
                   
                 YFC 
                   
               
               
                   
                   
                   
                 (75) 
                   
               
               
                   
               
               
                 mAb4 
                 QSVLTQPP 
                 WYQHLPG 
                 GVPDRFSG 
                 FGGGTKL 
               
               
                   
                 SASGTPG 
                 TAPKLLIH 
                 SKSGTSAS 
                 TVLGQP 
               
               
                   
                 LRVTISC 
                 (78) 
                 LAISGLQS 
                 (80) 
               
               
                   
                 (77) 
                   
                 EDEADYYC 
                   
               
               
                   
                   
                   
                 (79) 
               
               
                   
               
            
           
         
       
     
     Light Chain Framework Region Sequences Neutralising Antibodies of the Invention 
       
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 FR1 
                 FR2 
                 FR3 
                 FR4 
               
               
                   
                 (SEQ ID 
                 (SEQ ID 
                 (SEQ ID 
                 (SEQ ID 
               
               
                 Antibody 
                 NO) 
                 NO) 
                 NO) 
                 NO) 
               
               
                   
               
             
            
               
                 mAbl6 
                 AIRMTQSP 
                 WYQQKPGK 
                 GVPSRFSG 
                 FGQGTKV 
               
               
                   
                 STLSASVG 
                 APNLLIS 
                 SGSGTEF 
                 EIRRTV 
               
               
                   
                 DRVTITC 
                 (74) 
                 TLTISSLQ 
                 (76) 
               
               
                   
                 (73) 
                   
                 PDDFATY 
                   
               
               
                   
                   
                   
                 FC 
                   
               
               
                   
                   
                   
                 (75) 
                   
               
               
                   
               
               
                 mAb4 
                 QSVLTQPP 
                 WYQHLPGT 
                 GVPDRFSG 
                 FGGGTKL 
               
               
                   
                 SASGTPG 
                 APKLLIH 
                 SKSGTSAS 
                 TVLGQP 
               
               
                   
                 LRVTISC 
                 (78) 
                 LAISGLQS 
                 (80) 
               
               
                   
                 (77) 
                   
                 EDEADYYC 
                   
               
               
                   
                   
                   
                 (79) 
               
               
                   
               
            
           
         
       
     
     VH and VL Sequences for Neutralising Antibodies of the Invention 
       
                                                 Antibody   VH (SEQ ID NO)   VL (SEQ ID NO)                          mAb   QVQLVQSGAEVKKPG   AIRMTQSPSTLSASV           16   ASVRVSCKASGYTFT   GDRVTITCRASQSIN               SYGISWVRQAPGQGL   TWLAWYQQKPGKAPN               EWMGWISGYDGNTNY   LLISKASSLESGVPS               AQKLQGRVTMTTDTS   RFSGSGSGTEFTLTI               TSTAYMELRSLRSDD   SSLQPDDFATYFCQQ               TAVYYCARDGPQVGD   YNSYLYTFGQGTKVE               FDWQVYYYYGMDWGQ   IRRTV (82)               GTTVTVSS(81)                           mAb   EVQLVQSGAEVKKPG   QSVLTQPPSASGTPG           4   SSVKVSCKASGGTFS   LRVTISCSGSSSNIG               NYAINWVRQAPGQGL   SNTVNWYQHLPGTAP               EWMGGIIPIFATTNY   KLLIHSNNQRPSGVP               AQKFQGRVTITADES   DRFSGSKSGTSASLI               TSTAYMELSSLRSED   SGLQSEDEADYYCAA               TAVYFCARDKHSWSY   WDDSLNGWVFGGGTK               AFDIWGQGTMVTVSS   LTVLGQP               (83)   (84)                        
Epitope for the Neutralising Antibody mAb4 of the Invention: SEQ ID NO: 85
 
     
       
         
           
               
               
            
               
                   
                 KSNNNFCNRY HYDEYIHKLN IWR 
               
            
           
         
       
     
     This is a non-linear epitope corresponding to residues Lys196 to Ser197; Asn347 to Asn352; Arg357 to His365; and Lys443 to Arg448 of SEQ ID NO: 1 or 2 
     Epitope for the Neutralising Antibody mAb16 of the Invention: SEQ ID NO: 86 
     
       
         
           
               
               
            
               
                   
                 GKCIAVDAFI KKINETYDKK ICMDMKNY 
               
            
           
         
       
     
     This is a non-linear epitope corresponding to residues Gly201 to Lys219 and Lys327 to Tyr335 of SEQ ID NO: 1 or the corresponding residues in SEQ ID NO: 2 
     The Neutralising Antibody Red Epitope Bin Sequence: SEQ ID NO: 87 
       
     
       
         
           
               
             
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
            
           
         
       
     
     This is a non-linear epitope corresponding to residues Gly201 to Lys219 and Lys327 to Gln342 of SEQ ID NO: 1 or the corresponding residues in SEQ ID NO: 2 
     The Neutralising Antibody Blue Epitope Bin Sequence: SEQ ID NO: 88 
       
                            KSYNNNFCNT NKLNIWRTFQ K            
This is a non-linear epitope corresponding to residues Lys196, Ser 197, Tyr346 to Asn354 and Lys443 to Lys452 of SEQ ID NO: 1 or the corresponding residues in SEQ ID NO: 2
 
RH5.1 amino acid sequence—based on residues E26-Q526 of the 3D7 clone reference sequence, together with a C-terminal four-amino acid purification tag (C-tag: EPEA—underlined) and four mutations to delete N-linked glycosylation sequons (T40A, T216A, T286A and T299A, each in bold), i.e. residue 1 corresponds to residues 26 of SEQ ID NO: 1 (SEQ ID NO: 89)
 
                        ENAIKKTKNQ ENNL A LLPIK STEEEKDDIK NGKDIKKEID NDKENIKTNN AKDHSTYIKS  60                   YLNTNVNDGL KYLFIPSHNS FIKKYSVFNQ INDGMLLNEK NDVKNNEDYK NVDYKNVNFL 120               QYHFKELSNY NIANSIDILQ EKEGHLDFVI IPHYTFLDYY KHLSYNSIYH KSSTYGKCIA 180               VDAFIKKINE  A YDKVKSKCN DIKNDLIATI KKLEHPYDIN NKNDDSYRYD ISEEIDDKSE 240               ETDDETEEVE DSIQDTDSNH  A PSNKKKNDL MNR A FKKMMD EYNTKKKKLI KCIKNHENDF 300               NKICMDMKNY GTNLFEQLSC YNNNFCNTNG IRYHYDEYIH KLILSVKSKN LNKDLSDMTN 360               ILQQSELLLT NLNKKMGSYI YIDTIKFIHK EMKHIFNRIE YHTKIINDKT KIIQDKIKLN 420               IWRTFQKDEL LKRILDMSNE YSLFITSDHL RQMLYNTFYS KEKHLNNIFH HLIYVLQMKF 480               NDVPIKMEYF QTYKKNKPLT Q EPEA                                        505            
PfRH5 (PfRH5FL) sequence of amino acids E26-Q526 of the 3D7 clone  P. falciparum  reference sequence, with two mutations to delete N-linked glycosylation (N38Q and N214Q, shown in bold) and with an additional C-terminal AviTag™ and Strep-II® tag in tandem (SEQ ID NO: 90)
 
                        ENAIKKTKNQ EN Q LTLLPIK STEEEKDDIK NGKDIKKEID NDKENIKTNN AKDHSTYIKS  60                   YLNTNVNDGL KYLFIPSHNS FIKKYSVFNQ INDGMLLNEK NDVKNNEDYK NVDYKNVNFL 120               QYHFKELSNY NIANSIDILQ EKEGHLDFVI IPHYTFLDYY KHLSYNSIYH KSSTYGKCIA 180               VDAFIKKI Q E TYDKVKSKCN DIKNDLIATI KKLEHPYDIN NKNDDSYRYD ISEEIDDKSE 240               ETDDETEEVE DSIQDTDSNH TPSNKKKNDL MNRTFKKMMD EYNTKKKKLI KCIKNHENDF 300               NKICMDMKNY GTNLFEQLSC YNNNFCNTNG IRYHYDEYIH KLILSVKSKN LNKDLSDMTN 360               ILQQSELLLT NLNKKMGSYI YIDTIKFIHK EMKHIFNRIE YHTKIINDKT KIIQDKIKLN 420               IWRTFQKDEL LKRILDMSNE YSLFITSDHL RQMLYNTFYS KEKHLNNIFH HLIYVLQMKF 480               NDVPIKMEYF QTYKKNKPLT QGSASGLNDI FEAQKIEWHE WSHPQFEK              528            
PfRH5ΔNL amino acid sequence based on the 3D7 clone  P. falciparum  reference sequence—residues K140-K247 and N297-Q526 with two mutations to delete N-linked glycosylation sequons (T216A and T299A, each in bold) and with the addition of a C-terminal C-tag (underlined) (SEQ ID NO: 91).
 
                        KNVNFLQYHF KELSNYNIAN SIDILQEKEG HLDFVIIPHY TFLDYYKHLS YNSIYHKSST  60                   YGKCIAVDAF IKKINE A YDK VKSKCNDIKN DLIATIKKLE HPYDINNKNR  A FKKMMDEYN 120               TKKKKLIKCI KNHENDFNKI CMDMKNYGTN LFEQLSCYNN NFCNTNGIRY HYDEYIHKLI 180               LSVKSKNLNK DLSDMTNILQ QSELLLTNLN KKMGSYIYID TIKFIHKEMK HIFNRIEYHT 240               KIINDKTKII QDKIKLNIWR TFQKDELLKR ILDMSNEYSL FITSDHLRQM LYNTFYSKEK 300               HLNNIFHHLI YVLQMKFNDV PIKMEYFQTY KKNKPLTQ EP EA                     342            
PfCyRPA based on the 3D7 clone  P. falciparum  sequence and comprised amino acids D29-E362 with three mutations introduced to ablate N-linked glycosylation (S147A, T324A and T340A, in bold) and also included a C-terminal CD4 tag comprising rat domains 3 and 4 (CD4d3+4) tag followed by a hexahistidine (His6) tag (SEQ ID NO: 92)
 
                        DSRHVFIRTE LSFIKNNVPC IRDMFFIYKR ELYNICLDDL KGEEDETHIY VQKKVKDSWI  60                   TLNDLFKETD LTGRPHIFAY VDVEEIIILL CEDEEFSNRK KDMTCHRFYS NDGKEYNN A E 120               ITISDYILKD KLLSSYVSLP LKIENREYFL ICGVSPYKFK DDNKKDDILC MASHDKGETW 180               GTKIVIKYDN YKLGVQYFFL RPYISKNDLS FHFYVGDNIN NVKNVNFIEC THEKDLEFVC 240               SNRDFLKDNK VLQDVSTLND EYIVSYGNDN NFAECYIFFN NENSILIKPE KYGNT A AGCY 300               GGTFVKIDEN R A LFIYSSSQ GIYNIHTIYY ANYEGAPSTS ITAYKSEGES AEFSFPLNLG 360               EESLQGELRW KAEKAPSSQS WITFSLKNQK VSVQKSTSNP KFQLSETLPL TLQIPQVSLQ 420               FAGSGNLTLT LDRGILYQEV NLVVMKVTQP DSNTLTCEVM GPTSPKMRLI LKQENQEARV 480               SRQEKVIQVQ APEAGVWQCL LSEGEEVKMD SKIQVLSKGL NSGSLHHILD AQKMLWNHRD 540               RNLPPLAPLG PHHHHHH                                                557            
PfCyRPA based on the 3D7 clone  P. falciparum  sequence and comprised amino acids D29-E362 with three mutations introduced to ablate N-linked glycosylation (S147A, T324A and T340A, shown in bold) and also included a C-terminal 4-amino acid C-tag (EPEA, underlined) separated from the PfCyRPA sequence by a GGGGS linker (SEQ ID NO: 93)
 
                        DSRHVFIRTE LSFIKNNVPC IRDMFFIYKR ELYNICLDDL KGEEDETHIY VQKKVKDSWI  60                   TLNDLFKETD LTGRPHIFAY VDVEEIIILL CEDEEFSNRK KDMTCHRFYS NDGKEYNN A E 120               ITISDYILKD KLLSSYVSLP LKIENREYFL ICGVSPYKFK DDNKKDDILC MASHDKGETW 180               GTKIVIKYDN YKLGVQYFFL RPYISKNDLS FHFYVGDNIN NVKNVNFIEC THEKDLEFVC 240               SNRDFLKDNK VLQDVSTLND EYIVSYGNDN NFAECYIFFN NENSILIKPE KYGNT A AGCY 300               GGTFVKIDEN R A LFIYSSSQ GIYNIHTIYY ANYEGGGGS E PEA                    343            
PfP113Nt, encoding amino acids Y23-K219 of PfP113 (3D7) was expressed encoding C-terminal tags comprising CD4d3+4, a biotin acceptor peptide and a His6 tag in tandem. Mutations to deleted glycosylation are shown in bold. (SEQ ID NO: 94)
 
                        YVHNDVIKFG EENSLKCSQG NLYVLHCEVQ CLNGNNEIIH KRCNDDIEKK CNGNNKCIYF  60                   FEYELRKKTQ SFRNKNSIEI SECVESEQNE VKTSTTCLLS NSFILDEAFI QYFFFIKNKN 120               EEPVICKDGN INIKSALLHS PFCEIKLKDI SEYIRKKCDN NKECLIDPLD VQKNLLNEED 180               PCYINN A YVS VNVVCNKGAP STSITAYKSE GESAEFSFPL NLGEESLQGE LRWKAEKAPS 240               SQSWITFSLK NQKVSVQKST SNPKFQLSET LPLTLQIPQV SLQFAGSGNL TLTLDRGILY 300               QEVNLVVMKV TQPDSNTLTC EVMGPTSPKM RLILKQENQE ARVSRQEKVI QVQAPEAGVW 360               QCLLSEGEEV KMDSKIQVLS KGLNSGSLHH ILDAQKMLWN HRDRNLPPLA PLGPHHHHHH 420            
PfRH5Nt encoding amino acids F25-K140 of PfRH5 (3D7) was expressed encoding C-terminal tags comprising CD4d3+4, a biotin acceptor peptide and a His6 tag in tandem. Mutations to deleted glycosylation are shown in bold. (SEQ ID NO: 95)
 
                        FENAIKKTKN QENNL A LLPI KSTEEEKDDI KNGKDIKKEI DNDKENIKTN NAKDHSTYIK  60                   SYLNTNVNDG LKYLFIPSHN SFIKKYSVFN QINDGMLLNE KNDVKNNEDY KNVDYKGAPS 120               TSITAYKSEG ESAEFSFPLN LGEESLQGEL RWKAEKAPSS QSWITFSLKN QKVSVQKSTS 180               NPKFQLSETL PLTLQIPQVS LQFAGSGNLT LTLDRGILYQ EVNLVVMKVT QPDSNTLTCE 240               VMGPTSPKMR LILKQENQEA RVSRQEKVIQ VQAPEAGVWQ CLLSEGEEVK MDSKIQVLSK 300               GLNSGSLHHI LDAQKMLWNH RDRNLPPLAP LGPHHHHHH            
The present invention will now be described with reference to the following non-limiting Examples.
 
     EXAMPLES 
     Materials and Methods 
     Generation of Monoclonal Antibodies 
     Plasmablast Isolation and Sorting 
     Volunteers from a Phase Ia safety and immunogenicity clinical trial were bled seven days after the second immunization using MVA (modified vaccinia virus Ankara) encoding PfRH5FL. Blood was collected from volunteers in heparinized tubes and centrifuged in Leucosep tubes (Greiner Bio one) to separate the peripheral blood mononuclear cells (PBMC). The PBMC were enriched for B cells using a human pan-B cell enrichment kit (Easysep) and resuspended in DMEM before staining with a CD19 + , CD10 − , CD21 − , CD27 + , CD20 − , CD38 + , IgG fluorophore-conjugated antibody panel. Plasmablasts were single-cell sorted using a MoFlo cell sorter (Dako cytomation) into 96-well PCR plates containing 10 μL of 10 mM Tris HCl buffer containing 40 U/mL of RNase inhibitor (Promega). The study received ethical approval from the Oxfordshire Research Ethics Committee A in the UK (REC reference 14/SC/0120). The volunteers signed written consent forms and consent was verified before each vaccination. 
     Antibody Variable Gene Amplification 
     In each well of a 96-well plate containing a single antibody-secreting cell (ASC), a two-step RT-PCR was carried out with a first reverse transcription (RT) step using a Sensiscript RT kit (Qiagen) and degenerate primers 1-17 (see Table 1). 
     Next, a first PCR was performed on 1 μL of the RT reaction product using the same set of primers used before (1-17) which cover the diversity of all Vγ, Vκ and Vλ sequences using Phusion HF master mix (New England Biolabs). Following this, a nested PCR was performed using primers 18-51, also using Phusion HF master mix, (see Table 1) on 1 μL of the previous product diluted 1:100 to amplify inserts which contain plasmid-homologous extensions designed for circular polymerase extension cloning (CPEC). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 primers for antibody variable 
               
               
                 gene amplification 
               
            
           
           
               
               
               
            
               
                   
                 Primer number 
                 Primer sequence 
               
               
                   
                 (SEQ ID NO) 
                 (5′ to 3′) 
               
               
                   
                   
               
               
                   
                  1 (96) 
                 ACAGGTGCCCACTCCCAGGT 
               
               
                   
                   
                 GCAG 
               
               
                   
                   
               
               
                   
                  2 (97) 
                 AAGGTGTCCAGTGTGARGTG 
               
               
                   
                   
                 CAG 
               
               
                   
                   
               
               
                   
                  3 (98) 
                 CCCAGATGGGTCCTGTCCCA 
               
               
                   
                   
                 GGTGCAG 
               
               
                   
                   
               
               
                   
                  4 (99) 
                 CAAGGAGTCTGTTCCGAGGT 
               
               
                   
                   
                 GCAG 
               
               
                   
                   
               
               
                   
                  5 (100) 
                 GGAAGGTGTGCACGCCGCTG 
               
               
                   
                   
                 GTC 
               
               
                   
                   
               
               
                   
                  6 (101) 
                 ATGAGGSTCCCYGCTCAGCT 
               
               
                   
                   
                 GCTGG 
               
               
                   
                   
               
               
                   
                  7 (102) 
                 CTCTTCCTCCTGCTACTCTG 
               
               
                   
                   
                 GCTCCCAG 
               
               
                   
                   
               
               
                   
                  8 (103) 
                 ATTTCTCTGTTGCTCTGGAT 
               
               
                   
                   
                 CTCTG 
               
               
                   
                   
               
               
                   
                  9 (104) 
                 GTTTCTCGTAGTCTGCTTTG 
               
               
                   
                   
                 CTCA 
               
               
                   
                   
               
               
                   
                 10 (105) 
                 GGTCCTGGGCCCAGTCTGTG 
               
               
                   
                   
                 CTG 
               
               
                   
                   
               
               
                   
                 11 (106) 
                 GGTCCTGGGCCCAGTCTGCC 
               
               
                   
                   
                 CTG 
               
               
                   
                   
               
               
                   
                 12 (107) 
                 GCTCTGTGACCTCCTATGAG 
               
               
                   
                   
                 CTG 
               
               
                   
                   
               
               
                   
                 13 (108) 
                 GGTCTCTCTCSCAGCYTGTG 
               
               
                   
                   
                 CTG 
               
               
                   
                   
               
               
                   
                 14 (109) 
                 GTTCTTGGGCCAATTTTATG 
               
               
                   
                   
                 CTG 
               
               
                   
                   
               
               
                   
                 15 (110) 
                 GGTCCAATTCYCAGGCTGTG 
               
               
                   
                   
                 GTG 
               
               
                   
                   
               
               
                   
                 16 (111) 
                 GAGTGGATTCTCAGACTGTG 
               
               
                   
                   
                 GTG 
               
               
                   
                   
               
               
                   
                 17 (112) 
                 CACCAGTGTGGCCTTGTTGG 
               
               
                   
                   
                 CTTG 
               
               
                   
                   
               
               
                   
                 18 (113) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCGAGG 
               
               
                   
                   
                 TGCAGCTGGTGCAG 
               
               
                   
                   
               
               
                   
                 19 (114) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCTGAGG 
               
               
                   
                   
                 TGCAGCTGGTGGAG 
               
               
                   
                   
               
               
                   
                 20 (115) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCCAGG 
               
               
                   
                   
                 TGCAGCTGCAGGAG 
               
               
                   
                   
               
               
                   
                 21 (116) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCTGAGG 
               
               
                   
                   
                 TGCAGCTGTTGGAG 
               
               
                   
                   
               
               
                   
                 22 (117) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCCAGG 
               
               
                   
                   
                 TGCAGCTACAGCAGTG 
               
               
                   
                   
               
               
                   
                 23 (118) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCCAGG 
               
               
                   
                   
                 TTCAGCTGGTGCAG 
               
               
                   
                   
               
               
                   
                 24 (119) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCCAGG 
               
               
                   
                   
                 TCCAGCTGGTACAG 
               
               
                   
                   
               
               
                   
                 25 (120) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCTGAAG 
               
               
                   
                   
                 TGCAGCTGGTGGAG 
               
               
                   
                   
               
               
                   
                 26 (121) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCCAGG 
               
               
                   
                   
                 TACAGCTGCAGCAG 
               
               
                   
                   
               
               
                   
                 27 (122) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCCCAGC 
               
               
                   
                   
                 TGCAGCTGCAGGAG 
               
               
                   
                   
               
               
                   
                 28 (123) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCTCAGG 
               
               
                   
                   
                 TGCAGCTGGTGGAG 
               
               
                   
                   
               
               
                   
                 29 (124) 
                 GATGGGCCCTTGGTCGACGC 
               
               
                   
                   
                 TGAGGAGACGGTGACCAG 
               
               
                   
                   
               
               
                   
                 30 (125) 
                 GATGGGCCCTTGGTCGACGC 
               
               
                   
                   
                 TGAAGAGACGGTGACCATTG 
               
               
                   
                   
               
               
                   
                   
               
               
                   
                 31 (126) 
                 GATGGGCCCTTGGTCGACGC 
               
               
                   
                   
                 TGAGGAGACGGTGACCGTG 
               
               
                   
                   
               
               
                   
                 32 (127) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCTGACA 
               
               
                   
                   
                 TCCAGATGACCCAGTC 
               
               
                   
                   
               
               
                   
                 33 (128) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCAGACA 
               
               
                   
                   
                 TCCAGTTGACCCAGTCT 
               
               
                   
                   
               
               
                   
                 34 (129) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTGTGCCA 
               
               
                   
                   
                 TCCGGATGACCCAGTC 
               
               
                   
                   
               
               
                   
                 35 (130) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATGGGGATA 
               
               
                   
                   
                 TTGTGATGACCCAGAC 
               
               
                   
                   
               
               
                   
                 36 (131) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATGGGGATA 
               
               
                   
                   
                 TTGTGATGACTCAGTC 
               
               
                   
                   
               
               
                   
                 37 (132) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCAGAAA 
               
               
                   
                   
                 TTGTGTTGACACAGTC 
               
               
                   
                   
               
               
                   
                 38 (133) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCAGAAA 
               
               
                   
                   
                 TAGTGATGACGCAGTC 
               
               
                   
                   
               
               
                   
                 39 (134) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCAGAAA 
               
               
                   
                   
                 TTGTGTTGACGCAGTCT 
               
               
                   
                   
               
               
                   
                 40 (135) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTGTACATTCGGACA 
               
               
                   
                   
                 TCGTGATGACCCAGTC 
               
               
                   
                   
               
               
                   
                 41 (136) 
                 ATGGTGCAGCCACCGTACGT 
               
               
                   
                   
                 TTGATYTCCACCTTGGTC 
               
               
                   
                   
               
               
                   
                 42 (137) 
                 ATGGTGCAGCCACCGTACGT 
               
               
                   
                   
                 TTGATATCCACTTTGGTC 
               
               
                   
                   
               
               
                   
                 43 (138) 
                 ATGGTGCAGCCACCGTACGT 
               
               
                   
                   
                 TTAATCTCCAGTCGTGTC 
               
               
                   
                   
               
               
                   
                 44 (139) 
                 ATGGTGCAGCCACCGTACGT 
               
               
                   
                   
                 CTGATTTCCACCTTGGTC 
               
               
                   
                   
               
               
                   
                 45 (140) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTTCCTGGGCCCAGT 
               
               
                   
                   
                 CTGTGCTGACKCAG 
               
               
                   
                   
               
               
                   
                 46 (141) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTTCCTGGGCCCAGT 
               
               
                   
                   
                 CTGCCCTGACTCAG 
               
               
                   
                   
               
               
                   
                 47 (142) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTTCTGTGACCTCCT 
               
               
                   
                   
                 ATGAGCTGACWCAG 
               
               
                   
                   
               
               
                   
                 48 (143) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTTCTCTCTCSCAGC 
               
               
                   
                   
                 YTGTGCTGACTCA 
               
               
                   
                   
               
               
                   
                 49 (144) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTTCTTGGGCCAATT 
               
               
                   
                   
                 TTATGCTGACTCAG 
               
               
                   
                   
               
               
                   
                 50 (145) 
                 CTTTTTCTAGTAGCAACTGC 
               
               
                   
                   
                 AACCGGTTCCAATTCYCAGR 
               
               
                   
                   
                 CTGTGGTGACYCAG 
               
               
                   
                   
               
               
                   
                 51 (146) 
                 GGCTTGAAGCTCCTCACTCG 
               
               
                   
                   
                 AGGGYGGGAACAGAGTG 
               
               
                   
                   
               
            
           
         
       
     
     Cloning 
     The AbVec-hIgG1/AbVec-hIgKappa/AbVec-hIgLambda expression plasmids were a kind gift from Patrick C. Wilson (University of Chicago). These plasmids were 5′ digested using BshTI and at the 3′ using SalI (AbVec-hIgG1), XhoI (AbVec-hIgLambda) and Pfl23II (AbVec-hIgKappa) to yield linear products. CPEC assembly was done by mixing 100 ng of a 1:1 molar ratio of insert:plasmid in 20 μL containing 1× Phusion HF polymerase master mix and assembled using an 8-cycle CPEC protocol (8 cycles: 98° C. 10 s, slow ramp anneal 70° C.  55° C. at 0.1° C./s, 72° C. 35 s). Full nicked plasmids were subsequently transformed into Zymo 5a Mix &amp; go competent  Escherichia coli  (Zymo Research) according to manufacturer&#39;s instructions, streaked on LB agar petri dishes containing 100 μg/mL carbenicillin and grown at 37° C. overnight in a static incubator. Colonies were screened by PCR for inserts of the correct size. 
     Screening 
     Exponential growth-phase adherent HEK293 cells were resuspended in DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% ultra-low IgG foetal bovine serum (FBS) (all from Thermo Fischer Scientific) and seeded at 4×10 4  cells/well in 100 μL 24 h prior to transfection in Costar 96-well cell culture plates (Corning). On the day of transfection, for each well, 50 μL of 60 μg/mL linear 25 kDa PEI (Alfa Aesar) was mixed with 200 ng of cognate heavy- and light-chain coding plasmid in a volume of 50 μL and shaken at 20° C. for 30 min. The DNA-PEI complexes were then added to the HEK293 cells. The next day, an additional 50 μL of supplemented DMEM (as described above) was added to each well. Supernatants were screened for PfRH5FL binding by indirect ELISA as described in the ELISA methods section. 
     Recombinant Protein Constructs, Expression and Purification 
     The recombinant PfRH5 sequence used in all experiments except for those involving BLI ( FIGS. 1C and 4A ) was based on the 3D7 clone  P. falciparum  reference sequence and encoded amino acids E26-Q526. The sequence also encoded a C-terminal four-amino acid purification tag (C-tag: EPEA) and four mutations to delete N-linked glycosylation sequons (T40A, T216A, T286A and T299A) and was named “PfRH5FL” (this recombinant protein is also known as “RH5.1”, SEQ ID NO: 89). This protein was expressed as secreted protein by a stable monoclonal  Drosophila  S2 cell line and affinity purified using CaptureSelect™ C-tag affinity matrix (Thermo Fischer Scientific). A further size-exclusion chromatography (SEC) polishing step was done on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) to separate monomers from oligomers and contaminants as well as to buffer-exchange the protein into 20 mM Tris, 150 mM NaCl, pH 7.5. 
     The recombinant PfRH5 sequence (also named “PfRH5FL” for simplicity) used in BLI experiments ( FIGS. 1C and 4A ) also encoded amino acids E26-Q526 of the 3D7 clone  P. falciparum  reference sequence, with only two mutations to delete N-linked glycosylation (N38Q and N214Q). This sequence is identical to the vaccine sequence and was expressed with an additional C-terminal AviTag™ and Strep-II® tag in tandem (SEQ ID NO: 90). This protein was expressed in HEK293F cells as a secreted, monobiotinylated protein as described previously by Bushell et al., 2008 (Genome research, 18(4), pp. 622-630). 
     The recombinant PfRH5ΔNL sequence used was based on the 3D7 clone  P. falciparum  reference sequence and encoded amino acids K140-K247 and N297-Q526 with two mutations to delete N-linked glycosylation sequons (T216A and T299A) and with the addition of a C-terminal C-tag (SEQ ID NO: 91). PfRH5ΔNL was expressed as secreted protein from stably-transfected polyclonal  Drosophila  S2 cells. Its purification is detailed in the X-ray crystallography experimental procedures section. 
     The expression construct used for monomeric PfCyRPA production in  FIG. 4C  and for rat vaccination in  FIG. 6I  was based on the 3D7 clone  P. falciparum  sequence and comprised amino acids D29-E362 with three mutations introduced to ablate N-linked glycosylation (S147A, T324A and T364A) and also included a C-terminal GGGS linker followed by a 4-amino acid C-tag (EPEA), SEQ ID NO: 93. The PfCyRPA immunogen sequence used for rabbit vaccination in  FIG. 6E  was identical to that described above with the C-tag replaced by a C-terminal CD4 tag comprising rat domains 3 and 4 (CD4d3+4) tag followed by a hexahistidine (His6) tag (SEQ ID NO: 92). The protein was expressed as secreted protein from HEK293F cells using the Expi293™ Expression System (Thermo Fischer Scientific) according to the manufacturer&#39;s recommendations and purified by C-tag affinity chromatography followed by SEC on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare). Basigin protein comprised of immunoglobulin domains 1 and 2 of the short isoform (residues A22-H205) and was expressed from  E. coli  and purified by Ni 2+ -affinity and size exclusion chromatography. 
     PfP113Nt, encoding amino acids Y23-K219 of PfP113 (3D7) (SEQ ID NO: 94) and PfRH5Nt encoding amino acids F25-K140 of PfRH5 (3D7) (SEQ ID NO: 95), were expressed encoding C-terminal tags comprising CD4d3+4, a biotin acceptor peptide and a His6 tag in tandem. 
     Recombinant monoclonal antibodies were transiently expressed in HEK293F cells using the Expi293™ Expression System (Thermo Fischer Scientific) according to the manufacturer&#39;s recommendations. Cognate heavy and light chain-coding plasmids were co-transfected at a 1:1 ratio. Supernatants were harvested by centrifuging the culture at 2500×g for 15 min and filtering the supernatant with a 0.22 μm vacuum filter. All mAbs were purified using a 5 mL Protein G HP column (GE Healthcare) on an ÄKTA start FPLC system or an AKTA Pure FPLC system (both GE Healthcare). Equilibration and wash steps were performed with Dulbecco&#39;s PBS and mAbs were eluted in 0.1 M glycine pH 2.7. The eluates were pH equilibrated to 7.4 using 1.0 M Tris HCl pH 9.0 and immediately buffer-exchanged into Dulbecco&#39;s PBS and concentrated using an Amicon® ultra centrifugal concentrator (Millipore) with a molecular weight cut-off of 30 kDa. 
     IgG from serum in  FIGS. 3B , C, D and  FIGS. 6C , E was purified on drip columns packed with Pierce Protein G agarose resin (Thermo Fisher Scientific). Pierce protein G IgG binding buffer (Thermo Fisher Scientific) was used to dilute the serum 1:1 before loading as well as for equilibration and wash steps. Bound IgG was subsequently eluted, neutralized and concentrated as above. Bispecific DVD-Ig 1611 was constructed by cloning R5.016 variable regions upstream (5′) of R5.011 variable regions, separated by ASTKGPSVFPLAP and TVAAPSVFIFPP linkers for the heavy and light chains, respectively. 1611 DVD-Ig expression and purification was conducted as described above for monospecific mAbs. 
     X-Ray Crystallography 
     Complex Preparation 
     PfRH5ΔNL was purified from  Drosophila  S2 culture supernatant using C-tag affinity chromatography and glycosylated contaminants were removed by a subsequent lectin chromatography step with a HiTrap ConA 4B column (GE Healthcare). Disordered regions were trimmed by an overnight incubation at 20° C. with endoproteinase gluC (New England Biolabs) at a final concentration of 1 μg/mL. Fab fragments were generated by papain digestion using a Pierce™ Fab Preparation Kit (Thermo Fischer Scientific) following the manufacturer&#39;s recommendations. Complexes were prepared by mixing each Fab fragment with PfRH5ΔNL at a 1:1 molar ratio and were methylated with 1 M ABC (Borane dimethylamine complex) and 1 M formaldehyde (both Sigma-Aldrich) (Walter et al., 2006). The methylated complexes were subjected to SEC on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) at 4° C. in 20 mM Tris pH 7.4, 150 mM NaCl. The complex-containing fractions were pooled and concentrated using an Amicon® ultra centrifugal concentrator (Millipore) with a molecular weight cut-off of 30 kDa. 
     Crystallisation, Data Collection and Processing 
     Crystallization was achieved using vapour diffusion in sitting drops. Crystals were obtained for the Fab fragments of mAbs R5.004, R5.011 and R5.016 alone, as well as for complexes consisting of PfRH5ΔNER5.004:R5.016 and PfRH5ΔNL:R5.011:R5.016. In each case, a TTP Labtech Mosquito LCP robot was employed to mix 100 nL of each protein complex at a concentration of 10 mg/mL with 100 nL of well solutions from commercially available crystal screens. 
     Crystals of R5.004 Fab fragments were obtained in the JCSG-plus crystallization screen (Molecular Dimensions) and were optimized with a final well solution of 0.2 M lithium sulfate, 0.1 M NaAc pH 4.5, 30% PEG 8000 and 0.33% hexamminecobalt(III) chloride, 0.06 M MES monohydrate, 0.06 PIPES, 0.02 M HEPES chloride. They were cryo-protected by transfer into well solution supplemented with 25% glycerol, then cryo-cooled by plunging into liquid nitrogen. Data were collected on beamline 1-03 at Diamond Light Source (Harwell, UK), leading to a complete data set at a resolution of 1.7 Å. 
     Crystals of R5.016 Fab fragments were obtained in the JCSG-plus crystallization screen (Molecular Dimensions), with a final well solution of 0.2 M lithium sulfate, 0.1 M NaAc pH 4.5, 50% PEG 400. Crystals were cryo-cooled directly in the well solution by plunging into liquid nitrogen. Data were collected on beamline 104-1 at Diamond Light Source (Harwell, UK), leading to a complete data set at a resolution of 2.1 Å. 
     Crystals of R5.011 Fab fragments were obtained in the JCSG-IV crystallization screen (Qiagen), with a final well solution of 0.16 M ZnAc, 0.108 M Na cacodylate pH 6.5, 14.4% PEG 8000, 20% glycerol. They were cryo-protected by transfer into well solution supplemented with 25% glycerol, then cryo-cooled by plunging into liquid nitrogen. Data were collected on beamline PROXIMA-1 at SOLEIL (Saint-Aubin, France), leading to a complete data set at a resolution of 2.3 Å. 
     Crystals of the PfRH5ΔNL:R5.004:R5.016 complex were obtained in the JCSG-plus crystallization screen (Molecular Dimensions), with a final well solution of 0.15 M DL malic acid, 20% PEG 3350. They were cryo-protected by transfer into well solution supplemented with 25% glycerol, then cryo-cooled by plunging into liquid nitrogen. Data were collected on beamline 104 at Diamond Light Source (Harwell, UK), leading to a complete data set at a resolution of 4.0 Å. 
     Crystals of the PfRH5ΔNL:R5.011:R5.016 complex were obtained in the Morpheus crystallization screen (Molecular Dimensions), with a final well solution of 10% PEG 20000, 20% PEG 550MME, 0.02 M amino acids, 0.1 M MES/imidazole pH 6.5. Crystals were cryo-cooled directly in the well solution by plunging into liquid nitrogen. Data were collected on beamline ID23 at the European Synchrotron Radiation Facility (Grenoble, France), leading to a complete data set at a resolution of 3.6 Å. 
     In each case, data reduction was performed using XDS. Molecular replacement solutions were found for each individual Fab fragment, using PHASER with the most closely related Fab fragment structure in the PDB, split into their constant and variable domains, as search models (4KQ3 for R5.004, 4HK0 for R5.011 and 5K90 for R5.016). This led to a cycle of model building and refinement using COOT and BUSTER. For R5.004, this resulted in a complete model for the Fab fragment. For R5.011, this resulted in a complete model for the Fab fragment, with the exception of residues 143-148 in the heavy chain, which were disordered in the electron density map. For R5.016, this resulted in a complete model for the Fab fragment, with the exception of residues 103-108 in the heavy chain and 1-7, 25-29 and 55-58 in the light chain, all of which were disordered in the electron density map. 
     The structure of the PfRH5ΔNL:R5.011:R5.016 complex was obtained to 3.6 Å resolution using PHASER with the structure of PfRH5ΔNL (PDB code: 4U0R) and those of Fab fragments obtained above, split into their constant and variable domains, as search models. All domains were well resolved, although significant changes in conformation were observed in CDR H3 of both R5.011 and R5.016, as a result of PfRH5ΔNL binding. COOT and BUSTER were used for refinement, including application of restraints that derived from the higher resolution structures of PfRH5ΔNL and the shared regions of the Fab fragments. This allowed the production of a final model in which all of the Fab fragment domains, and all of the CDR loops, were clearly resolved. 
     The structure of the PfRH5ΔNL:R5.004:R5.016 complex was obtained at 4 Å resolution, using PHASER. The structure of PfRH5ΔNL bound to the variable domain of R5.016, obtained from the structure of the PfRH5ΔNL:R5.011:R5.016 complex, and the structure of the variable domain of R5.004 obtained above, were used as search models in PHASER. No significant changes were observed in the CDR loops of the Fab fragment of R5.004. Placement of the constant domains of R5.016 and R5.004 was challenging, due to anisotropy resulting from disorder within the crystal. These domains were therefore placed using real space docking after refinement in BUSTER of a structure consisting of PfRH5ΔNL and the variable domains of R5.004 and R5.016. This gave unambiguous electron density for the regions of the constant domains which lie close to the contact with the variable domains, but, after refinement, electron density is still absent for the PfRH5ΔNL-distal parts of the constant domains. Refinement in COOT and BUSTER allowed the production of a final model in which the Fab fragment domains, and all of the CDR loops, were clearly resolved. 
     Humanized Mouse Passive Transfer 
       P. falciparum  Sporozoite Production and Mouse Infection 
     The luciferase expressing strain  P. falciparum  NF54HT-GFP-luc was maintained in RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 50 μM hypoxanthine, 10% human serum and sub-cultured in 5% O+ human erythrocytes. Briefly, asexual cultures were inoculated at 1 parasitemia with no further sub-culturing and daily media changes to induce gametocytogenesis. Mature gametocytes were fed to 4-day old  Anopheles stephensi  mosquitoes to initiate infection. Mosquitoes were incubated at 27° C. and 75% humidity for 14 d and given 8% dextrose+PABA to foster parasite growth. Liver humanized FRGN KO mice were purchased from Yecuris Corp. and showed human hepatocyte repopulation levels above 70% determined by human serum albumin levels. Liver humanized mice were cycled on NTBC once a month for 3 d at 8 μg/mL in the drinking water to maintain health. Animals did not receive NTBC three weeks prior to and during the infection study. For mosquito bite infection, 3-5 liver humanized FRGN mice were anesthetised and placed on top of a mosquito cage containing 150-250 infected mosquitoes for 20 min. 
       P. falciparum  Liver-to-Blood Stage Transition 
     On the day of challenge, liver humanized FRGN mice were injected both in the retro-orbital plexus and the peritoneal cavity with 50 μL clodronate-containing liposomes (Clophosome®-A), 100 mg/kg cyclophosphamide (Sigma-Aldrich). Five days after challenge, the animals were bled a volume of approximately 200 μL, and 500 μL hRBC was injected in the retro-orbital plexus (70% O+ human erythrocytes in RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 50 μM hypoxanthine and 10% human serum). The next day, mice were bled 200 μL and received an intraperitoneal injection of 700 μL hRBC. mAb transfer was done on day 6 to precede the establishment of blood-stage infection. Dosage was 15 mg total rabbit IgG per mouse formulated in PBS and delivered intravenously. The clodronate liposome and cyclophosphamide injections were repeated on days 5, 9, 11, 13. Human RBC were injected daily in a volume of 300-700 μL to keep the percentage of hRBC stable around 50-60%. If the percentage reached more than 70%, the mice were not injected with hRBC to limit morbidity. On days 9, 11 and 13 each mouse was bled from the retro-orbital plexus to sample antibody levels. 
     Quantification of Parasite Burden 
     Luciferase activity was measured in the mice using the IVIS Lumina II animal imager (Perkin Elmer). The abdomen of mice was shaved to enhance detection. Mice were injected intraperitoneally with 100 μL of luciferase substrate RediJect D-Luciferin (Perkin Elmer) to quantitate specific enzymatic activity. Animals were anesthetized and imaged within 5 min of substrate injection. Signal was acquired for 5 min using a field of view of 10 cm and medium binning factor. Living Image 3.0 software was used to measure total flux (photons/second) of a region of interest, which was placed around each mouse. 
     This study was carried out in accordance with the recommendations of the NIH Office of Laboratory Animal Welfare standards (OLAW welfare assurance #A3640-01). The protocol was approved by the Center for Infectious Disease Research Institutional Animal Care and Use Committee (IACUC) under protocol SK-16. 
     PfCyRPA/PfP113/BSG SPR Blocking Assays 
     Data were collected on a Biacore X100 (GE Healthcare). All data used were reference subtracted from a non-immobilized flow cell (Fc 2-1). Binding values were measured manually in the Biacore X100 control software. 
     PfRH5-Basigin Binding 
     Experiments were performed at 25° C. in SPR running buffer (PBS+0.05% Polysorbate-20, GE Healthcare). Approximately 670 RU of basigin was amine-coupled to a CMS chip (GE Healthcare) on flow cell 2 (Fc 2) using standard NHS/EDC chemistry. PfRH5FL-mAb complexes were made by mixing PfRH5FL and mAb to a final concentration of 0.5 μM PfRH5FL and 1 μM mAb in SPR running buffer. Complexes were injected over Fc 1 and Fc 2 at a flow rate of 10 μL/min for 30 s and the surface was regenerated with 10 mM glycine pH 1.5 for at a flow rate of 10 μL/min for 60 s. Between experiments, one injection of 0.5 μM PfRH5FL was made to assess basigin degradation caused by regeneration. 
     PfRH5-PfCyRPA Binding 
     Experiments were performed at 25° C. in SPR running buffer (PBS+0.05% Polysorbate-20, GE Healthcare) using a sensor chip protein A (GE Healthcare). mAb was injected at a concentration of 20 nM at a flow rate of 5 μL/min for 35 s over Fc 2 on a protein A coated chip (GE Healthcare). Next, PfRH5FL was injected over Fc 1 and Fc 2 at a concentration of 50 nM at a flow rate of 10 μL/min for 120 s before injecting PfCyRPA at a concentration of 1 μM for 120 s, also at a flow rate of 10 μL/min. The chip surface was regenerated with 10 mM glycine pH 1.5 for at a flow rate of 10 μL/min for 60 s. 
     PfRH5-PfP113 Binding 
     Experiments were performed at 37° C. in SPR running buffer (PBS+0.05% Polysorbate-20, GE Healthcare) using a Sensor chip CAP (GE Healthcare). The whole experiment was run at a flow rate of 5 μL/min. Approximately 1500 RU of CAP reagent (GE Healthcare) was captured on Fc 1 and Fc 2. On Fc 1, approximately 500 RU of a biotinylated control CD4d3+4-tagged protein was immobilized. On Fc 2, each experiment consisted of capturing 1500 RU of CAP reagent in an 80 s injection followed by approximately 1800 RU of PfP113Nt in an 80 s injection at a concentration of 20 μg/mL. After this, PfRH5FL-mAb complex (both at 1 μM) was flowed over Fc 1 and Fc 2. Flow cell 2 was regenerated between experiments in a 110 s injection of 6 M guanidine+250 mM NaOH, as per the manufacturer&#39;s instructions. 
     Bio-Layer Interferometry (BLI) 
     All BLI was carried out on an OctetRED384 (Pall ForteBio) using streptavidin-coated biosensors (Pall ForteBio) to immobilize PfRH5FL enzymatically monobiotinylated on a C-terminal AviTag™. Assays were carried out in 96-well format in black plates (Greiner). For assaying mAb binding to PfRH5FL variants ( FIG. 1C ), the experiment followed a four-step sequential assay: Baseline (PBS, 30 s); Protein immobilization (neat supernatant, 180 s); Wash (PBS, 60 s); and mAb binding (150 nM mAb, 120 s). Graphed data show the fold-change in binding of each mAb to the 3D7 PfRH5FL reference protein relative to the binding of each mAb to each mutant protein after correction for PfRH5FL immobilization level on each biosensor. The fold-change values which were inferior to 1 were plotted as their inverse to avoid skewing in their data representation compared to fold-change values greater than 1. For epitope binning studies ( FIG. 4A ) the experiment followed a six-step sequential assay: Baseline (PBS, 30 s); Protein immobilization (neat supernatant, 120 s); Wash (PBS, 60 s); mAb1 binding (300 nM mAb1, 120 s); Wash (PBS, 60 s); mAb2 binding (150 nM mAb2, 120 s). “Relative binding” was calculated giving the ratio (Signal mAb2  with mAb1 bound)/(Signal mAb2  with no mAb1) where “Signal mAb2 ” was normalized for the amount of PfRH5FL bound to the biosensor such that “Signal mAb2 ”=the raw signal in “mAb2 binding” divided by the raw signal in the “Protein immobilization” phase. To establish the epitope bins, binding profiles between each mAb pair was correlated using a person product-moment correlation coefficient. mAb pairs whose binding profile correlation was &gt;0.7 were grouped into the same epitope bin. 
     ELISA 
     Qualitative mAb binding ELISAs such as those used in  FIG. 3A  were carried out by coating PfRH5FL, PfRH5ΔNL or PfRH5Nt on Maxisorp flat-bottom 96-well ELISA plates (Nunc) at 2 μg/mL in 50 μL at 4° C. overnight. To examine linear epitopes, PfRH5FL was heat-treated by incubation at 90° C. for 10 min. Plates were then washed twice with PBS-Tween 20 and blocked with 200 μL of Blocker™ Casein (Thermo Fischer Scientific) for 1 h. Next, wells were incubated with 1000 ng/mL of mAb for approximately 45 min at 20° C. then washed 4 times with PBS-Tween 20 before the addition of 504 of goat anti-human gamma-chain alkaline phosphatase-conjugated secondary antibody (goat anti-mouse IgG alkaline phosphatase-conjugated secondary antibody for QA1) (both Sigma-Aldrich) for 45 min at 20° C. Wells were then washed 6 times with PBS-Tween 20 and developed with 100 μL of PNPP substrate at 1 mg/mL (Sigma-Aldrich) and read at 405 nm. 
     Peptide ELISAs were carried out with a set of sixty-two custom-synthesized biotinylated 20-mer peptides of PfRH5 overlapping by 12 amino acids (Mimotopes). The list of peptide sequences was described previously by Payne et al., 2017 and can be found in supplementary table 4. Briefly, the peptides were designed to be oriented and tethered by their N-terminal biotinylated linker peptide (biotin-SGSG) with the exception the first peptide which contained the biotinylated linker at its C-terminus to preserve potential binding activity to the most N-terminal residues. These peptides were coated to the bottom wells of a streptavidin-coated 96-well plate in 50 4. The next day wells were blocked with 200 μL of Blocker™ Casein (Thermo Fisher) and washed five times with PBS-Tween 20. Bound mAbs were detected using an anti-human gamma-chain-specific alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich) or the mouse equivalent for QA1 in 504 for 30 min at a dilution of 1:1000 in PBS and washed six times with PBS-Tween 20 before being developed with 100 μL of PNPP alkaline phosphatase substrate for 20 min and read at 405 nm. To determine total PfRH5FL-specific IgG as in  FIGS. 3C, 3D and 3F , standardized methodology was used as described previously (Sheehy et al., 2011; Payne et al., 2017). Responses measured in AU are reported in μg/mL following generation of a conversion factor by calibration-free concentration analysis (CFCA). For the calculation of the conversion factor, see Williams et al., 2012. 
     Animal Vaccination Immunization Regimes 
     In  FIGS. 3  C, D two groups of six outbred New Zealand White rabbits were immunized three times three weeks apart and terminally exsanguinated two weeks following the final vaccination. Vaccines were formulated as equimolar doses (29 μg of PfRH5FL or 20 μg of PfRH5ΔNL per dose) in 50% v/v AddaVax™ adjuvant (InvivoGen), a squalene-based oil-in-water nano-emulsion, and administered by intramuscular (i.m.) route by GenScript (Piscataway, N.J., USA). In  FIGS. 6E and 6I , anti-PfAMA1 and anti-PfMSP1 antisera were generated in rabbits as previously reported (Douglas et al., 2011). For PfCyRPA, rabbit immunizations to generate the IgG used in  FIG. 6E  were carried out by Cambridge Research Biochemicals, UK, in compliance with the UK Animals (Scientific Procedures) 1986 Act (ASPA). New Zealand white female rabbits (n=2) were immunized i.m. on day 0 with 100 μg of PfCyRPA protein formulated in AddaVax™ adjuvant (InvivoGen) followed by two i.m. booster immunizations on days 28 and 56. Serum was collected pre-immunization (day 0) and 1 week after the final immunization on day 63. To generate the PfCyRPA-reactive IgG used in  FIG. 6I , a single Sprague Dawley rat was immunized three times on day 0, day 28, day 56 and terminally exsanguinated two weeks following the final vaccination. Vaccines were formulated as 50 μg doses in complete Freund&#39;s adjuvant for the initial vaccination and incomplete Freund&#39;s adjuvant for the following two, and administered i.m by GenScript (Piscataway, N.J., USA). GenScript holds a valid and current Animal Welfare Assurance in compliance with the Public Health Service (PHS) Policy on humane Care and Use of Laboratory Animals as granted by the Office of Laboratory Animal Welfare (OLAW). 
     Affinity Determination by SPR 
     Data were collected on a Biacore X100 (GE Healthcare). Experiments were performed at 25° C. in Dulbecco&#39;s PBS+0.05% Polysorbate-20 (GE Healthcare). A sensor chip protein A (GE Healthcare) was used to capture 50-100 RU of purified mAb diluted in SPR running buffer at a flow rate of 5 μL/min. Next, an appropriate range (typically 20 nM-0.625 nM) of six 2-fold dilutions with one replicate of PfRH5FL was injected for 90 s at 60 μL/min and dissociation was measured for 1600 s (7200 s when necessary). The PfRH5FL analyte was &gt;95% pure as assessed by SDS-PAGE. Specific binding of the PfRH5FL protein to mAb was obtained by reference-subtracting the response of a blank surface from that of the mAb-coated surface. The sensor surface was regenerated with a 60 s pulse of 10 mM glycine-HCl pH 1.5 (GE Healthcare). Sensorgrams were fitted to a global Langmuir 1:1 interaction model, allowing determination of the kinetic association and dissociation rate constants using Biacore X100 evaluation software. 
     AVEXIS Blocking Assay 
     All AVEXIS assays were conducted at the Wellcome Trust Sanger Institute, Cambridge, UK. Biotinylated monomeric bait protein was captured on streptavidin-coated flat-bottomed 96-well microtiter plates in 50 μL volumes for 1 h at 20° C. Plates were then washed 3 times with PBS and incubated with 50 μL of human PfRH5-specific mAb for 1 h at 20° C. The plates were washed 3 more times in PBS before the addition of 50 μL of pentameric β-lactamase-tagged prey proteins for a further 1 h at 20° C. All wells were subsequently washed twice with PBS-Tween 20 and then twice with PBS before the addition of 150 μL/well of the β-lactamase substrate nitrocefin. Absorbance readings were made at 485 nm. The protein constructs were all full-length ectodomains with threonine to alanine mutations to remove N-linked glycosylation sequons (except for basigin) as previously reported (Crosnier et al., 2011; Galaway et al., 2017): PfRH5 amino acids F25-Q526; PfCyRPA amino acids D29-E362; PfP113 amino acids Y23-K942; and basigin isoform 2 amino acids M1-A23 followed by G140-L322. All bait and prey proteins were expressed as fusion proteins N-terminal of a CD4d3+4 tag, a biotin acceptor peptide and a His6 tag. Prey proteins were expressed with a C-terminal with a collagen oligomeric matrix protein (COMP) peptide to promote pentamerization and a β-lactamase enzyme for quantification purposes. 
     Assay of GIA 
     All mAb GIA assays in  FIGS. 2A , B, C and  3 B were performed at the GIA Reference Center, NIAID, NIH. Test mAbs were buffer exchanged against RPMI 1640 (KD Medical) and concentrated to 6 mg/mL. The one-cycle GIA was performed at indicated concentrations of mAbs in duplicate wells and a biochemical measurement using a  P. falciparum  lactate dehydrogenase assay was used to quantify parasitemia which has been described previously (Malkin et al., 2005). GIA assays performed with rabbit polyclonal antibody and mAb combinations in  FIGS. 3C , D and  6  were performed at the Jenner Institute, Oxford using identical protocols and procedures to those at the NIAID but in triplicate wells. 
     Dot Blot 
     Briefly, 1.5 4 of anti-PfRH5 mAb, a human anti-Zaire Ebolavirus GP IgG1 mAb, recombinant PfRH5FL (all at 1 mg/mL) and PBS were spotted onto 0.2 μm nitrocellulose membrane and air-dried for 10 min. Afterwards, the membrane was blocked in 3% BSA+3% skimmed milk in PBS for 1 h and washed in PBS. It was then immersed in  P. falciparum  3D7 culture supernatant for 1 h and washed again in PBS. Bound PfRH5FL was detected by incubating the membrane in PfRH5FL-immunized rabbit serum diluted 2000-fold in PBS followed by an alkaline phosphatase-conjugated anti-rabbit IgG mAb (clone RG-96, Sigma-Aldrich) also diluted 1:2000, separated by two wash steps in PBS. After a final series of five PBS washes, the dot blot was developed with Sigmafast BCIP/NBT alkaline phosphatase substrate at 1 mg/mL (Sigma-Aldrich). 
     Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) 
     HDX-MS was performed using a Waters HDX platform composed of a liquid handling robotic setup (LEAP technologies) for sample preparation and a nano-Acquity UPLC coupled to a Synapt G2-Si (Waters) mass spectrometer. Samples were prepared by 11-fold dilutions from 7 μM of apo PfRH5FL or PfRH5FL-mAb complex in deuterated or non-deuterated 20 mM HEPES, 150 mM NaCl pH 7.4 buffer. The pH of the sample was brought down to 2.3 by adding 50% vol/vol 150 mM HCl. Non-deuterated and deuterated samples were loaded by the robot. The apo PfRH5FL protein or PfRH5FL-mAb complex was digested in-line using a pepsin-immobilized column at 20° C. The peptides generated from pepsin digestion were trapped on a micro peptide trap for 2 min for the removal of salts at a flow rate of 200 μL/min and then separated using a C18 column with a linear gradient of 5-80% acetonitrile (CH 3 CN) and water both supplemented with 0.1% formic acid for 12 min at flow rate of 40 μL/min. The liquid chromatography temperature was set at 0° C. to reduce back-exchange. Sequence coverage and deuterium uptake were analysed by using ProteinLynx Global Server (Waters) and DynamX (Waters) programmes, respectively. Peptide mapping was obtained by using nondeuterated samples in triplicates and only unique peptides present in all three data files were selected for deuterium uptake data analysis. Leucine enkephalin at a continuous flow rate of 5 μL/min was sprayed as a lock mass for mass correction. Apo PfRH5FL protein digests provided a list of 2056 peptides, after applying several selection filters and manual inspection only 127 peptides were selected for analysis. These peptides provided &gt;93% sequence coverage with many overlapping peptides. The samples were labelled for 20 s, 10 min and 2 h. All HDX-MS experiments were performed in duplicate. 
     Statistical Analyses 
     Data were analysed using GraphPad Prism version 6.07 for Windows (GraphPad Software Inc.). All tests used were described in the figure legends. In  FIGS. 2B and 3C , a four-parameter sigmoidal dose-response curve was fitted to the relationship between Log 10 (antibody concentration) and percentage GIA for each dataset and used to interpolate EC 50  values. In  FIG. 4D , the nonparametric Spearman&#39;s rank correlation coefficient (p) was used to assess a correlation between the variables K on /K off /K D  and GIA EC 30 . A two-tailed value of P&lt;0.05 was considered significant. 
     Example 1—Vaccine-Induced Human mAbs to PfRH5 
     Anti-PfRH5 mAbs were isolated from single-cell sorted plasmablasts of immunized volunteers enrolled in a first-in-human Phase Ia clinical trial of a PfRH5-based vaccine delivered using recombinant chimpanzee adenovirus and poxvirus viral-vectors (see Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Vaccination details of the three volunteers 
               
               
                 from which anti-PfRH5FL mAbs were isolated. 
               
            
           
           
               
               
               
               
               
            
               
                 VAC057 
                   
                   
                 ChAd63 PfRH5FL 
                 MVA PfRH5FL 
               
               
                 Volunteer # 
                 Trial Group 
                 Time-point 
                 Prime Dose (vp) 
                 Boost Dose (pfu) 
               
               
                   
               
               
                 1017 
                 2C 
                 Day 63 (7 days 
                 5 × 10 10   
                 2 × 10 8   
               
               
                   
                   
                 post-boost) 
               
               
                 1019 
                 2B 
                 Day 63 (7 days 
                 5 × 10 10   
                 1 × 10 8   
               
               
                   
                   
                 post-boost) 
               
               
                 2207 
                 2C 
                 Day 63 (7 days 
                 5 × 10 10   
                 2 × 10 8   
               
               
                   
                   
                 post-boost) 
               
               
                   
               
            
           
         
       
     
     Variable region (VR)-coding genes were isolated by RT-PCR and PCR and cloned into a human IgG1 scaffold. Cognate heavy-chain and light-chain plasmids were co-transfected in HEK293 cells and PfRH5-specificity was confirmed by supernatant reactivity to full-length PfRH5 protein comprising amino acids E26-Q526 (PfRH5FL) by ELISA. Seventeen genetically distinct mAbs were isolated ( FIG. 1A ). Alignment with the most similar germline VR genes in IgBLAST revealed little non-germline sequence, suggesting that PfRH5 is readily recognized with high affinity by germline B cell receptors in humans. The monovalent binding affinity of each mAb was assessed by surface plasmon resonance (SPR), with anti-PfRH5 mouse or chimeric (c) mAbs c2AC7, c4BA7, c9AD4 and QA1 included because of their extensive previous characterisation (Douglas et al., 2014, Nature communications, 2, p. 601; Wright et al., 2014, Nature, 515(7527), p. 427) ( FIG. 1B  and 
                     TABLE 3                  These affinities were generally in the       low nanomolar to high picomolar range.                                         K on     K off     K D             mAb   (M −1 s −1 )   (s −1 )   (M)                       R5.001   1.02E+06   1.65E−03   1.62E−09           R5.002   3.86E+05   4.91E−03   1.27E−08           R5.003   1.70E+06   1.86E−03   1.09E−09           R5.004   1.71E+06   1.09E−03   6.36E−10           R5.006   6.77E+05   2.34E−04   3.46E−10           R5.007   3.78E+05   3.26E−04   8.63E−10           R5.008   7.22E+05   6.39E−04   8.86E−10           R5.009   5.12E+05   2.72E−04   5.31E−10           R5.010   7.83E+05   3.63E−04   4.63E−10           R5.011   1.61E+06   1.67E−05   1.03E−11           R5.013   7.73E+05   6.02E−05   7.79E−11           R5.014   3.15E+06   &lt;2.91E−06    &lt;9.21E−13            R5.015   6.29E+05   4.23E−05   6.73E−11           R5.016   1.14E+06   4.88E−04   4.28E−10           R5.017   1.44E+06   2.91E−04   2.01E−10           R5.018   4.25E+05   6.71E−06   1.58E−11           R5.019   3.07E+06   4.72E−04   1.54E−10           c2AC7   5.16E+06   1.62E−04   3.14E−11           c4BA7   5.89E+05   2.55E−05   4.33E−11           c9AD4   3.63E+06   5.04E−04   1.39E−10           QA1   2.95E+05   7.27E−04   2.46E−09                        
Table 3: List of anti-PfRH5 mAb binding properties, related to  FIG. 1 . Association-rate (K on ), dissociation-rate (K off ) and affinity (K D ) are shown.
 
     The effect of PfRH5 polymorphism on mAb recognition was determined by measuring binding to recombinant PfRH5FL variants, each carrying one of the five most common amino acid substitutions ( FIG. 1C ). In each case, except for the C203Y substitution, the generated PfRH5FL variants carried the minor allele. All global minor allele frequencies were below 0.19 (MalariaGEN v4.0). In addition, using a dot blot against  P. falciparum  3D7 clone in vitro culture supernatant, all mAbs were shown to be able to bind parasite-expressed PfRH5 (not shown) which consists of a processed ˜45 kDa form. 
     Example 2—the Neutralizing Capacity of Human mAbs Binding PfRH5 
     To characterize the ability of the human mAbs to block merozoite entry into the RBC, they were all tested for in vitro GIA against 3D7 clone  P. falciparum . An initial screen was carried out at high concentration to broadly determine which mAbs are capable of neutralization ( FIG. 2A ). They were grouped into three categories: “GIA-high” (GIA≥75%), “GIA-low” (75%&lt;GIA&gt;25%) and “GIA-negative” (GIA≤25%). Dilution curves of GIA-high mAbs were made against the 3D7 clone parasites to determine a hierarchy of potency ( FIG. 2B ). The two most potent mAbs (R5.016 and R5.004) had EC 50  values comparable to the most potent anti-merozoite mouse-derived mAbs previously described (Douglas et al., 2014; Ord et al., 2014). A strong correlation was also observed between the association-rate (K on ) of the neutralizing mAbs (nAb) and GIA (data not shown), indicating that mAb-PfRH5 binding in the context of merozoite invasion is likely time-limited. The GIA assays were repeated against six heterologous strains and isolates originating from diverse geographical locations ( FIG. 2C ), revealing some strain-dependent differences in anti-PfRH5 mAb potency, albeit with very similar hierarchies. Indeed, GB4 was more easily neutralized by all mAbs relative to 3D7, whilst M-Camp was less easily neutralized. The reason for these in vitro differences, and their relevance to in vivo neutralization, remains to be investigated. Notably the M-Camp isolate has previously been shown to be as susceptible as 3D7 to PfRH5 vaccine-induced polyclonal human IgG from the same origin as the mAbs tested here. One notable exception was R5.017, which showed a conspicuous lack of efficacy against the FVO strain and Cp845 isolate. Sequencing the rh5 gene in these six parasites revealed that only these two carry the S197Y polymorphism ( FIG. 2D ), a substitution known to reduce binding of R5.017 from data in  FIG. 1C . This sequencing also revealed that this selection of parasites contains all five of the most common PfRH5 polymorphisms ( FIG. 2D ). 
     Example 3—all Major Neutralizing PfRH5 Epitopes are Contained in Truncated PfRH5ΔNL Protein 
     Previous crystallization of PfRH5 protein was aided by removal of two sections of disordered sequence resulting in a truncation lacking 188 of 526 residues (M1-Y139 and N248-M296), termed PfRH5ΔNL. Notably this truncated protein lacks the N-terminal region of PfRH5 (PfRH5Nt), including amino acids K33-K51 previously reported to be the minimal PfP113 binding region and thus a potential site for antibody-mediated neutralization. 
     Interestingly, all of the GIA-high mAbs and two of three GIA-low mAbs bound PfRH5ΔNL, suggesting that this construct contains the major neutralizing epitopes ( FIG. 3A ). Only R5.007 and c4BA7 were unable to bind this construct. Their binding site was mapped by ELISA to the internal disordered loop and not the N-terminal region, using an array of overlapping peptides; both of these mAbs bound the same linear PfRH5 peptide (amino acids Y242-D261) (data not shown), consistent with their recognition of heat-treated PfRH5FL protein (data not shown). Furthermore, no mAbs bound recombinant PfRH5Nt by ELISA (amino acids F25-K140) (data not shown). 
     To investigate this further, the effect of PfRH5FL or PfRH5ΔNL proteins on the GIA of total polyclonal IgG purified from sera of PfRH5FL-immunized human vaccines was assessed. The in vitro GIA could be reversed by adding 0.5 μM of recombinant PfRH5FL or PfRH5ΔNL into the IgG sample. In samples from all seven vaccinated volunteers, the GIA of their IgG was completely reversed following the addition of either PfRH5 variant protein ( FIG. 3B ), a finding which also repeated when using purified IgG from rabbits immunized with PfRH5FL (data not shown). Notably vaccine-induced IgG against PfRH5Nt was previously reported in the serum of these volunteers by ELISA, suggesting minimal or no contribution of these responses to overall GIA. 
     To confirm that PfRH5ΔNL vaccination is also capable of eliciting antibody to the most functionally relevant neutralizing epitopes present in PfRH5FL, two groups of rabbits were immunized with equimolar doses of PfRH5FL or PfRH5ΔNL. The resulting purified IgG showed comparable potency in the GIA assay across both groups, indicating that no major neutralizing epitopes are lost in PfRH5ΔNL ( FIGS. 3C , D). Passive transfer of PfRH5ΔNL-immunized rabbit IgG into humanized mice, carrying human hepatocytes and erythrocytes and challenged by  P. falciparum -infected mosquito bites, resulted in a significant reduction of blood-stage parasite burden, further serving to highlight that vaccine-induced IgG to this PfRH5 truncation is sufficient to efficaciously retard or arrest blood-stage parasitemia in vivo ( FIG. 3E ). Analysis of PfRH5-specific pAb concentrations in the serum of passively immunized mice ( FIG. 3F ) were consistent with the levels that led to control of blood-stage parasitemia, but not sterilizing immunity, in PfRH5FL-vaccinated  Aotus  monkeys (Douglas et al., 2015, Nature communications, 2, p. 601). The rigid α-helical core of PfRH5, lacking PfRH5Nt, is thus likely to contain all major neutralizing epitopes and is sufficient to recapitulate antibody raised to the full-length PfRH5 antigen. 
     Example 3—Clustering of PfRH5-Specific mAbs into Functional Groups 
     In an effort to better understand the relationship between mAb binding site and function, all mAbs were tested in pairs for their ability to bind simultaneously to monobiotinylated PfRH5FL by BLI (data not shown). This defined seven distinct epitope bins, with each bin containing mAbs that bind overlapping epitopes ( FIG. 4A ). The resulting bins strongly correlated with GIA, with the red and blue bins containing the most potent anti-PfRH5 mAbs and the purple, yellow, green and orange bins exclusively containing GIA-low or GIA-negative antibodies ( FIG. 4B ). The epitope bins also proved successful in clustering together those mAbs whose binding blocked in vitro interactions between PfRH5FL and the invasion complex protein PfCyRPA as well as the RBC receptor BSG ( FIG. 4C ). Indeed, the blue and olive bins contained all the mAbs capable of blocking BSG binding by both SPR and AVEXIS; whilst the purple, yellow and orange bins contained all the mAbs capable of blocking PfCyRPA binding in the SPR assay and the most potent blockers in the AVEXIS assay. Some incomplete blockade of the PfRH5-BSG and PfRH5-PfCyRPA interactions was seen in the AVEXIS assay for some other mAbs, with a similar result for the PfP113-PfRH5 interaction; however, these results were not replicated using SPR. Incomplete PfP113 blockade is unsurprising since no mAbs bind PfRH5 near the PfP113 binding site (data not shown). Collectively, these data highlight the vicinity of the BSG binding site, and not the PfCyRPA binding site, as the key target of growth inhibitory mAbs, and that the most potent nAbs bind to these regions with the highest association-rates ( FIG. 4D ). 
     Example 4—Identifying the Epitopes for Key Neutralizing mAbs 
     The binding modes of R5.016 (also referred to as mAb16) and R5.004 (also referred to as mAb4), the two most potent known human anti-PfRH5 nAbs, were next determined by X-ray crystallography. The structure of a co-crystal containing PfRH5ΔNL, one R5.016 Fab fragment and one R5.004 Fab fragment diffracted to a resolution of 4.0 Å ( FIG. 5A  and Table 3). High-resolution structures of unbound R5.016 and R5.004 Fab fragment crystals were also determined, with clear electron density observed for all CDR loops in the bound states, facilitating determination of the complex structure (Table 4). The R5.004 and R5.016 binding sites in solution were corroborated by hydrogen-deuterium exchange mass spectrometry (HDX-MS) using full-length PfRH5 and parental R5.016 and R5.004 mAbs ( FIG. 5B ). R5.004 binds PfRH5 towards the tip of the “kite-like” structure, contacting the N-terminus of helix 4 and each of the three loops that link the converging helices at this apex of PfRH5. These interactions are mediated by five of the CDR loops of the antibody, with only L2 not participating (Table 5). R5.016 binds predominantly to the N-terminus of helix 2 of PfRH5. The major contact is mediated by the H3 loop, which lies along the groove between helices 2 and 3. Additional interactions are mediated by the H1, H2 and L2 loops (Table 4). While the CDR loops of R5.004 are not altered upon binding (RMSD=0.58 Å aligning 59/68 CDR α-carbon atoms), four of the CDR loops of R5.016 (H3, L1, L2 and L3) show significant rearrangement upon binding (data not shown). 
     Superimposition of the R5.004 and R5.016 Fab structures on the structure of PfRH5ΔNL-BSG (PDB entry 4U0Q) reveals major overlap between the BSG binding site and R5.004 ( FIG. 5C ). In contrast, of the two copies of BSG in the PfRH5ΔNL:BSG 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Crystallographic data collection and refinement statistics of Fab fragments and complexes, related to FIGS. 5 and 7 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 R5.004 Fab 
                 R5.011 Fab 
                 R5.016 Fab 
                 PfRH5ΔNL:R5.004:R5.016 
                 PfRH5ΔNL:R5.011:R5.016 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Data 
                   
                   
                   
                   
                   
               
               
                 collection 
               
            
           
           
               
               
               
               
               
               
            
               
                 Space 
                 P1 
                 P2 1 2 1 2 1   
                 P4 1 2 1 2 
                 C 1 2 1 
                 P2 1 2 1 2 1   
               
               
                 group 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Cell 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 dimensions 
               
            
           
           
               
               
               
               
               
               
            
               
                 a, b, c 
                 55.21, 70.18, 
                 47.86, 86.38, 
                 80.08, 80.08, 
                 235.15, 58.78, 
                 140.99, 150.99, 
               
               
                 (Å) 
                 73.26 
                 127.4 
                 162.69 
                 116.79 
                 163.97 
               
               
                 α, β, γ 
                 101.42, 92.97, 
                 90.0, 90.0, 
                 90.0, 90.0, 
                 90.0, 106.41, 
                 90.0, 90.0, 
               
               
                 (°) 
                 112.47 
                 90.0 
                 90.0 
                 90.0 
                 90.0 
               
               
                 Wavelength 
                 0.9999 
                 0.9786 
                 0.92819 
                 0.9795 
                 0.9763 
               
               
                 (Å) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Resolution 
                 42.45-1.66 
                 (1.69-1.66) 
                 47.86-2.28 
                 (2.36-2.28) 
                 15.24-2.10 
                 (2.14-2.10) 
                 46.92-4.01 
                 (4.07-4.01) 
                 49.16-3.58 
                 (3.73-3.58) 
               
               
                 (Å) 
               
               
                 Total 
                 389497 
                 (18845) 
                 316738 
                 (27746) 
                 39702 
                 (1666) 
                 42257 
                 (1687) 
                 281346 
                 (41777) 
               
               
                 observations 
               
               
                 Total unique 
                 111530 
                 (5493) 
                 24770 
                 (2284) 
                 31807 
                 (1564) 
                 13131 
                 (522) 
                 30867 
                 (4579) 
               
               
                 R pim   
                 3.9 
                 (83.9) 
                 7.0 
                 (35.2) 
                 2.9 
                 (55.5) 
                 8.1 
                 (50.8) 
                 13.6 
                 (51.4) 
               
               
                 R merge  (%) 
                 6.2 
                 (131.1) 
                 17.1 
                 (86.3) 
                 10.0 
                 (173.1) 
                 12.3 
                 (78.1) 
                 30.3 
                 (114.6) 
               
               
                 R meas  (%) 
                 7.3 
                 (156.3) 
                 18.5 
                 (90.6) 
                 10.5 
                 (182.5) 
                 14.8 
                 (93.4) 
                 35.8 
                 (135.0) 
               
               
                 CC 1/2  (%) 
                 99.7 
                 (49.9) 
                 99.5 
                 (85.3) 
                 99.9 
                 (53.6) 
                 98.8 
                 (64.1) 
                 99.1 
                 (86.3) 
               
               
                 I/s(I) 
                 8.6 
                 (1.0) 
                 11.0 
                 (3.5) 
                 16.4 
                 (1.3) 
                 5.9 
                 (1.4) 
                 4.7 
                 (1.6) 
               
               
                 Com- 
                 95.7 
                 (94.5) 
                 99.3 
                 (92.7) 
                 100 
                 (100) 
                 99.0 
                 (80.7) 
                 99.7 
                 (98.4) 
               
               
                 pleteness 
               
               
                 (%) 
               
               
                 Multiplicity 
                 3.5 
                 (3.4) 
                 12.8 
                 (12.1) 
                 12.5 
                 (10.7) 
                 3.2 
                 (3.2) 
                 6.7 
                 (6.7) 
               
               
                 Refinement 
               
            
           
           
               
               
               
               
               
               
            
               
                 Reflections 
                 111570 
                 23457 
                 31616 
                 13016 
                 41714 
               
               
                 R work /R free   
                 18.3/20.4 
                 18.6/22.7 
                 20.4/22.3 
                 32.6/37.7 
                 27.4/31.0 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Number of 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 residues 
               
            
           
           
               
               
               
               
               
               
            
               
                 Protein 
                 872 
                 431 
                 413 
                 1175 
                 2343 
               
               
                 Water 
                 833 
                 238 
                 203 
                 0 
                 0 
               
               
                 Ligands 
                 7 
                 2 
                 3 
                 0 
                 0 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 R.m.s 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 deviations 
               
            
           
           
               
               
               
               
               
               
            
               
                 Bond lengths 
                 0.01 
                 0.01 
                 0.01 
                 0.01 
                 0.01 
               
               
                 (Å) 
               
               
                 Band angles 
                 1.09 
                 1.23 
                 1.11 
                 1.19 
                 1.2 
               
               
                 (°) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Ram- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 achandran 
               
               
                 plot 
               
            
           
           
               
               
               
               
               
               
            
               
                 Favored (%) 
                 97.8 
                 96.7 
                 97.3 
                 92.9 
                 93.6 
               
               
                 Allowed (%) 
                 2.2 
                 3.3 
                 2.7 
                 7.1 
                 6.4 
               
               
                 Outliers (%) 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
            
           
         
       
     
                     TABLE 5               Proposed interactions between R5.004 and PfRH5 and between R5.016 and PfRH5, related to FIG. 5                                            R5.004   PfRH5                                             FWR/CDR region   Residue (chain)   Group   Structural element   Residue (chain)   Group   Interaction type               CDR H1   Asn 31 (B)   Side chain   Helix 4   His 365 (A)   Side chain   Hydrogen       CDR H1   Asn 31 (B)   Side chain   Helix 4   Asp 361 (A)   Side chain   Hydrogen       CDR H2   Ile 52 (B)   Side chain   ⅚ loop   Trp 447 (A)   Side chain   Hydrophobic       CDR H2   Phe 55 (B)   Side chain   Helix 5   Lys 443 (A)   Side chain   Hydrophobic       CDR H2   Thr 57 (B)   Side chain   Helix 5   Lys 443 (A)   Side chain   Hydrogen       CDR H3   Asp 99 (B)   Side chain   Helix 4   Arg 357 (A)   Side chain   Hydrogen       CDR H3   His 101 (B)   Side chain   Helix 4   Glu 362 (A)   Side chain   Hydrogen       CDR H3   Tyr 105 (B)   Side chain   Helix 4   Asp 361 (A)   Side chain   Hydrogen       CDR L1   Ser 31 (C)   Side chain   ½ loop   Lys 196 (A)   Side chain   Hydrogen       CDR L1   Ser 31 (C)   Main chain   ½ loop   Ser 197 (A)   Side chain   Hydrogen       CDR L3   Trp 92 (C)   Side chain   ¾ loop   Asn 352 (A)   Side chain   Hydrogen       CDR L3   Asp 94 (C)   Side chain   ¾ loop   Asn 347 (A)   Side chain   Hydrogen       CDR L3   Asn 97 (C)   Side chain   ⅚ loop   Trp 447 (A)   Main chain   Hydrogen       CDR L3   Asn 97 (C)   Side chain   ⅚ loop   Arg 448 (A)   Main chain   Hydrogen                                 R5.016   PfRH5                                             FWR/CDR region   Residue (chain)   Group   Structural element   Residue (chain)   Group   Interaction type               CDR H1   Thr 28 (E)   Side chain   Helix 2   Lys 211 (A)   Side chain   Hydrogen       CDR H1   Ser 31 (E)   Side chain   Helix 2   Asp 207 (A)   Side chain   Hydrogen       CDR H1   Tyr 32 (E)   Side chain   Helix 2   Ala 208 (A)   Main chain   Hydrogen       CDR H2   Tyr 54 (E)   Side chain   Helix 2   Ile 204 (A)   Side chain   Hydrophobic       Heavy chain FWR3   Arg 98 (E)   Side chain   Helix 2   Glu 215 (A)   Side chain   Hydrogen       CDR H3   Pro 101 (E)   Side chain   Helix 2   Ile 204 (A)   Side chain   Hydrophobic       CDR H3   Gln 102 (E)   Side chain   Helix 2   Gly 201 (A)   Main chain   Hydrogen       CDR H3   Asp 105 (E)   Side chain   Helix 2   Lys 202 (A)   Side chain   Hydrogen       CDR H3   Tyr 111 (E)   Side chain   Helix 2   Lys 202 (A)   Side chain   Hydrophobic       CDR H3   Tyr 111 (E)   Main chain   Helix 3   Tyr 335 (A)   Side chain   Hydrogen       CDR H3   Tyr 113 (E)   Side chain   Helix 3   Asp 331 (A)   Side chain   Hydrogen       CDR H3   Tyr 113 (E)   Side chain   Helix 2   Phe 209 (A)   Side chain   Hydrophobic       CDR H3   Asp 117 (E)   Side chain   Helix 2   Lys 212 (A)   Side chain   Hydrogen       CDR L2   Leu 54 (D)   Main Chain   Helix 3   Lys 327 (A)   Side chain   Hydrogen       CDR L2   Glu 55 (D)   Side chain   Helix 2   Lys 212 (A)   Side chain   Hydrogen       CDR L2   Ser 56 (D)   Side chain   Helix 2   Lys 219 (A)   Side chain   Hydrogen                    
structure, one overlaps with R5.016 while one does not. This suggests that, while simultaneous binding is possible, the proximity of R5.016 to the BSG binding site is likely to lead to steric occlusion in the context of an intact IgG antibody and membrane attachment of both components. In comparison to previously studied murine mAbs, R5.016 shares much of its binding site with 9AD4, while R5.004 binds to a distinct epitope which shows some overlap with that of QA1. Indeed, 9AD4 and QA1 compete for binding with R5.016 and R5.004, respectively ( FIG. 4A ). These data serve to further confirm the BSG binding area and the helical face composed of helices 2 and 3 as being the two major sites of antibody-mediated neutralization on PfRH5, and identify epitopes for neutralizing antibodies that are close to human germline that are readily elicited following vaccination.
 
     Example 5—a Class of Non-Neutralizing mAb Potentiates the Effect of Anti-PfRH5, -PfCyRPA and -PfAMA1 Antibodies 
     Having observed one example of antagonism, functional assessment was continued in relation to alternative mAb pairs that bind non-overlapping epitopes on PfRH5 and which do not apparently compete for binding. Combinations of mAbs were tested in the GIA assay to determine whether such mAb combinations lead to improved inhibition of invasion either additively or synergistically. 
     Remarkably, we determined that mAb R5.011 (also referred to as mAb11) potentiated the inhibitory effect of all eight of the most potent nAbs against 3D7 clone parasites, showing a clear synergistic effect despite showing no neutralizing capacity when tested alone. This effect did not extend to GIA-low mAbs R5.001 and R5.015 or any GIA-negative mAbs ( FIG. 6B  and see  FIG. 2A ). 
     R5.011 shares the same green epitope bin as R5.014 and R5.010 ( FIG. 4A ). Notably, R5.014 was also capable of synergy, potentiating the effect of a nAb (tested here with R5.016). It is likely R5.011 and R5.014 represent a distinct type of clone that bind within the green epitope bin. 
     The synergistic effect of mAb R5.011 was investigated further. Interestingly, this was maintained when using a nAb Fab fragment or a R5.011 Fab fragment, ruling out mechanisms linked to IgG bivalency or the Fc ( FIG. 6F ). Furthermore, the dose-response curve of the nAb+R5.011 IgG combination is altered to resemble the curve shape of nAb Fab fragment alone. These data suggest a degree of self-competition of nAb IgG binding to merozoite-bound PfRH5 at higher concentrations, which appears to be relieved in the presence of mAb R5.011 ( FIG. 6F ). Synergistic effects were also seen upon addition of R5.011 mAb to polyclonal IgG from PfRH5-vaccinated human volunteers ( FIG. 6C ), as well as rabbits (data not shown), indicating that this potentiating phenomenon is far from being maximized in these naturally-elicited vaccine-induced pAb. 
     Dilutions of nAb in the presence of a large excess of R5.011 revealed the maximum possible synergistic effect. Under these conditions, the EC 80  of R5.016 is reduced from 500 μg/mL to ˜55 μg/mL and, likewise, the EC 80  of R5.004 is reduced from 550 μg/mL to ˜55 μg/mL ( FIG. 6D ). Furthermore, titrating R5.011 into several fixed concentrations of nAb showed that this maximal potentiating effect is achieved from ˜200 μg/mL of R5.011 ( FIG. 6G ). The optimal ratio of nAb:R5.011 across the concentration range was approximately 4:1, with nAb-biased ratios being more effective at lower concentrations and all ratios roughly equivalent at concentrations above 100 μg/mL ( FIG. 6H ). 
     It was also investigated whether the effect of R5.011 was PfRH5-specific and the potential for synergy with antibodies targeting merozoite invasion ligands involved in chronologically distinct steps of the RBC invasion process. The addition of R5.011 mAb to polyclonal IgG from rabbits or rats immunized with PfRH4, PfRipr, PfAMA1 or PfCyRPA leads to substantially increased GIA ( FIGS. 6E and 8  A and B), whereas its addition to anti-PfMSP1 IgG had no effect ( FIGS. 6E and 8A  and B). These data suggest R5.011-like antibodies can act synergistically with other functional antibodies targeting the PfRH5 invasion complex antigens as well as downstream targets such as PfRH4 and PfAMA1 involved in tight junction formation; whereas this effect is not observed for antibodies targeting antigens that function upstream of the PfRH5-BSG interaction, such as PfMSP1 involved with initial RBC attachment events. Supporting this conclusion, the addition of R5.011 mAb to polyclonal IgG from rabbits immunized with PfRipr also leads to substantially increased GIA ( FIGS. 6E and 8 ). 
     Finally, it was investigated whether the functionality of R5.016 and R5.011 can be combined into a single molecule, by production of a bispecific dual variable domain immunoglobulin (DVD-Ig™) containing both the variable domains of R5.011 and R5.016. To achieve high levels of growth inhibition, a R5.016+R5.011 mAb combination required fewer total molecules than R5.016 alone ( FIG. 6I ). Furthermore, these data show that approximately half the molar concentration of the 1611 DVD-Ig is required to achieve the same levels of GIA as compared to the parental R5.016+R5.011 mAb combination, hinting to a bivalent binding mode of this DVD-Ig™ molecule. It is believed that this DVD-Ig™ shows the lowest reported EC 80  for an anti-merozoite antibody-like molecule. Because high levels of merozoite neutralization are known to be required for protection (Douglas et al., 2015), the antibody EC 80  or EC 90  may prove to be a more useful measure of efficacy than the more widely used EC 50 . 
     Overall, these data identify R5.011 as an example of a class of antimalarial mAb that displays a new functionality by potentiating the effect of all tested anti-PfRH5 nAbs and pAb, as well as pAb directed against other merozoite antigens, despite having no intrinsic neutralizing properties when tested alone. This raises the critical importance of inducing such antibodies in next-generation PfRH5-based vaccination strategies. 
     Example 6—the Epitope for Potentiating mAb R5.011 Lies at the N-Terminus of PfRH5ΔNL 
     To identify the R5.011 epitope, a crystal structure of PfRH5ΔNL bound to one R5.011 Fab fragment and one R5.016 Fab fragment was determined to a resolution of 3.6 Å, with all CDR loops clearly resolved ( FIG. 7A  and Table 4). A high-resolution structure of the unbound R5.011 Fab fragment was also determined and used, together with structures of R5.016 Fab fragment and PfRH5ΔNL, to provide model restraints (Table 4). R5.011 binds PfRH5 primarily at the interface between the disordered N-terminus and the rigid α-helical core at residues Y155-L162, a finding also confirmed by HDX-MS ( FIG. 7A  lower left inset box). Residues F144-N159 of PfRH5 are variably ordered in different crystal structures but most frequently show disorder. R5.011 binds to these residues and constrains and orders their conformation as they emerge from the α-helical core of PfRH5 ( FIG. 7C ). All of the CDR loops except L3 make contact to PfRH5, with the predominant interaction mediated by H3 (Table 6). Indeed, this is accompanied by a major change in the conformation of the H3 CDR loop of R5.011, which packs against residues Y155-D162 upon binding ( FIG. 7D ). 
     The position of R5.016 in PfRH5ΔNL:R5.011:R5.016 is equivalent to that in PfRH5ΔNL:R5.004:R5.016. There is no structural change in the binding site of R5.016 or R5.004 when R5.011 is bound, ruling out an allosteric mechanism for potentiation.  FIG. 7B  shows an overlay of R5.004, R5.011 and R5.016 Fab fragments bound to PfRH5ΔNL, radiating in different directions. Thus R5.011 accesses an epitope largely devoid of secondary structure at a new site on PfRH5. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Proposed interactions between R5.011 and PfRH5, related to FIG. 7 
               
            
           
           
               
               
               
            
               
                 R5.011 
                 PfRH5 
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 FWR/CDR region 
                 Residue (chain) 
                 Group 
                 Structural element 
                 Residue (chain) 
                 Group 
                 Interaction type 
               
               
                   
               
               
                 CDR H1 
                 Thr 30 (C/H) 
                 Main chain 
                 N-terminal coil 
                 Asn 154 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H1 
                 Ser 31 (C/H) 
                 Side chain 
                 N-terrninal coil 
                 Ser 153 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 CDR H1 
                 Ser 31 (C/H) 
                 Main chain 
                 N-terminal coil 
                 Tyr 155 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 CDR H1 
                 Tyr 32 (C/H) 
                 Side chain 
                 N-terminal coil 
                 Tyr 155 (A/F) 
                 Side chain 
                 Hydrophobic 
               
               
                 CDR H2 
                 Asn 52 (C/H) 
                 Side chain 
                 N-terminal coil 
                 Asn 156 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H2 
                 Thr 53 (C/H) 
                 Main chain 
                 N-terminal coil 
                 Asn 156 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H2 
                 Thr 53 (C/H) 
                 Side chain 
                 N-terminai coil 
                 Asn 156 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H2 
                 Asn 54 (C/H) 
                 Main chain 
                 N-terminal coil 
                 Asn 156 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Asn 102 (C/H) 
                 Main chain 
                 N-terminai coil 
                 Asn 159 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Tyr 104 (C/H) 
                 Main chain 
                 Beta-strand 1 
                 Ile 161 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Ser 106 (C/H) 
                 Main chain 
                 Beta-strand 1 
                 Ile 161 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Ser 106 (C/H) 
                 Side chain 
                 Beta-strand 1 
                 Ile 163 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Ser 107 (C/H) 
                 Side chain 
                 Helix 3 
                 Lys 311 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Tyr 109 (C/H) 
                 Side chain 
                 Helix 3 
                 Asn 308 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Ser 111 (C/H) 
                 Side chain 
                 Helix 3 
                 Lys 319 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR H3 
                 Tyr 114 (C/H) 
                 Side chain 
                 Helix 3 
                 Lys 319 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR L1 
                 Gly 28 (B/G) 
                 Main chain 
                 Helix 3 
                 Lys 312 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR L1 
                 Gly 28 (B/G) 
                 Main chain 
                 Helix 3 
                 Asn 308 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR L1 
                 Ser 29 (B/G) 
                 Side chain 
                 Helix 3 
                 Asn 308 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR L2 
                 Asp 49 (B/G) 
                 Side chain 
                 Helix 3 
                 Lys 316 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR L2 
                 Asp 50 (B/G) 
                 Side chain 
                 Helix 3 
                 Lys 312 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 CDR L2 
                 Asp 52 (B/G) 
                 Main chain 
                 Helix 3 
                 Lys 316 (A/F) 
                 Main chain 
                 Hydrogen 
               
               
                 Light chain FWR3 
                 Asn 65 (8/G) 
                 Side chain 
                 Helix 3 
                 Lys 312 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                 Light chain FWR3 
                 Gly 67 (B/G) 
                 Main chain 
                 Helix 3 
                 Asp 305 (A/F) 
                 Side chain 
                 Hydrogen 
               
               
                   
               
            
           
         
       
     
     Example 6—mAb R5.011 Potentiates Anti-Merozoite nAbs by Increasing the Time Required for Invasion 
     Examples 4 demonstrates the ability of R5.011 to potentiate the activity of nAbs which bind to various merozoite invasion proteins, and Example 2 demonstrates that antibody on-rate is likely a critical determinant of neutralizing efficacy. Therefore, it was next decided to investigate whether this mAb slows down the invasion process, thus giving longer for these many different nAbs to reach their binding sites. 
     Live-cell imaging revealed that the time taken for merozoites to invade RBC is significantly longer (around three-fold) in the presence of R5.011 versus a control mAb targeting Ebolavirus surface protein ( FIG. 9A, 10A ) and versus non-neutralizing, non-potentiating anti-PfRH5 mAb R5.009 ( FIG. 10B ). Concentrations of R5.016 around the EC50 did not slow the invasion of those merozoites which were able to invade, whereas high concentrations of R5.016 blocked invasion altogether ( FIG. 10B ). The observed delay is largely attributed to the phase of invasion preceding merozoite penetration ( FIG. 9B, 10A ), when invasion ligands PfRH4, PfRH5, PfCyRPA, PfRipr and PfAMA1 are thought to act. Therefore R5.011 lengthens the exposure window of critical merozoite targets to nAbs by slowing RBC entry, thus increasing their opportunity to bind and prevent invasion. 
     Discussion 
     The Examples herein determine the features of a desirable human antibody response to the most advanced blood-stage malaria vaccine candidate antigen, by relating the functions of mAbs to their binding sites on PfRH5. The existence of different classes of anti-PfRH5 mAb are revealed including highly-neutralizing mAbs and a novel class of synergistic non-neutralizing mAbs with the unexpected ability to potentiate the invasion inhibition properties of all other neutralizing mAbs, including others that target different invasion proteins such as PfCyRPA and PfAMA1, by slowing invasion. 
     All of these mAb clones were elicited by PfRH5FL vaccination in humans using a modestly immunogenic viral-vectored vaccine delivery platform. Indeed, the panel of mAbs was limited in size as a result of relatively low B cell induction, a feature typical of viral-vectored vaccination. It is therefore possible that not all mAb classes have been explored and, furthermore, that more potent members of each mAb class may exist, as determined by their association-rate within that class. However, more potent mAbs may be uncommon, as K on  values for the mAbs identified here approach the kinetic limits commonly seen in normal B cell development post-vaccination and there is no particular inbuilt in vivo mechanism to select for B cells expressing ultra-high association-rate mAbs (or indeed ultra-slow dissociation-rate mAbs) during affinity maturation. The fact that antibody association-rate, as opposed to the often-measured dissociation-rate, emerged as a key indicator of antibody potency implies highly dynamic processes are involved in invasion. This relationship has been noted before for a viral pathogen; nevertheless, this is an often-neglected parameter that appears highly relevant to malaria vaccine design. Minimal deviation from germline sequences was noted in these mAb clones, potentially related to: i) their isolation from plasmablasts 7 days post-booster vaccination rather than the memory B cell pool, or ii) their isolation after only two PfRH5 vaccinations, and/or iii) their induction by a modestly immunogenic vaccine delivery platform. 
     Three epitope bins contained GIA-high mAbs: red, blue and olive. The mechanism of action of those contained in the blue and olive bins is undoubtedly blockade of BSG binding, supported by in vitro SPR and AVEXIS assays as well as X-ray crystallography data for exemplar mAb R5.004. However, the crystal structure showed that, unlike R5.004, mAb R5.016 (from the more potent red bin) and BSG can simultaneously bind PfRH5 in solution. The question of why nAbs such as R5.016 are more potent than overt BSG-blocking nAbs such as R5.004 leaves room for additional biological mechanisms to be defined. Experiments in  FIG. 4C  were designed to answer this question, and revealed that mAbs of the red epitope bin interfere little with binding to BSG, PfCyRPA or PfP113. Without being bound by theory, for now the most likely explanation for their efficacy seems linked to their close proximity of binding on PfRH5 to the BSG binding site. With both PfRH5 and BSG constrained through attachment to their cognate membrane, R5.016 whole IgG molecules and other similar antibodies probably act by preventing PfRH5 from binding to BSG. If so, their improved potency over the BSG-blocking nAbs could be due to increased ease of epitope access on merozoite-bound PfRH5. This is supported by the data which demonstrate a three-fold lengthening of invasion time correlates with a potentiation of nAb activity. 
     R5.011 is not inhibitory in the time scale of a GIA experiment (one single cycle of blood-stage invasion and growth), it probably mediates its function by increasing the likelihood of nAb binding to PfRH5. This effect is not due to an R5.011-induced increase in the binding affinity for nAbs, as the crystal structures revealed that the R5.004 and R5.016 binding sites are unaltered (&lt;0.5 Å RMSD aligned over 39 and 30 α-carbon atoms, respectively) by the presence of R5.011. Furthermore, the binding affinity and K on  of nAbs R5.004 and R5.016 for PfRH5 are unchanged by R5.011 binding, dismissing structural allostery as a mechanism of anti-PfRH5 nAb potentiation. Thus, R5.011 accesses an epitope largely devoid of secondary structure at a new site on PfRH5. Moreover, the effect occurs for all GIA-high nAbs tested as well as R5.007, and these bind four distinct epitope regions on PfRH5.Instead R5.011 reduces the rate of RBC invasion by the merozoite, such that nAbs have longer to access to their epitopes on PfRH5 and are thus more likely to mediate their function. In agreement with this view, R5.011 enhances the effect of antibodies binding merozoite proteins thought to act at the time of PfRH5-BSG binding or afterwards, but not before, consistent with R5.011 causing a retardation in the merozoite invasion process. This is supported by the data which demonstrate that R5.011 significantly slows the invasion process, allowing more time for nAb to bind to their epitopes on various merozoite surface proteins. The ability to delay parasite invasion would represent a highly novel and attractive means of improving vaccine-induced neutralizing antibody efficacy. 
     One possible mechanism is suggested by the proximity of the R5.011 binding site to the predicted N-terminal cleavage site of PfRH5, the processing of which yields the ˜45 kDa fragment. Indeed, if proteolytic cleavage of PfRH5 is necessary for successful merozoite invasion and if R5.011 binding reduces this cleavage rate but not below the rate necessary for successful invasion, protease occlusion would be a plausible mechanism to explain this synergistic cooperation between antibodies (again, not being bound by this theory). Cooperativity between pairs of mAbs targeting a single pathogen protein has been described before, whereby two mAbs specific for the same protein display synergistic neutralizing effects. For instance, cooperativity involving non-neutralizing mAbs to Ebolavirus glycoprotein has been described, as it has previously been with  Neisseria meningitidis  fHBP. However, to the inventors&#39; knowledge, non-neutralizing antibodies like R5.011 that specifically potentiate multiple neutralizing antibodies against the same and other antigenic targets have not been described for other pathogens. 
     Whatever the mechanism (and the present invention is not bound by theory in this regard), R5.011-like mAbs may explain the high potency of anti-PfRH5 pAb. Indeed the 3D7  P. falciparum  GIA EC 50  of anti-PfRH5-specific polyclonal IgG has previously been reported to be ˜9 μg/mL across eight vaccinated volunteers, equivalent to that of the most potent of all known anti-PfRH5 mAbs (red epitope bin) and substantially lower than reported for historical vaccine candidates such as PfMSP1 and PfAMA1. The possibility of ultra-potent serum antibody clones contributing most of the GIA at low concentrations in all eight volunteers cannot be excluded, however, synergistic functional interactions between mAbs in the polyclonal IgG seems a more likely explanation. Given that two of the seventeen mAbs in this study displayed potentiating activity, the induction of such clones does not appear to be a rare event following human immunization with PfRH5. Notably, the results herein demonstrate there was still substantial room for improvement through spiking in mAb R5.011 to all the vaccine-induced human pAb samples, suggesting new avenues to maximize the potency of next-generation PfRH5-based vaccines destined for clinical development. 
     Overall, the data presented here support the view that vaccine-induced anti-PfRH5 pAb growth inhibition represents a synthesis between the neutralizing effect of nAb and synergy from R5.011-type antibodies. Immuno-focusing on these key epitope regions will be critical for next-generation PfRH5 vaccine design, and the ability of the GIA assay to correlate with in vivo protection in NHP, as well as identify anti-PfRH5 mAb clones with in vivo efficacy against  P. falciparum  in a humanized mouse model, further underpins this strategy. 
     Notably, we demonstrated there was still substantial room for improvement through spiking in mAb R5.011 to all the vaccine-induced human pAb samples, suggesting new avenues to maximize the potency of next-generation PfRH5-based vaccines destined for clinical development. Although relatively high concentrations (around 200 μg/mL) of R5.011 are needed to leverage this effect to its fullest, antigen-specific antibody titers of this magnitude can be achieved in humans. Alternatively, delivering potentiating antibody clones alongside a traditional vaccine using vectored immunoprophylaxis could be a viable alternative. 
     The development of synergistic and functionally important “non-neutralizing” epitopes challenges the paradigm of structural vaccinology which traditionally sees the advancement of epitopes against which overtly neutralizing antibodies are directed. It is therefore important that future immunogen design strategies for the development of PfRH5-based vaccines use the structural insights obtained here to focus the human immune response to generate both neutralizing and potentiating antibodies. These data thus provide a strategy to design effective PfRH5-based blood-stage malaria vaccines that provide functional anti-merozoite immunity at lower overall concentrations of PfRH5-specific human IgG.