POLYPEPTIDES FIELD OF THE INVENTION The present invention relates to polypeptides 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 ~620,000 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 immuno-prophylaxis development (e.g., vaccines or antibody therapeutics). There are only two malarial vaccines approved by the WHO, RTS,S/AS01 (Mosquirix™) and R21/Matrix M, both of which are based on the malarial circumsporozoite protein and act by blocking the pre-erythrocytic stage of P. falciparum. However, the RTS,S/AS01 (Mosquirix™) vaccine has achieved only partial efficacy (~30-50% in phase II/III clinical trials), with the R21/Matrix M vaccine achieving higher efficacy (~75% in a phase IIb trial). There continues to be an urgent need for improved vaccine immunogens as the world aims for malaria eradication. 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. Research has also been ongoing to identify other candidate malarial antigens for use as an anti-malarial immuno-prophylactic therapy. 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 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 other erythrocyte antigens and remain effective at lower concentrations of immunoglobulin. In addition, PfRH5 induces antibodies which are effective against genetically diverse strains of the Plasmodium falciparum parasite. Therefore, PfRH5 is a promising target antigen for an anti-malarial immuno-prophylactic.
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. The inventors previously manufactured an insect cell expressed recombinant protein vaccine, known as RH5.1. This vaccine works by eliciting polyclonal antibodies that block parasite invasion of red blood cells and has demonstrated a reduction in parasite growth in vivo and a delay to the time of diagnosis following a human blood-stage malaria challenge in Phase I/IIa clinical trials in UK adults. This result is the first blood-stage malaria vaccine to demonstrate a reduction in parasite growth in humans and hence it is of significant interest to the malaria field to develop new vaccines that can improve upon RH5.1. The present invention addresses one or more of the above needs by providing a polypeptide, conjugate, isolated nucleic acid, vector and composition comprising a Reticulocyte-binding protein Homologue 5 (PfRH5) epitope presented in a correctly folded form on a scaffold, which demonstrate that a highly focused single epitope vaccine higher elicits quality antibody response in comparison to wild-type PfRH5. SUMMARY OF THE INVENTION As described herein, the present inventors have for the first time designed a synthetic immunogen on which an epitope which elicits antibodies with high growth inhibitory activity against Plasmodium falciparum is presented on a polypeptide scaffold. The inventors have demonstrated that this synthetic immunogen is correctly folded and induces antibodies that are growth inhibitory at concentrations orders of magnitude lower than those required when equivalent antibody responses are induced using the full-length antigen. In particular, the present inventors have developed synthetic immunogens each comprising an epitope mimic for one of numerous growth inhibitory PfRH5 antibodies, including R5.034. In the synthetic immunogen, the epitope is presented on a helical scaffold, which comprises three α-helices, two of which are re-surfaced to present the epitope. The remainder of the immunogen has been re-designed to ensure that the residues which form the epitope adopt a structure which matches that of the epitope in intact PfRH5. The inventors have found that these synthetic immunogens give rise to high quality antibody responses, with the immunogen comprising the R5.034 epitope mimic induing PfRH5-targeting antibodies that inhibit parasite growth at a thousand-fold lower concentration that those induced through immunisation with full-length PfRH5. The inventors have also demonstrated that priming with the synthetic immunogen
comprising a focus epitope, followed by boosting with full-length PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth- inhibitory response. Accordingly, the present invention provides a polypeptide comprising a Reticulocyte- binding protein Homologue 5 (PfRH5) epitope and a scaffold; wherein: (a) the epitope comprises or consists of amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1; and (b) the scaffold comprises at least two α-helices. Said epitope may be grafted onto the scaffold. Said PfRH5 epitope may specifically bind at least one antibody selected from the group selected from: R5.034, 9AD4 and R5.016, preferably wherein the epitope specifically binds to R5.034, 9AD4 and R5.016. Said PfRH5 epitope as presented on the scaffold protein may comprise or consist of a consensus sequence of SEQ ID NO: 3 and a consensus sequence of SEQ ID NO: 4. Said polypeptide, wherein (a) the consensus sequence of SEQ ID NO: 3 may correspond to a first portion of the epitope presented on the first α-helix of the scaffold; and (b) the consensus sequence of SEQ ID NO: 4 may correspond to a second portion of the epitope presented on the second α-helix of the scaffold. Said polypeptide may comprise or consist of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 5. Said polypeptide may comprise at least two cysteine residues positioned such that they form a disulphide bridge in the polypeptide. Said at least two cysteine residues may be positioned at (a) residue 9 and residue 90 of the polypeptide; or (b) residue 44 and residue 116 of the polypeptide; or (c) residue 9 residue 90, residue 44 and residue 116 of the polypeptide. Said polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. Said at least two α-helices may have been resurfaced to present the PfRH5 epitope on the scaffold such that the amino acid residues of the PfRH5 epitope have the same relative three-dimensional conformation as in PfRH5. Said scaffold may comprise three α-helices, and wherein said amino acid residues of the PfRH5 epitope may be presented on two of the three α-helices and have the same relative three-dimensional conformation as in PfRH5. Said polypeptide may be multimerized, preferably trimerized.
The invention further provides a conjugate comprising a polypeptide according to the invention which is conjugated to a virus-like particle. Said virus-like particle may be directly conjugated to the polypeptide according to the invention. Said virus-like particle may be I53-50. Said virus-like particle may be conjugated to the polypeptide via a spy-tag, preferably wherein the virus-like particle is attached to the N-terminus of the polypeptide via a spy-tag. Said polypeptide according to the invention or conjugate according to the invention may induce PfRH5-specific antibodies that have a growth inhibitory activity (GIA) of at least 50%, preferably wherein the EC30 GIA value of said antibodies is lower than 100 ng/mL. Said polypeptide according to the invention or conjugate according to the invention may induce antibodies that have a growth inhibitory quality which is at least 100-fold greater than wild-type PfRH5, preferably at least 500-fold greater than wild-type PfRH5, more preferably wherein said polypeptide or conjugate induces antibodies that have a growth inhibitory quality that is at least 1000-fold greater than wild-type PfRH5. Said polypeptide according to the invention or conjugate according to the invention may induce PfRH5-specific antibodies which specifically bind to the PfRH5 epitope with a binding affinity in the range of 50 pM to 200 nM range. Said polypeptide according to the invention or conjugate according to the invention may have a melting temperature of greater than 75°C. The invention further provides an isolated nucleic acid sequence, encoding the polypeptide according to the invention, or the conjugate according to the invention. The invention further provides a vector encoding a polypeptide according to the invention, or the conjugate according to the invention, optionally wherein said vector comprises a nucleic acid sequence according to the invention. Said vector may be capable of expression in a mammalian cell. Said vector may be capable of expression in a heterologous protein expression system. Said vector may be a viral vector, optionally wherein 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 further provides a host cell comprising an isolated nucleic acid sequence according to the invention or a vector according to the invention. The invention further provides a composition comprising (a) one or more polypeptides according to the invention; (b) one or more conjugates according to the invention; and/or one or more vector according to the invention; and optionally comprising one or more of a pharmaceutically acceptable excipient, diluent or carrier.
Said composition may comprise one or more vector selected from a viral vector, RNA vaccine, or DNA plasmid. The invention further provides a polypeptide, conjugate, nucleic acid, vector or composition according to the invention for use in therapy. The invention further provides a polypeptide, conjugate, nucleic acid, vector or composition according to the invention for use in treating and/or preventing malaria. Said polypeptide, conjugate, nucleic acid, vector or composition according to the invention, wherein the treatment and/or prevention may comprise (a) administering the polypeptide, conjugate, nucleic acid, vector or composition to a patient; and (b) subsequently administering at least one dose of a full-length PfRH5 antigen which comprises or consists of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1, wherein preferably the full- length PfRH5 antigen is a thermostable form of PfRH5, optionally comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 17. The invention further provides the use of a polypeptide, conjugate, nucleic acid, or vector according to the invention in the manufacture of a medicament for the prevention and/or treatment of malaria. Said use of the polypeptide, isolated DNA molecule, vector or composition according to the invention, wherein the treatment and/or prevention may comprise (a) administering the polypeptide, conjugate, nucleic acid, vector or composition to a patient; and (b) subsequently administering at last one dose of a full-length PfRH5 antigen which comprises or consists of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1, wherein preferably the full- length PfRH5 antigen is a thermostable form of PfRH5, optionally comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 17. The invention further provides a polypeptide, conjugate, nucleic acid, vector or composition according to the invention for use in immunising a subject, wherein the polypeptide, conjugate, nucleic acid, vector or composition results in anti-PfRH5 antibodies with a growth inhibitory activity (GIA) of at least 50% against the blood-stage Plasmodium parasite; wherein optionally, the Plasmodium parasite is Plasmodium falciparum. DESCRIPTION OF THE FIGURES Figure 1 - Design of a synthetic epitope mimic displaying the 9AD4 antibody epitope a) The structure of PfRH5 bound to monoclonal antibody 9AD4. b) Surface representation of
PfRH5. In the right-hand panel, residues coloured dark grey are those which directly contact 9AD4 while the left-hand panel shows residues defined as forming the broader 9AD4 epitope. c) The left-hand panel shows the structure of PfRH5 with the epitope residues coloured dark grey. The right-hand panel shows the designed synthetic immunogen with the grafted residues in dark grey. d) The location of two disulphide bonds introduced to stabilise the synthetic immunogen are labelled CC1 and CC2. e) Size exclusion chromatography traces obtained for the twelve designs. f) KD values obtained from surface plasmon resonance analysis of the binding of the twelve synthetic designs to immobilised antibody 9AD4 with error bars representing the span of the values measured. Figure 2 - The epitope mimic binds growth-neutralising antibodies 9AD4, R5.016 and R5.034. a) The structure of PfRH5 bound to the Fab fragment of growth-inhibitory monoclonal antibody R5.034. The inset shows a close-up on the epitope region. b) An alignment of the structures of PfRH5 bound to the Fab fragments of 9AD4, R5.016 and R5.034 as surfaces. Each antibody is shown in surface representation. The right-hand panel shows that all three antibodies bind to the two helices recapitulated in the epitope mimic, while approaching it from different angles. c) The left-hand panels shows a circular dichroism trace of RH5-34EM. The right-hand panel shows the effect of increasing temperature on the ellipticity at 208nm of RH5DNL, thermally stabilised RH5_HS1 and RH5- 34EM, showing temperature stability. Surface plasmon resonance traces of PfRH5 and RH5- 34EM binding to d) 9AD4; e) R5.016; f) R5.034. Figure 3 - Structures of RH5-34EM bound to antibodies R5.016 and R5.034. Crystal structures of RH5-34EM bound to the scFv fragment of antibody a) R5.016 and b) R5.034 viewed from two directions. c) Alignments of antibody-bound structures of RH5-34EM and PfRH5. The left-hand panel shows an alignment of the R5.016-bound forms of RH5-34EM and PfRH5. Side chains that contact R5.016 are shown as sticks and are labelled according to the numbering of RH5-34EM. The right-hand panel shows an alignment of the R5.034- bound forms of RH5-34EM and PfRH5. Side chains that contact R5.034 are shown as sticks and are labelled according to the numbering of RH5-34EM. The central panels showing the same alignments, viewed from different angles and showing bound antibodies. Figure 4 - comparison of antibodies induced through immunisation with RH5-34EM and PfRH5. a) Immunisation scheme for rat immunisations with RH5-34EM and PfRH5. Blood samples were taken on days -2, 27, 55 and 70 and immunisations were conducted on days 0, 28 and 56. b) Total IgG purified from rats immunised with RH5-34EM and PfRH5 were assessed for their binding to immobilised PfRH5 by ELISA. c) Total IgG purified from
for rats immunised with RH5-34EM and PfRH5 were assessed for their binding to immobilised PfRH5 by ELISA. d) Comparison of the binding of sera raised through immunisation with RH5-34EM to immobilised PfRH5 with the binding of sera raised through immunisation with PfRH5 to immobilised RH5-34EM, as measured by ELISA. e) Growth inhibitory activity of different concentrations of total IgG purified from sera raised by immunisation of rats with RH5-34EM and PfRH5. f) The EC30 of IgG from e. g) Growth inhibitory activity of different concentrations of PfRH5 specific IgG purified from sera raised by immunisation of rats with RH5-34EM and PfRH5. h) The EC30 of IgG from g. Analysed using a two-tailed Mann-Whitney test. Figure 5 - Comparison of different prime-boost regimes for RH5-34EM and PfRH5. RH5-34EM (E) and PfRH5 (R) were used in different prime-boost regimes, using the same dosing schedule as Figure 4a. The three-letter code used in this figure gives the dosing regimen: for example, RER is a PfRH5 dose on day 0, a RH5-34EM dose on day 28 and a PfRH5 dose on day 56. Data for RRR and EEE is the same as that shown in Figure 4. Total IgG purified from serum samples were assessed for binding to a) RH5-34EM and b) PfRH5 by ELISA. c) Growth-inhibitory activity (left) and its EC30 (right) for PfRH5-specific IgG. D) Growth-inhibitory activity (left) and its EC30 (right) for total IgG. Analysed using two-tailed Mann-Whitney test. Figure 6 - Characterisation of epitope mimic designs. a) SDS PAGE gel for the twelve designs, stained with Coomassie. b) Surface Plasmon Resonance traces for the twelve designs. In each case, antibody 9AD4 was captured on the chip surface and a dilution series of each epitope mimic, from a maximum concentration of 500nM, was flowed over this surface. Figure 7 - Circular dichroism traces for the twelve designs. Circular dichroism measurements for the twelve different epitope mimic designs. Figure 8 - ELISA data for different time-points during immunisation. Absorbance measurements from ELISA against RH5-34EM and PfRH5 at day -2 (before immunisation) and days 27, 55 and 70 (after the first, second and third vaccine doses). The plots show the eight different vaccine regimens. Figure 9 - TB4 immunogen. a) Crystal structure of TB4, with the R5.016 epitope region b) Crystal structure of TB4 bound to scFv fragments from R5.016 shown as surface representation. c) The TB4 immunogen and RH5-34EM were each conjugated to the HepB
surface antigen (HBsAg) particle through the SpyTag-SpyCatcher system, were mixed with the adjuvant Matrix M and were used to immunise rats. This figure shows the ELISA titres against PfRH5 and against RH5-34EM for sera from these rats. TB4 gives about 2.5-fold higher titres of PfRH5-specific antibodies and around 5-fold lower titres of antibodies against the backbone of the immunogen. d) Comparison of the growth-inhibitory activity of the IgG purified from the rats described in c). The y axis shows the EC50 for these IgG in preventing Plasmodium falciparum from growing in human erythrocytes. IgG raised with TB4 is effective at four-fold lower concentrations than that elicited using RH5-34EM. DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. 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. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts described herein to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
The headings provided herein are not limitations of the various aspects or embodiments of this disclosure. As used herein, the term "capable of' when used with a verb, encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaves, "capable of binding" also means binds and "capable of specifically targeting…" also means specifically targets. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. As used herein, the articles "a" and “an” may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article. 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. “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term “about” shall be understood herein as plus or minus (±) 5%, preferably ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used. The term "consisting of'' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the invention. As used herein the term "consisting essentially of'' refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non- immunogenic ingredients).
Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of” and/or “consisting essentially of” such features. Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the target molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996), the contents of which are herein incorporated by reference for this purpose. In addition, as used herein, an epitope can comprise one or more monosaccharide units of a polysaccharide to which an antibody specifically binds. In specific aspects, an epitope can be a conformational epitope. See, e.g., Thompson et al., 2009, J. of Biol. Chem. 51: 35621- 35631, the contents of which are herein incorporated by reference for this purpose. A "vector" or "construct" (sometimes referred to as gene delivery or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A vector can be a linear or a circular molecule. A vector of the invention may be viral or non-viral. All disclosure herein in relation vectors of the invention applies equally to viral and non-viral vectors unless otherwise stated. All disclosure in relation to viral vectors of the invention applies equally and without reservation to adenovirus vectors or pox virus vectors. As used herein, the term "plasmid", refers to a common type of non-viral vector. A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. Preferably a plasmid is circular and may be double stranded. The terms "nucleic acid cassette”, “nucleic acid construct", "expression cassette" and "nucleic acid expression cassette" are used interchangeably to mean a nucleic acid molecule that is capable of directing transcription. A nucleic acid cassette includes, at the least, a promoter or a structure functionally equivalent to a promoter and a nucleic acid sequence to be transcribed. Thus, a nucleic acid cassette includes, at the least, a promoter or a structure functionally equivalent to a promoter and a nucleic acid sequence encoding a protein of
interest. In the present invention, a nucleic acid cassette includes, at the least, a promoter or a structure functionally equivalent to a promoter, and a nucleic acid encoding a therapeutic protein. A nucleic acid cassette may include additional elements, such as an enhancer, and/or a transcription termination signal. As used herein, the term “virus-like particle” (VLP) refers to a particle which resembles a virus, but which does not contain viral nucleic acid and is therefore non-infectious. VLPs commonly contain one or more virus capsid or envelope proteins which are capable of self- assembly to form the VLP. VLPs have been produced from components of a wide variety of virus families (Noad and Roy (2003), Trends in Microbiology, 11:438-444; Grgacic et al., (2006), Methods, 40:60-65). Some VLPs have been approved as prophylactic vaccines, for example Engerix-B (for hepatitis B), Cervarix and Gardasil (for human papilloma viruses). As used herein, “conjugate group” means a group of atoms that is directly attached to a polypeptide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the polypeptide. As used herein, “conjugate linker” means a single bond or a group of atoms comprising at least one bond that connects a conjugate moiety to a polypeptide. As used herein, “conjugate moiety” means a group of atoms that modifies one or more properties of a molecule compared to the identical molecule lacking the conjugate moiety, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. As used herein, the terms “transduced” and “modified” are used interchangeably to describe cells which have been modified to express a transgene of interest, particularly an antigen as defined herein. Typically, the modification occurs through transduction of the cells. As used herein, the terms “titre” and “yield” are used interchangeably to mean the amount of viral vector produced by a method of the invention. Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogues, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but
usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing. As used herein, the terms “polynucleotides”, "nucleic acid" and "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analogue thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides. The terms “transgene” and “gene” are also used interchangeably, and both terms encompass fragments or variants thereof encoding the target protein, specifically an antigen of the invention. Minor variations in the amino acid sequences of the invention are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to the amino acid sequence of the invention or a fragment thereof as defined anywhere herein. The term homology is used herein to mean identity. As such, the sequence of a variant or analogue sequence of an amino acid sequence of the invention may differ on the basis of substitution (typically conservative substitution) deletion or insertion. Proteins comprising such variations are referred to herein as variants. Proteins of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. Variants of protein molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure/property-activity relationships [see for example, Wold, et al. Multivariate data analysis in chemistry. Chemometrics- Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6] quantitative activity-property relationships of proteins can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition and classification [see for example Norman et al. Applied Regression Analysis. Wiley-lnterscience; 3rd edition (April 1998) ISBN: 0471170828; Kandel, Abraham et al. Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May
11, 1995), ISBN: 0133418847; Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN:1558605525; Denison David G. T. (Editor) et al Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Probability and Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369; Ghose, Arup K. et al. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications in Drug Discovery. ISBN: 0-8247-0487-8]. The properties of proteins can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of proteins sequence, functional and three-dimensional structures and these properties can be considered individually and in combination. Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Amino acid residues at non-conserved positions may be substituted with conservative or non-conservative residues. In particular, conservative amino acid replacements are contemplated. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in a protein of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.
“Non-conservative amino acid substitutions” include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). “Insertions” or “deletions” are typically in the range of about 1, 2, or 3 amino acids. The variation allowed may be experimentally determined by systematically introducing insertions or deletions of amino acids in a protein using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more than routine experiments for a skilled person. A “fragment” of a polypeptide comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or more of the original polypeptide. For example, a fragment may comprise at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400 or more amino acids of the protein from which it is derived. Preferably a fragment may comprise no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 150, no more than 200, no more than 250, no more than 300, no more than 350, or no more than 400 amino acids of the protein from which it is derived. A fragment may be continuous or discontinuous, preferably continuous. The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually, the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant, or other eukaryotic cell lines. The polynucleotides of the present invention may also be produced by chemical synthesis, e.g., by the phosphonamidite method or the tri-ester method and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an appropriate primer sequence. When applied to a nucleic acid sequence, the term “isolated” in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators) and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment. In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below: Amino Acid Codons Degenerate Codon Cys TGC TGT TGY Ser AGC AGT TCA TCC TCG TCT WSN Thr ACA ACC ACG ACT ACN Pro CCA CCC CCG CCT CCN Ala GCA GCC GCG GCT GCN Gly GGA GGC GGG GGT GGN Asn AAC AAT AAY Asp GAC GAT GAY Glu GAA GAG GAR Gln CAA CAG CAR His CAC CAT CAY Arg AGA AGG CGA CGC CGG CGT MGN Lys AAA AAG AAR Met ATG ATG Ile ATA ATC ATT ATH Leu CTA CTC CTG CTT TTA TTG YTN Val GTA GTC GTG GTT GTN Phe TTC TTT TTY Tyr TAC TAT TAY Trp TGG TGG Ter TAA TAG TGA TRR Asn/ Asp RAY Glu/ Gln SAR Any NNN
One of ordinary skill in the art will appreciate that flexibility exists when determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention. It will be understood by those skilled in the art that the numbering of amino acid residues in any of the sequences provided herein may include or exclude an initial methionine (also referred to as Met or M) residue, depending on the context. In cases where the sequence does not explicitly contain an "M" at the start, it is an option to include an initial methionine residue at the beginning of the sequence for the purpose of numbering the residues. Where an initial methionine amino acid residue is indicated in any of the amino acid sequences (with their corresponding SEQ ID Nos) herein, the inclusion of such a residue is optional. Thus, numbering may proceed either from the first amino acid in the sequence as presented, or from an initial methionine if included, with the numbering adjusted accordingly. In other words, for an amino acid sequence of the invention: (a) an initial methionine may be included in the sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) an initial methionine may excluded in the sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. Thus, it will be understood that the key residues identified in the sequences, when calculated from one perspective (e.g., including the initial methionine residue), are equally valid when calculated from the alternative perspective (e.g., excluding the initial methionine residue). Both approaches to residue numbering are consistent with the invention and may be used interchangeably, depending on the context or specific requirement. Similarly, for a nucleic acid sequence encoding an amino acid sequence of the invention: (a) a codon encoding an initial methionine may be included in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) a codon encoding an initial methionine may excluded in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. A “variant” nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is “substantially homologous” (or “substantially identical”) to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more% of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.
Alternatively, a “variant” nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the “variant” and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions. Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter. Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST (as described below). One of ordinary skill in the art appreciates that different species exhibit “preferential codon usage”. As used herein, the term “preferential codon usage” refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different codons may be preferential. Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. A “fragment” of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide. By way of example, a “fragment” of a polynucleotide of interest may comprise (or consist of) at least 600 consecutive nucleotides from the sequence of said polynucleotide (e.g. at least 600, 650, 700, 750, 800 850, 900, or 950 consecutive nucleic acid residues of said polynucleotide). Typically, a fragment as defined herein retains the same function as the full-length polynucleotide. The terms “decrease” "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. The terms "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g.
the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" encompasses a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition (i.e. abrogation) as compared to a reference level. The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. The terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more compared with a reference level, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level. In the context of a yield or titre, an "increase" is an observable or statistically significant increase in such level. The terms "individual”, "subject”, and "patient”, are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and/or therapy optimisation is desired. The mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In a preferred embodiment, the individual, subject, or patient is a human. An “individual” may be an adult, juvenile or infant. An “individual” may be male or female. A "subject in need" of treatment for a particular condition can be an individual having that condition, diagnosed as having that condition, or at risk of developing that condition. A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications or symptoms related to such a condition, and optionally, have already undergone treatment for a condition as defined herein or the one or more complications or symptoms related to said condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition as defined herein or one or more or symptoms or complications related to said condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more or symptoms or complications related to said condition or a subject who does not exhibit risk factors.
As used herein, the term “healthy individual” refers to an individual or group of individuals who are in a healthy state, e.g. individuals who have not shown any symptoms of the disease, have not been diagnosed with the disease and/or are not likely to develop the disease e.g. malaria. Preferably said healthy individual(s) is not on medication affecting malaria and has not been diagnosed with any other disease. The one or more healthy individuals may have a similar sex, age, and/or body mass index (BMI) as compared with the test individual. Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels. Herein the terms “control” and “reference population” are used interchangeably. The term “pharmaceutically acceptable” as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. Disclosure related to the various methods of the invention are intended to be applied equally to other methods, therapeutic uses or methods, the data storage medium or device, the computer program product, and vice versa. Reticulocyte-binding protein Homologue 5 (PfRH5) epitope The present invention provides a polypeptide comprising a PfRH5 epitope. The PfRH5 epitope of the present invention is a fragment of the full length PfRH5 antigen. Typically, the PfRH5 epitope of the present invention is a discontinuous fragment of the full length PfRH5 antigen. An exemplary full-length PfRH5 protein sequence is given in SEQ ID NO: 1. The PfRH5 epitope typically induces an immune response (e.g. an antibody response) against the blood-stage Plasmodium falciparum parasite. Preferably, the PfRH5 epitope induces an improved immune response (e.g. an antibody response) against the blood-stage Plasmodium falciparum parasite compared with the full-length PfRH5 (e.g. SEQ ID NO: 1). All disclosure herein in relation to PfRH5 epitopes of the invention applies equally and without reservation to polypeptides of the invention comprising said epitope, and vice versa, unless expressly stated to the contrary. Although an exemplary, and in some instances preferred, full-length length PfRH5 protein sequence is given in SEQ ID NO: 1, it will be appreciated that any disclosure of full- length length PfRH5 herein is not limited to SEQ ID NO: 1. Other sequences may be used as examples of a full-length length PfRH5 protein sequence. For example, another exemplary full-length length PfRH5 protein sequence is given in SEQ ID NO: 66. It should be noted that
any reference to full-length length PfRH5 refers to any suitable full-length length PfRH5 protein sequence such as those full-length length PfRH5 protein sequences given in SEQ ID NO: 1 and SEQ ID NO: 66. The Reticulocyte binding Homologue 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. ln vitro cleaved fragments of approximately 45 KDa and 28 KDa have been reported. The PfRH5 epitope of the invention is comprised within a fragment of PfRH5 which is referred to as the PfRH5ΔNL fragment. The PfRH5ΔNL lacks both the signal peptide (corresponding to residues 1 to 23 of SEQ ID NO: 1); 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) of PfRH5) and the flexible loop region corresponding to amino acid residues 248 to 296 of SEQ ID NO: 1). The PfRH5ΔNL fragment was characterised in more detail by the present inventors in WO2016/016651, which is herein incorporated by reference in its entirety. The amino acid sequence of PfRH5ΔNL is given in SEQ ID NO: 2. The amino acid sequence of a thermostable version of this fragment is given in SEQ ID NO: 18. The PfRH5 epitope of the invention may be the same as or overlap with an epitope for any PfRH5 antibody, particularly any PfRH5 antibody which binds to an epitope within the basigin-binding site of PfRH5. The crystal structure of PfRH5 and its receptor, basigin (BSG) was previously determined by the present inventors as described in WO2016/016651, which is herein incorporated by reference in its entirety. Examples of such PfRH5 antibodies include 9AD4 and QA1, both of which are commercially available (e.g. from Absolute Antibody). By way of non-limiting example, the functional epitope for the 9AD4 antibody consists of PfRH5 residues 202, 205, 209, 212, 213, 331, 334, 335, 338, 339, 341 and 342 (e.g. of SEQ ID NO: 1). Thus, a PfRH5 epitope of the invention may comprise or consist of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or all 12 of the amino acids of the PfRH5 functional epitope for 9AD4 (i.e. the amino acid residues within PfRH5 which contact the 9AD4 antibody). In other words, a PfRH5 epitope of the invention may comprise or consist of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or all 12 of PfRH5 residues 202, 205, 209, 212, 213, 331, 334, 335, 338, 339, 341 and 342 (e.g. of SEQ ID NO: 1). The inventors have previously identified PfRH5 epitope bins which elicit antibodies with strong growth inhibitory activity (GIA) against P. falciparum. These epitope bins are
described in WO2016/016651 and GB Patent Application No. GB 2313544.5 (filed on 5 September 2023), both of which are herein incorporated by reference in their entirety. Thus, a PfRH5 epitope of the invention may be the same as or overlap with a PfRH5 epitope bin comprising or consisting of the amino acid sequence GKCIAVDAFIKKINETYDKKICMDMKNYGTNLFEQ (SEQ ID NO: 19, which is the neutralising epitope bin of the R5.016 antibody as described in WO2016/016651). In other words, a PfRH5 epitope of the invention may comprise or consist of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15¸ at least 16, at least 17¸ at least 18¸ at least 19¸ at least 20¸ at least 21¸ at least 22¸ at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more of the amino acids of SEQ ID NO: 19. A PfRH5 epitope of the invention may have 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 to the epitope bin of SEQ ID NO: 19, or a fragment thereof. A PfRH5 epitope of the invention may be the same as or overlap with a PfRH5 epitope comprising or consisting of the amino acid sequence GKCIAVDAFIKKINETYDKKICMDMKNY (SEQ ID NO: 20, which is the neutralising epitope of the R5.016 antibody as described in WO2016/016651). In other words, a PfRH5 epitope of the invention may comprise or consist of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15¸ at least 16, at least 17¸ at least 18¸ at least 19¸ at least 20¸ at least 21¸ at least 22¸ at least 23, at least 24, at least 25, at least 26, at least 27, or all 28 of the amino acids of SEQ ID NO: 20. A PfRH5 epitope of the invention may have 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 to the epitope of SEQ ID NO: 20, or a fragment thereof. In GB Patent Application No. GB 2313544.5 (herein incorporated by reference in its entirety), the contact residues for the R5.016 antibody were identified as ILE 193, THR 199, GLY 201, LYS 202, CYS 203, ILE 204, ALA 205, VAL 206, ASP 207, ALA 208, PHE 209, LYS 211, LYS 212, ILE 213, GLU 215, THR 216, ASP 331, ASN 334, TYR 335, ASN 338, LEU 339, and GLN 342 of SEQ ID NO: 66 for the heavy chain and LYS 212, THR 216, LYS 219, VAL 220, LYS 327, ILE 328, and ASP 331 of SEQ ID NO: 66 for the light chain. A PfRH5 epitope of the invention may comprise or consist of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15¸ at least 16, at least 17¸ at least 18¸ at least 19¸ at least 20¸ at least 21, at least 22¸ at least 23, at least 24, at least 25, at least 26, or all 27 of ILE 193, THR 199, GLY 201, LYS 202, CYS 203, ILE 204, ALA 205, VAL 206, ASP 207, ALA 208, PHE 209, LYS 211, LYS 212, ILE 213, GLU 215, THR 216, LYS
219, VAL 220, ASP 331, ASN 334, TYR 335, LYS 327, ILE 328, ASP 331, ASN 338, LEU 339, and GLN 342 of SEQ ID NO: 66. Alternatively, KSYNNNFCNTNKLNIWRTFQK (SEQ ID NO: 21, which is the neutralising epitope bin of the R5.004 antibody as described in WO2016/016651). In other words, a PfRH5 epitope of the invention may comprise or consist of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15¸ at least 16, at least 17¸ at least 18¸ at least 19¸ at least 20 or all 21 of the amino acids of SEQ ID NO: 21. A PfRH5 epitope of the invention may have 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 to the epitope bin of SEQ ID NO: 21, or a fragment thereof. Preferably, the PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain CDR1 sequence of GFTFNTYW (SEQ ID NO: 22), the heavy chain CDR2 sequence of IQQDGSEK (SEQ ID NO: 23), the heavy chain CDR3 sequence of ARDNPASAVAFDV (SEQ ID NO: 24), and the light chain CDR1 sequence of SSNIGNNA (SEQ ID NO: 25), the light chain CDR2 sequence of FDD (SEQ ID NO: 26), and the light chain CDR3 sequence of AAWDDRLNGVV (SEQ ID NO: 27). Preferably, the PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain variable region sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMSWVRQAPGKGLEWVANIQQDGSEKD YLNSVRGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARDNPASAVAFDVWGQGAMVTVS S (SEQ ID NO: 28) and the light chain variable region sequence of QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPQLLIYYDDLLPSGVSD RFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGLVFGGGTKLTVL (SEQ ID NO: 29). The PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain CDR1 sequence of GYTFTSYG (SEQ ID NO: 38), the heavy chain CDR2 sequence of ISGYDGNT (SEQ ID NO: 39), the heavy chain CDR3 sequence of ARDGPQVGDFDWQVYYYYGMDV (SEQ ID NO: 40), and the light chain CDR1 sequence of QSINTW (SEQ ID NO: 41), the light chain CDR2 sequence of KAS (SEQ ID NO: 42), and the light chain CDR3 sequence of QQYNSYLYT (SEQ ID NO: 43). The PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain variable region sequence of QVQLVQSGAEVKKPGASVRVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISGYDGNTN YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGPQVGDFDWQVYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 44) and the light chain variable region sequence of AIRMTQSPSTLSASVGDRVTITCRASQSINTWLAWYQQKPGKAPNLLISKASSLESGVPSRF SGSGSGTEFTLTISSLQPDDFATYFCQQYNSYLYTFGQGTKVEIR (SEQ ID NO: 45).
The PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain CDR1 sequence of GFTFSDYG (SEQ ID NO: 30), the heavy chain CDR2 sequence of ISNMAYSI (SEQ ID NO: 31), the heavy chain CDR3 sequence of TRAIFDYAGYWYFDV (SEQ ID NO: 32), and the light chain CDR1 sequence of ESVEYYGTSL (SEQ ID NO: 33), the light chain CDR2 sequence of GAS (SEQ ID NO: 34), and the light chain CDR3 sequence of QQSTKVPWT (SEQ ID NO: 35). The PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain variable region sequence of MGWSWIFLFLLSGTAGVHSEVKLVESGGGVVQPGGSRKLSCAASGFTFSDYGMAWVRQA PGKGPEWVTFISNMAYSIYYADTVTGRFTISRENAKNTLHLEMSSLRSEDTAMYYCTRAIFD YAGYWYFDVWGAGTTVTVS (SEQ ID NO: 36) and the light chain variable region sequence of MVSTPQFLVFLLFWIPASRGDIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWFQQ KPGQPPRLLIHGASNVQSGVPARFSGSGSGTDFSLNIHPVEEDDFAMYFCQQSTKVPWTF GGGTKLEI (SEQ ID NO: 37). The PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain CDR1 sequence of NYAIN (SEQ ID NO: 46), the heavy chain CDR2 sequence of GIIPIFATTNYAQKFQG (SEQ ID NO: 47), the heavy chain CDR3 sequence of DKHSWSYAFDI (SEQ ID NO: 48), and the light chain CDR1 sequence of SGSSSNIGSNTVN (SEQ ID NO: 49), the light chain CDR2 sequence of SNNQRPS (SEQ ID NO: 50), and the light chain CDR3 sequence of AAWDDSLNGWV (SEQ ID NO: 51). The PfRH5 epitope may be the same epitope as that bound by an antibody having the heavy chain variable region sequence of EVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAINWVRQAPGQGLEWMGGIIPIFATTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARDKHSWSYAFDIWGQGTMVTVSS (SEQ ID NO: 52) and the light chain variable region sequence of QSVLTQPPSASGTPGLRVTISCSGSSSNIGSNTVNWYQHLPGTAPKLLIHSNNQRPSGVPD RFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQP (SEQ ID NO: 53). Binding of an antibody to an epitope, may be determined using any appropriate technique, examples of which are well known in the art, including PEPSCAN-based enzyme- linked immunoassays, hydrogen/deuterium exchange (HDX), electron microscopy and crystallography. Binding of an antibody to a discontinuous epitope, may be determined using any appropriate technique, examples of which are well known in the art, including, hydrogen/deuterium exchange (HDX), electron microscopy and crystallography.
In some preferred embodiments, a PfRH5 epitope of the present invention comprises or consists of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15¸ at least 16, at least 17¸ at least 18¸ at least 19¸ at least 20¸ at least 21¸ at least 22¸ at least 23, at least 24 or all 25 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1 (full-length PfRH5). In some particularly preferred embodiments, a PfRH5 epitope of the present invention comprises or consists of all 25 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1 (full-length PfRH5). The PfRH5 epitope may comprise one or more additional amino acid substitutions. By way of non-limiting example, a PfRH5 epitope may comprise 1, 2, 3, 4, 5 amino acid substitutions. Each amino acid substitution may be independently selected. Each amino acid substitution may be a conservative or non-conservative amino acid substitution, preferably a conservative amino acid substitution. The PfRH5 epitope of the invention is grafted onto a scaffold. Suitable scaffolds are described in more detail below. A scaffold of the invention typically comprises at least two α- helices, such that when the PfRH5 epitope is grafted onto the scaffold it is presented in an immunogenic conformation. As used herein, the terms “grafted onto a scaffold” and “presented on a scaffold” can therefore be used interchangeably. A PfRH5 epitope presented on a scaffold of the invention typically adopts the same relative three-dimensional conformation as in native (full-length) PfRH5, such as in SEQ ID NO: 1. Adopting the same relative conformation of the epitope as in native PfRH5 is advantageous as it facilitates a strong specific immune response. Preferably, the amino acids residues of the PfRH5 epitope as defined herein (i.e. the at least 5 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1, and preferably all 25 of these residues) are presented on two α helices of the scaffold. Again, typically these residues are presented in the same three-dimensional configuration as the equivalent residues on PfRH5. The relative conformation of a molecule, such as a PfRH5 epitope, can be assessed via any suitable technique, such techniques are well known in the art, including X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. The relative conformation of a molecule, such as a PfRH5 epitope, can be described by any suitable measurement
including co-ordinates, angles (e.g. torsional and dihedral), Ramachandran plots and root mean square deviation (commonly abbreviated as RMSD or RMS deviation). By way of a non-limiting example, the relative three-dimensional coordinates of any PfRH5 epitope residue in a polypeptide of the invention may be compared with the equivalent three-dimensional coordinates of the same epitope residue within PfRH5. The root mean square deviation the relative three-dimensional coordinates of any PfRH5 epitope residue in a polypeptide of the invention compared with the equivalent three-dimensional coordinates of the same epitope residue in PfRH5 is typically 2 Å or less. Preferably, the root mean square deviation the relative three-dimensional coordinates of any one of PfRH5 epitope residue in a polypeptide of the invention compared with the three-dimensional coordinates of the same epitope residue in PfRH5 is less than 1.5 Å. Typically, the amino acids residues of the PfRH5 epitope as defined herein (i.e. the at least 5 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1, and preferably all 25 of these residues) are exposed when presented on a scaffold of the invention. Preferably, the amino acids residues of the PfRH5 epitope as defined herein (i.e. the at least 5 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1, and preferably all 25 of these residues) are fully exposed. Any suitable technique may be used to measure how exposed the surface of a molecule, such as an epitope, is, examples of which are well known in the art. Typically, the amino acids residues of the PfRH5 epitope as defined herein (i.e. the at least 5 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1, and preferably all 25 of these residues) may have a Relative Solvent Accessibility (RSA) of at least 80%. For example, the amino acids residues of the PfRH5 epitope as defined herein (i.e. the at least 5 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1, and preferably all 25 of these residues) have a Relative Solvent Accessibility (RSA) of at least 80%, at least 85%, at least 90% or at least 95%. Without being bound by theory, it is believed that exposure of the amino acids residues of the PfRH5 epitope as defined herein (i.e. the at least 5 amino acids corresponding to residues K202, I204, A205, A208, F209, K211, K212, I213, E215, A216, D218, K219, V220, K327, I328, M330, D331, K333, N334, Y335, T337, N338, L339, E341 and Q342 of SEQ ID NO: 1, and preferably all 25 of these residues) allows the PfRH5 epitope presented thereon to be available for antibody binding and/or elicitation. In
particular, it is believed that exposure of the PfRH5 epitope such that it is not blocked or constrained by the non-epitope portions of the scaffold, facilitates antibody binding and/or elicitation. As discussed herein, for an amino acid sequence of the invention, such as an epitope: (a) an initial methionine may be included in the sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) an initial methionine may excluded in the sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. Similarly, for a nucleic acid sequence encoding an epitope sequence of the invention: (a) a codon encoding an initial methionine may be included in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) a codon encoding an initial methionine may excluded in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. A PfRH5 epitope of the invention may bind to at least one antibody that is specific for PfRH5. Non-limiting examples of such PfRH5-specific antibodies include 9AD4, R5.034, R5.016 and R5.004, preferably R5.034. A PfRH5 epitope of the invention may bind to any one, two, three or all four of R5.034, 9AD4, R5.016 and R5.004. Thus, a PfRH5 epitope may bind to (i) R5.034; (ii) 9AD4; (iii) R5.016; (iv) R5.004; (v) R5.034 and 9AD4; (vi) R5.034 and R5.016; (viii) R5.034 and R5.004; (ix) 9AD4 and R5.016; (x) 9AD4 and R5.004; (xi) R5.0016 and R5.004; (xii) R5.034, 9AD4 and R5.016; (xiii) R5.034, 9AD4 and R5.004; (xiv) R5.034, R5.016 and R5.004; (xv) 9AD4, R5.016 and R5.004; or (xvi) R5.034, 9AD4, R5.016 and R5.004. Preferably, a PfRH5 epitope of the invention may bind to any one, two or all three of R5.034, 9AD4 and R5.016. Thus, a PfRH5 epitope may bind to (i) R5.034; (ii) 9AD4; (iii) R5.016; (iv) R5.034 and 9AD4; (v) R5.034 and R5.016; (vi) 9AD4 and R5.016; or (vii) R5.034, 9AD4 and R5.016. More preferably a PfRH5 epitope of the invention binds to R5.034, or any combination of antibodies including R5.034, such as (i) R5.034 and 9AD4; (ii) R5.034 and R5.016; or (iii) R5.034, 9AD4 and R5.016. Thus, a PfRH5 epitope of the invention may bind a PfRH5 antibody which has a heavy chain CDR1 sequence of GFTFNTYW (SEQ ID NO: 22), a heavy chain CDR2 sequence of IQQDGSEK (SEQ ID NO: 23), a heavy chain CDR3 sequence of ARDNPASAVAFDV (SEQ ID NO: 24), and a light chain CDR1 sequence of SSNIGNNA (SEQ ID NO: 25), a light chain CDR2 sequence of FDD (SEQ ID NO: 26), and a light chain CDR3 sequence of AAWDDRLNGVV (SEQ ID NO: 27). Said PfRH5 antibody may have a heavy chain variable region sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMSWVRQAPGKGLEWVANIQQDGSEKD YLNSVRGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARDNPASAVAFDVWGQGAMVTVS S (SEQ ID NO: 28) and a light chain variable region sequence of
QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPQLLIYYDDLLPSGVSD RFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGLVFGGGTKLTVL (SEQ ID NO: 29). Alternatively or in addition, a PfRH5 epitope of the invention may bind a PfRH5 antibody which has a functional epitope consisting of PfRH5 residues 202, 205, 209, 212, 213, 331, 334, 335, 338, 339, 341 and 342 (e.g. of SEQ ID NO: 1). Thus, a PfRH5 epitope of the invention may bind a PfRH5 antibody which has a heavy chain CDR1 sequence of GFTFSDYG (SEQ ID NO: 30), a heavy chain CDR2 sequence of ISNMAYSI (SEQ ID NO: 31), a heavy chain CDR3 sequence of TRAIFDYAGYWYFDV (SEQ ID NO: 32), and a light chain CDR1 sequence of ESVEYYGTSL (SEQ ID NO: 33), a light chain CDR2 sequence of GAS (SEQ ID NO: 34), and a light chain CDR3 sequence of QQSTKVPWT (SEQ ID NO: 35). Said PfRH5 antibody may have a heavy chain variable region sequence of MGWSWIFLFLLSGTAGVHSEVKLVESGGGVVQPGGSRKLSCAASGFTFSDYGMAWVRQA PGKGPEWVTFISNMAYSIYYADTVTGRFTISRENAKNTLHLEMSSLRSEDTAMYYCTRAIFD YAGYWYFDVWGAGTTVTVS (SEQ ID NO: 36) and a light chain variable region sequence of MVSTPQFLVFLLFWIPASRGDIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWFQQ KPGQPPRLLIHGASNVQSGVPARFSGSGSGTDFSLNIHPVEEDDFAMYFCQQSTKVPWTF GGGTKLEI (SEQ ID NO: 37). Further alternatively or in addition, a PfRH5 epitope of the invention may bind a PfRH5 antibody which has a heavy chain CDR1 sequence of GYTFTSYG (SEQ ID NO: 38), a heavy chain CDR2 sequence of ISGYDGNT (SEQ ID NO: 39), a heavy chain CDR3 sequence of ARDGPQVGDFDWQVYYYYGMDV (SEQ ID NO: 40), and a light chain CDR1 sequence of QSINTW (SEQ ID NO: 41), a light chain CDR2 sequence of KAS (SEQ ID NO: 42), and a light chain CDR3 sequence of QQYNSYLYT (SEQ ID NO: 43). Said antibody may have a heavy chain variable region sequence of QVQLVQSGAEVKKPGASVRVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISGYDGNTN YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGPQVGDFDWQVYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 44) and a light chain variable region sequence of AIRMTQSPSTLSASVGDRVTITCRASQSINTWLAWYQQKPGKAPNLLISKASSLESGVPSRF SGSGSGTEFTLTISSLQPDDFATYFCQQYNSYLYTFGQGTKVEIR (SEQ ID NO: 45). Further alternatively or in addition, a PfRH5 epitope of the invention may bind the which has a heavy chain CDR1 sequence of NYAIN (SEQ ID NO: 46), a heavy chain CDR2 sequence of GIIPIFATTNYAQKFQG (SEQ ID NO: 47), a heavy chain CDR3 sequence of DKHSWSYAFDI (SEQ ID NO: 48), and a light chain CDR1 sequence of SGSSSNIGSNTVN (SEQ ID NO: 49), a light chain CDR2 sequence of SNNQRPS (SEQ ID NO: 50), and a light chain CDR3 sequence of AAWDDSLNGWV (SEQ ID NO: 51). Said antibody may have a heavy chain variable region sequence of
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAINWVRQAPGQGLEWMGGIIPIFATTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARDKHSWSYAFDIWGQGTMVTVSS (SEQ ID NO: 52) and a light chain variable region sequence of QSVLTQPPSASGTPGLRVTISCSGSSSNIGSNTVNWYQHLPGTAPKLLIHSNNQRPSGVPD RFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQP (SEQ ID NO: 53). A PfRH5 epitope of the invention may specifically bind to at least one antibody that is specific for PfRH5, such as those described herein. Preferably, a PfRH5 epitope may specifically bind to one or more of R5.034, 9AD4, R5.016 and R5.004. For example, a PfRH5 epitope may specifically bind to (i) R5.034; (ii) A9AD4; (iii) R5.016; (iv) R5.004; (v) R5.034 and 9AD4; (vi) R5.034 and R5.016; (viii) R5.034 and R5.004; (ix) A9AD4 and R5.016; (x) A9AD4 and R5.004; (xi) R5.0016 and R5.004; (xii) R5.034, 9AD4 and R5.016; (xiii) R5.034, 9AD4 and R5.004; (xiv) R5.034, R5.016 and R5.004; (xv) 9AD4, R5.016 and R5.004; or (xvi) R5.034, 9AD4, R5.016 and R5.004. Preferably, a PfRH5 epitope may specifically bind to (i) R5.034; (ii) 9AD4; (iii) R5.016; (iv) R5.034 and 9AD4; (v) R5.034 and R5.016; (vi) 9AD4 and R5.016; or (vii) R5.034, 9AD4 and R5.016. More preferably a PfRH5 epitope of the invention specifically binds to R5.034, or any combination of antibodies including R5.034, such as (i) R5.034 and 9AD4; (ii) R5.034 and R5.016; or (iii) R5.034, 9AD4 and R5.016. As exemplified herein, the present inventors have shown that polypeptides of the invention comprising a PfRH5 epitope as described herein can interact with multiple monoclonal antibodies which are known in the art to have good GIA against P. falciparum, and which interact with overlapping epitopes. This indicates that the polypeptides of the invention have the potential to induce a highly focused immune response, eliciting a range of antibodies (e.g.9AD4, R5.016, R5.034 and R5.004, particularly 9AD4, R5.016 and R5.034) with strong GIA which target the epitope. This ability is advantageous as it indicates that the polypeptides of the invention have the potential to be clinically effective vaccines, as it increases the quality of the immune response, specifically eliciting neutralising antibodies, enhancing the vaccine’s overall efficacy. Malaria parasites exhibit genetic diversity, and different strains may have variations in key surface proteins. Without being bound by theory, an epitope recognized by multiple antibodies in in vitro assays indicates that polypeptides comprising said epitopes have the potential to elicit a range of antibodies when used to immunise a patient. Thus, the polypeptides of the invention may provide cross-strain protection against multiple P. falciparum strains, making the vaccine more broadly effective in diverse geographical regions where various strains may be prevalent.
As described herein, according to the present invention, a PfRH5 epitope is grafted onto a scaffold, such that it is presented in an immunogenic conformation. Typically, the PfRH5 epitope is presented on a scaffold such that it adopts the same relative three- dimensional conformation as in native (full-length) PfRH5, such as in SEQ ID NO: 1. To achieve this presentation, the amino acid residues of the PfRH5 epitope may be interspersed with one or more scaffold amino acid residues to ensure that the PfRH5 epitope adopts the same conformation as in native (full-length) PfRH5. Thus, any two amino acid residues of the PfRH5 epitope may be separated by one or more amino acid residue from the scaffold. Typically, any two amino acid residues of the PfRH5 epitope may be separated by 1, 2, 3, 4 or 5 amino acid residues from the scaffold. Preferably, any two amino acid residues of the PfRH5 epitope may be separated by at most two scaffold amino acids (i.e. by 2, 1 or 0 scaffold amino acids). The number of scaffold amino acids separating any two amino acids of the PfRH5 epitope may be determined independently. For example, one pair of amino acids of the PfRH5 epitope may be separated by 2 scaffold amino acids, another pair of amino acids of the PfRH5 epitope may be separated by 1 amino acid and still another pair of amino acids of the PfRH5 epitope may be separated by 0 scaffold amino acids. Alternatively, any two amino acid residues of the PfRH5 epitope in a polypeptide of the invention may be separated by the same distance as that observed between the corresponding two amino acid residues in the wild-type PfRH5. The distance between two amino acid residues may be measured in any appropriate units, examples of which are well known in the art, including angstroms (Å) or nanometers (nm). The similarity of the distances between any two amino acid residues of the PfRH5 epitope in a polypeptide of the invention and the corresponding two amino acid residues in the wild-type PfRH5 may be measured by any suitable technique, examples of which are well known in the art including protein crystallography or cryogenic electron microscopy, with the similarity in the distances being referred to as the Root Mean Square Deviation (RMSD). By way of non-limiting example, the RMSD of any two amino acid residues of the PfRH5 epitope in a polypeptide of the invention compared with the corresponding two amino acid residues in the wild-type PfRH5 is typically 2 Å or less. Preferably, the RMSD of any two amino acid residues of the PfRH5 epitope in a polypeptide of the invention compared with the corresponding two amino acid residues in the wild-type PfRH5 is less than 1.5 Å. As the nature of the scaffold amino acids separating any two amino acids of the PfRH5 epitope (where present) are typically not limiting provided that the PfRH5 epitope adopts the same relative three-dimensional conformation as in native (full-length) PfRH5, such as in SEQ ID NO: 1, a PfRH5 epitope sequence comprising one or more scaffold amino acids interspersed therein may be defined in terms of a consensus sequence. The term consensus sequence refers to a theoretical representative nucleotide or amino acid
sequence (in the case of a PfRH5 epitope, an amino acid sequence) in which each nucleotide or amino acid is the one which occurs most frequently at that site in the different sequences which occur in nature. A reference herein to a PfRH5 epitope of the invention can be used to refer to both the PfRH5 epitope sequence without any scaffold amino acids that may be present in the polypeptide of the invention (i.e. without any gaps in the PfRH5 epitope), and to a PfRH5 epitope sequence comprising the one or more scaffold amino acids interspersed therein (i.e. with any gaps in the PfRH5 epitope). A PfRH5 epitope of the invention may comprise or consist of a consensus sequence of KxIAxxAFxKKIxEAxDKV (SEQ ID NO: 3) and/or a consensus sequence of KIxMDxKNYxTNLxEQ (SEQ ID NO: 4), where in each instance “x” is any amino acid and each occurrence of x may be selected independently. In each of these consensus sequences, the “x” amino acid residues are typically scaffold residues required to ensure that the PfRH5 epitope is presented on a scaffold such that it adopts the same relative three- dimensional conformation as in native (full-length) PfRH5, such as in SEQ ID NO: 1. Thus, the “ungapped” consensus sequence corresponding to SEQ ID NO: 3 would be KIAAFKKIEADKV (SEQ ID NO: 54), and the “ungapped” consensus sequence corresponding to SEQ ID NO: 4 would be KIMDKNYTNLEQ (SEQ ID NO: 55). Preferably a PfRH5 epitope of the invention may comprise or consist of both a consensus sequence of KxIAxxAFxKKIxEAxDKV (SEQ ID NO: 3) and a consensus sequence of KIxMDxKNYxTNLxEQ (SEQ ID NO: 4). Whilst the “ungapped” consensus sequences may be useful in identifying key residues within the PfRH5 protein which are useful in a PfRH5 epitope of the invention, typically an “ungapped” consensus sequence is not used in determining the final amino acid sequence of a polypeptide of the invention, as one or more scaffold residues are typically to separate the PfRH5 amino acids to ensure that the PfRH5 epitope is presented on a scaffold such that it adopts the same relative three-dimensional conformation as in native (full-length) PfRH5. Therefore, in some preferred embodiments, references to a PfRH5 epitope or a PfRH5 consensus sequence of the invention refer to sequences which include the one or more scaffold amino acids required to ensure that the PfRH5 epitope is presented on a scaffold such that it adopts the same relative three- dimensional conformation as in native (full-length) PfRH5. Thus, a PfRH5 epitope as presented on a scaffold according to the invention may comprise or consist of a consensus sequence of KxIAxxAFxKKIxEAxDKV (SEQ ID NO: 3) and/or a consensus sequence of KIxMDxKNYxTNLxEQ (SEQ ID NO: 4). Preferably, a PfRH5 epitope as presented on a scaffold according to the invention may comprise or consist of both a consensus sequence of KxIAxxAFxKKIxEAxDKV (SEQ ID NO: 3) and a consensus sequence of KIxMDxKNYxTNLxEQ (SEQ ID NO: 4).
Typically, the consensus sequence of KxIAxxAFxKKIxEAxDKV (SEQ ID NO: 3) and the consensus sequence of KIxMDxKNYxTNLxEQ (SEQ ID NO: 4) are comprised in different α-helices within the scaffold to ensure that PfRH5 epitope is presented on a scaffold such that it adopts the same relative three-dimensional conformation as in native (full-length) PfRH5. The consensus sequence of SEQ ID NO: 3 may be presented on a first α-helix of the scaffold and the consensus sequence of SEQ ID NO: 4 may be presented on a second α- helix of the scaffold. As described herein, a scaffold may comprise a third α-helix to ensure that the polypeptide is correctly folded and the PfRH5 epitope is presented on a scaffold such that it adopts the same relative three-dimensional conformation as in native (full-length) PfRH5. When a polypeptide of the invention comprises a third (or further) α-helix, typically neither the SEQ ID NO: 3 or the consensus sequence of SEQ ID NO: 4 is presented on said third (or further) α-helix. Preferably, the consensus sequence of SEQ ID NO: 3corresponds to a first portion of the PfRH5 epitope presented on the first α-helix of the scaffold and the consensus sequence of SEQ ID NO: 4 corresponds to a second portion of the PfRH5 epitope presented on the second α-helix of the scaffold. Scaffold The present invention provides a polypeptide comprising a scaffold. The term scaffold refers to a supporting structure or framework that provides a physical or organizational framework for various components and thus maintaining the spatial arrangement of components and their interaction with molecules. As described herein, a scaffold may be a molecular structure designed to display or present epitopes in a specific conformation. Typically, the scaffold is a polypeptide. Any scaffold may be used provided that a PfRH5 epitope of the invention can be presented on said scaffold such that it adopts the same relative three-dimensional conformation as in native (full-length) PfRH5. Typically, the scaffold of the invention is an exogenous scaffold. In other words, typically the scaffold of the invention does not comprise or consist of an amino acid sequence from PfRH5. Thus, for the avoidance of doubt, native PfRH5 and fragments (continuous or discontinuous) of PfRH5, such as the ectodomain fragment of PfRH5, are not scaffolds according to the present invention. Thus, the PfRH5 sequences of SEQ ID NOs: 1, 2 and 18 herein are not exogenous scaffolds. Similarly, the PfRH5 sequences and fragments described in US 2018/0193440 and WO 2020/074908 are not exogenous scaffolds according to the present invention.
By extension, native PfRH5 and fragments (whether continuous or discontinuous) of PfRH5, such as the ectodomain fragment of PfRH5, are not polypeptides according to the present invention, because there is no exogenous scaffold within PfRH5 or such fragments. Again, and for the avoidance of doubt, the PfRH5 sequences of SEQ ID NOs: 1, 2 and 18 herein are not polypeptides according to the present invention. Similarly, the PfRH5 sequences and fragments described in US 2018/0193440 and WO 2020/074908 are not polypeptides according to the present invention. The scaffold of the invention may be an exogenous scaffold which is derived from a source other than Plasmodium falciparum. By way of non-limiting example, in the present Examples the inventors have generated an exemplary polypeptide of the invention using a three-helical bundle from the Escherichia coli ribosome recycling factor as a scaffold. The use of an exogenous scaffold offers several advantages, as it provides greater flexibility in designing polypeptides with improved stability, enhanced folding efficiency, and the potential for targeted immune responses. Moreover, an exogenous scaffold creates a unique structural environment that is not naturally found in Plasmodium falciparum proteins, allowing for more effective manipulation of the grafted epitope's conformation and functionality.All references herein to scaffolds of the invention relate to exogenous scaffolds unless expressly stated to the contrary. Typically, the invention relates to a scaffold which comprises at least two α-helices. The term α-helix refers to a common secondary structure found in proteins which is characterized by a right-handed helical arrangement of amino acid residues within a polypeptide chain. A scaffold of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 α- helices. Preferably, the scaffold comprises three α-helices. A scaffold of the invention typically comprises two α-helices which comprise a PfRH5 epitope of the invention, as described herein. Said two α-helices may be resurfaced such that the PfRH5 epitope can be grafted onto them, allowing the successful presentation of the PfRH5 epitope. In other words, wherein a scaffold comprises at least two α-helices, the at least two α-helices of the scaffold typically have the same three-dimensional arrangement as in PfRH5, such that the PfRH5 epitope of the invention is presented such that it has the same three-dimensional arrangement as in PfRH5. The similarity of the three-dimensional arrangement of the at least two α-helices of the scaffold of the invention compared to the equivalent three-dimensional arrangement as in wild-type PfRH5 may be measured by any suitable technique, examples of which are well known in the art including RMSD. By way of non-limiting example, the RMSD of the three-dimensional arrangement of the at least two α-helices of the scaffold of the invention (or any amino acid residues therein) compared to the equivalent three-dimensional arrangement as in wild-type PfRH5 (or the
corresponding amino acid residues therein) is typically 2 Å or less. Preferably, the RMSD of the three-dimensional arrangement of the at least two α-helices of the scaffold of the invention (or any amino acid residues therein) compared to the equivalent three-dimensional arrangement as in wild-type PfRH5 (or the corresponding amino acid residues therein) is less than 1.5 Å. Typically, the invention relates to a scaffold wherein the at least two α-helices are sufficiently long that the scaffold fully contains a PfRH5 epitope of the invention. Typically, an α-helix in a polypeptide of the invention is at least 15 amino acid residues in length. For example, an α-helix in a polypeptide of the invention may be at least 15 amino acid residues in length, at least 20 amino acid residues in length, at least 25 amino acid residues in length, at least 30 amino acid residues in length or at least 35 amino acid residues in length. The length of each α-helix in a polypeptide of the invention may be independently selected. Alternatively, the length of each α-helix in a polypeptide of the invention may be the same. Wherein a PfRH5 epitope of the invention is presented on two α-helices and a third α-helix is present to stabilise and/or multimerise the polypeptide as described herein, the lengths of each of the two α-helices presenting/comprising the PfRH5 epitope may be independently selected or may be the same. By way of non-limiting example, the first α-helix and/or the second α-helix may be about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids, about 33 amino acids, about 34 amino acids, about 35 amino acids, about 36 amino acids, about 37 amino acids, about 38 amino acids, about 39 amino acids or about 40 amino acids in length. In some preferred embodiments, the first α-helix may be 29 amino acids in length and the second α-helix may be 33 amino acids in length. The at least two α-helices are typically sufficiently long such that, when combined with the rest of the scaffold, they will stably adopt the correct three-dimensional shape. Typically, an α-helix in a polypeptide of the invention is at least 15 amino acid residues in length. For example, an α-helix in a polypeptide of the invention may be at least 15 amino acid residues in length, at least 20 amino acid residues in length, at least 25 amino acid residues in length, at least 30 amino acid residues in length or at least 35 amino acid residues in length. The length of each α-helix in a polypeptide of the invention may be independently selected. Alternatively, the length of each α-helix in a polypeptide of the invention may be the same. Wherein a PfRH5 epitope of the invention is presented on two α-helices and a third α-helix is present to stabilise and/or multimerise the polypeptide as described herein, the lengths of each of the two α-helices presenting/comprising the PfRH5 epitope may be independently selected or may be the same. By way of non-limiting
example, the first α-helix and/or the second α-helix may be about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids, about 33 amino acids, about 34 amino acids, about 35 amino acids, about 36 amino acids, about 37 amino acids, about 38 amino acids, about 39 amino acids or about 40 amino acids in length. In some preferred embodiments, the first α-helix may be 29 amino acids in length and the second α-helix may be 33 amino acids in length. Typically, the at two α-helices of the invention are sufficiently long such that, when combined with the rest of the scaffold, they will stably adopt the same three-dimensional arrangement as in PfRH5, such that the PfRH5 epitope of the invention is presented such that it has the same three-dimensional arrangement as in PfRH5. The similarity of the three- dimensional arrangement of the at least two α-helices of the scaffold of the invention compared to the equivalent three-dimensional arrangement as in wild-type PfRH5 may be measured by any suitable technique, examples of which are well known in the art including protein crystallography or cryogenic electron microscopy, with the similarity in the distances being referred to as the Root Mean Square Deviation (RMSD). By way of non-limiting example, the RMSD of the three-dimensional arrangement of the at least two α-helices of the scaffold of the invention compared to the equivalent three- dimensional arrangement as in wild-type PfRH5 is typically 2 Å or less. Preferably, the RMSD of the three-dimensional arrangement of the at least two α-helices of the scaffold of the invention compared to the equivalent three-dimensional arrangement as in wild-type PfRH5 is less than 1.5 Å. Preferably, a scaffold of the invention may comprise at least three α-helices, more preferably three α-helices. Of said three α-helices, typically two of the α-helices present the PfRH5 epitope of the invention (and may be resurfaced to facilitate this, as described herein), and the third α-helix facilitates multimerization of the polypeptide of the invention. Typically, the inclusion of a third α-helix stabilises the polypeptide, and wherein the polypeptide is multimerized, said third α-helix can facilitate multimerization. As a non-limiting example, said third α-helix may comprise a zipper motif, particularly an isoleucine zipper, to facilitate multimerization. The third α-helix may be resurfaced to comprise a zipper motif. A zipper motif refers to a repeating pattern of interactions, often involving hydrogen bonds or hydrophobic interactions, which stabilizes the association of two or more protein subunits. For example, a zipper motif may comprise 1, 2, 3, 4, 5 or more amino acid residues in a repeating pattern. Typically, a zipper motif comprised in an α-helix (particularly the third α- helix) of a scaffold of the invention may comprise a single amino acid residue repeating
pattern. For example, the single amino acid residue of the zipper motif may be isoleucine, leucine, or valine. Preferably, the single amino acid residue of the zipper motif is isoleucine. By way of non-limiting example, as exemplified herein, the present inventors identified an isoleucine zipper from Saccharomyces cerevisiae (such as that which can be found on the Protein Data Bank under the PDB ID: 1GCM (version 1.3, as accessed 7 February 2024). An exemplary isoleucine zipper motif is given in SEQ ID NO: 56. As well as the third α-helix comprising a zipper motif, said third α-helix may be further redesigned (e.g. using Rosetta) to facilitate correct folding of said third α-helix, and/or of the polypeptide comprising said third α-helix. In some preferred embodiments, a polypeptide of the invention comprises or consists of three α-helices, the first two of which comprise a PfRH5 antigen of the invention, the third of which comprises a zipper motif to facilitate multimerization, particularly trimerization, of the polypeptide. In some preferred embodiments, a polypeptide or scaffold comprises of the invention may comprise or consist of an amino acid sequence of SEQ ID NO: 57. Without being bound by theory, it is believed that the inclusion of a zipper motif, e.g. an isoleucine zipper on the third α-helix results in the multimerization of the polypeptide. Wherein a scaffold comprises at least 3 α-helices (e.g.3 α-helices), the amino acid residues of the PfRH5 epitope are typically presented on two of the α-helices. Again, without being bound by theory, it is believed that this better allows for the amino acid residues of the PfRH5 epitope to be presented with the same relative three-dimensional conformation as in PfRH5 and without other parts of the scaffold preventing antibody binding to this epitope. By way of non-limiting example, as exemplified herein, the present inventors identified a three- helical bundle from the Escherichia coli ribosome recycling factor with the correct helical topology to allow epitope grafting (PDB 3LHP, chain S). An exemplary sequence of a scaffold protein using said PDB 3LHP, chain S protein before resurfacing is given in SEQ ID NO: 59. In some preferred embodiments, therefore, the scaffold used to produce a polypeptide of the invention is said PDB 3LHP, chain S protein. Typically, the at least two α-helices of a scaffold of the invention may be resurfaced to optimise presentation of the PfRH5 epitope. Such resurfacing may comprise an initial in silico graft of the PfRH5 epitope onto the scaffold, followed by in silico mutagenesis of scaffold (not PfRH5 epitope) residues, energy minimisation and selection of suitable modified scaffolds. Typically, a comprehensive array of molecular designs is generated and systematically evaluated for their capacity to bind to specific antibodies that recognize the epitope. This extensive testing process aims to pinpoint designs that exhibit proper folding, ensuring that they effectively interact with the targeted antibodies. This process is exemplified in the context of the PDB 3LHP, chain S scaffold and PfRH5 epitope in the Examples below. Briefly, the exemplified PfRH5 epitope mimics were designed using the
Rosetta software suite. The region of PfRH5 containing the epitope was manually aligned with the scaffold structure (PDB: 3LHP) in Coot31, allowing a composite model to be generated in which residues 202-220 and 327-342 from PfRH5 were transplanted to the new epitope mimic. The resultant model was subjected to ab initio modelling using the energy minimisation function deployed in Rosetta. The folding process was carried out 10,000- times, scoring the output for stability. Next, the Rosetta package was used to perform computational site saturation mutagenesis for all residues in the synthetic immunogen, other than those specified as invariant due to their role in forming the epitope. This process generated 500 designs, which were scored for their Rosetta score as well as their root- mean-square-deviation from the desired design. The best designs were then analysed by evolutionary trace analysis in Jalview, allowing selection of designs which best represented sequence diversity for the designed proteins. Individual scaffold designs may incorporate multiple factors, such as folding and stability of the resulting polypeptide, and functional efficacy of the epitope when grafted onto the scaffold in order to maintain the immunogenicity of the epitope. Trimerization helix The present invention provides a trimerization helix that has been resurfaced to encode a zipper motif. Typically, this trimerization helix is the third helix in a polypeptide of the invention. By way of a non-limiting example, a zipper motif may comprise 1, 2, 3, 4, 5 or more amino acid residues in a repeating pattern. Typically, a zipper motif encoded on a trimerization helix may comprise of a single amino acid residue repeating pattern. The single amino acid residue may be any amino acid residue. For example, the single amino acid residue of the zipper motif may be isoleucine, leucine, or valine. Preferably, the single amino acid residue of the zipper motif is isoleucine. An exemplary isoleucine zipper sequences is given in SEQ ID NO: 56. Typically, a trimerization helix of the invention is an α-helix. A trimerization helix of the invention may be incorporated into the structure of an immunogen, such as the polypeptides of the invention. In some embodiments, the immunogen may comprise at least two α-helices in addition to the trimerization helix. To facilitate trimerization, each polypeptide monomer will typically comprise a trimerization helix which comprises the same zipper motif. Any suitable technique may be used to resurface the trimerization helix, examples of such techniques are well known in the art. Without being bound by theory, the isoleucine zipper of the trimerization helix facilitates the multimerization of said polypeptides, such as those of the invention.
Multimerization of immunogens, such as the polypeptides of the invention has advantages over monomeric immunogens. For example, multimeric immunogens may elicit a stronger immune response. Polypeptides The present invention provides a polypeptide which comprises a PfRH5 epitope and scaffold. Typically, the PfRH5 epitope is grafted onto the scaffold to produce a polypeptide of the invention. The scaffold may be a resurfaced scaffold, as described herein. The PfRH5 epitope may be any PfRH5 epitope as described herein. Thus, any disclosure herein in relation to a PfRH5 epitope of the invention may be combined with any disclosure in relation to a scaffold of the invention. By way of example, any disclosure herein in relation to a PfRH5 epitope of the invention may be combined with any disclosure in relation to the exemplified three-helical bundle from the Escherichia coli ribosome recycling factor scaffold). The polypeptides of the invention may be interchangeably referred to as immunogens, as they are capable of eliciting an immune response (e.g. an antibody response) when used to immunise a subject. The term "grafting," as used herein, refers to the process of attaching a specific molecule, such as an epitope, to a larger scaffold by interspersing the residues of the grafted molecule with those of the scaffold. Accordingly, the present invention provides a polypeptide in which the PfRH5 epitope is grafted onto the scaffold. In this context, "grafted" means that the epitope residues are interspersed with the scaffold amino acid residues ensuring that the PfRH5 epitope presented on the scaffold of the invention typically adopts a three dimensional conformation analogous to that of native (full-length) PfRH5, such as in SEQ ID NO: 1.Grafting of an PfRH5 epitope onto a scaffold may comprise interspersing individual amino acids from the PfRH5 epitope with one or more (e.g.1, 2, 3, 4, or more) scaffold amino acids. The number of scaffold amino acids between epitope amino acids may vary. By way of non-limiting example, Ep-Sc-Ep-Sc-Sc-Sc-Ep-Sc-Ep-Sc-Sc-Ep-Sc, where “Ep” is an amino acid residue from the PfRH5 epitope and “Sc” is an amino acid from the scaffold. Alternatively, or in addition, grafting of an PfRH5 epitope onto a scaffold may comprise interspersing two or more consecutive amino acids from the PfRH5 epitope (e.g.2, 3, or more) with one or more (e.g.1, 2, 3, 4, or more) scaffold amino acids. The number of scaffold amino acids between epitope amino acids may vary. By way of a further non-limiting example, Ep-Sc-Ep-Ep-Sc-Sc-Sc-Ep-Ep-Ep-Sc-Ep-Sc-Sc-Ep-Ep-Sc, where “Ep” is an amino acid residue from the PfRH5 epitope and “Sc” is an amino acid from the scaffold. By way of a non-limiting example, grafting of a PfRH5 epitope onto a scaffold may comprise interspersing no more than three consecutive amino acids from the PfRH5 epitope (e.g., 1, 2, or 3 consecutive residues) with one or more scaffold amino acids (e.g., 1, 2, 3, 4,
or more). Non-limiting examples of such consecutive epitope residues include the following: K57-I59, D64-V66, and K25-Y27, as exemplified in Table 1 herein.As discussed above, as the scaffold of the invention is typically exogenous, native PfRH5, or fragments thereof, such as the ectodomain of PfRH5 are not polypeptides according to the present invention. In some embodiments, a polypeptide of the invention may not comprise complete, uninterrupted PfRH5 epitope as seen in native (full-length) PfRH5, such as in SEQ ID NO: 1. In some further embodiments, a polypeptide of the invention may not comprise large runs of consecutive amino acids (e.g., 5 or more) from a PfRH5 epitope as seen in native (full- length) PfRH5, such as in SEQ ID NO: 1. Thus, the polypeptides of the invention are not necessarily identical to the native PfRH5 epitopes in terms of uninterrupted sequence or consecutive amino acid residues. Rather, the design of the polypeptides aims to maintain functionality and immune activity, even if the epitope is not presented in its native form or as a continuous stretch of consecutive amino acids. Any appropriate technique may be used variety of approaches can be used to graft a PfRH5 epitope onto scaffolds to produce a polypeptide of the invention. Non-limiting examples of such techniques include side chain grafting (McLellan, J. S. et al. J Mol Biol 409, 853-866, doi:10.1016/j.jmb.2011.04.044 (2011) and Schoeder, C. T. et al PLoS Pathog 18, e1010518, doi:10.1371/journal.ppat.1010518 (2022), both herein incorporated by reference). backbone grafting (Azoitei, M. L. et al. Science 334, 373-376, doi:10.1126/science.1209368 (2011), incorporated by reference) and building novel protein topologies to support a specific epitope (Sesterhenn, F. et al. Science 368, doi:10.1126/science.aay5051 (2020), incorporated by reference) and Rosetta-based methods or protein hallucination. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 5. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 5. The polypeptide of the invention may comprise at least two cysteine residues positioned such that they form a disulphide bridge in the polypeptide. For example, a polypeptide of the invention may comprise at least two, at least four, at least six, at least eight or at least ten cysteine residues positioned such that they form two, three, four or five disulphide bridges.
Disulphide bridges play a crucial role in maintaining the stability and structural integrity of proteins. These covalent bonds form between the sulphur atoms of two cysteine residues within a protein, creating a strong linkage. The presence of disulfide bridges contributes to the stabilization of a protein’s tertiary and quaternary structures by forming loops and connecting different regions of the polypeptide chain. This covalent linkage enhances the epitope’s resistance to denaturation, providing a level of structural support that is especially important in extracellular proteins exposed to fluctuating environmental conditions. Disulphide bridges contribute to the overall folding and maintenance of a protein’s native conformation, influencing its functional properties, durability, and resilience against unfolding or degradation over time. The at least two cysteine residues may be positioned at residue 9 and residue 90 of the polypeptide (e.g. SEQ ID NO: 5 or a variant thereof); at residue 44 and residue 116 (e.g. SEQ ID NO: 5 or a variant thereof); or at residue 9 residue 90, residue 44 and residue 116 (e.g. SEQ ID NO: 5 or a variant thereof). A polypeptide of the invention may comprise or consist of an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 98%, at least 99% or 100% sequence identity) to an amino acid sequence of selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57 and SEQ ID NO: 58. A polypeptide of the invention may comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 57 and SEQ ID NO: 58. Typically a polypeptide of the invention may comprise or consist of an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 98%, at least 99% or 100% sequence identity) to an amino acid sequence of selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. Preferably a polypeptide of the invention may comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. A polypeptide of the invention may comprise or consist of an amino acid sequence having one or more amino acid residues removed from the C-terminus and/or the N-terminus of the amino acid sequence. The number of amino acids that may be removed from the C- terminus and/or N-terminus is not limited, provided that the polypeptide retains the ability to fold correctly. The number of amino acids removed from the C-terminus and/or N-terminus may be determined independently. Thus: (i) one or more amino acids may be removed from
the C-terminus; (ii) one or more amino acids may be removed from the N-terminus; or (iii) one or more amino acids may be removed from the C-terminus and one or more amino acids may be removed from the N-terminus. A polypeptide of the invention may comprise or consist of an amino acid selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57 and SEQ ID NO: 58 in which the amino acid sequence has one or more amino acid residues removed from the C-terminus and/or N-terminus of the amino acid sequence. A polypeptide of the invention may comprise or consist of an amino acid sequence having up to 10 amino acid residues removed from the C-terminus and/or N-terminus of the amino acid sequence. For example, a polypeptide of the invention may comprise or consist of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues removed from the C-terminus and/or N-terminus of the amino acid sequence. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 6. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 6. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 7. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 7. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 7. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 8. Preferably the polypeptide may comprise or consists of an amino acid
sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 8. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 9. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 9. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 9. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 10. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 10. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 10. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 11. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 11. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 12. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 12. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13. For example, the polypeptide may comprise or consist of an amino acid sequence having at
least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 13. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 13. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 14. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 14. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 14. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 15. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 15. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 16. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 16. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 16. A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 57. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 57. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 57.
A polypeptide of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. For example, the polypeptide may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 58. Preferably the polypeptide may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 58. As discussed herein, for an amino acid sequence of the invention, such as a polypeptide: (a) an initial methionine may be included in the sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) an initial methionine may excluded in the sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. Similarly, for a nucleic acid sequence encoding a polypeptide sequence of the invention: (a) a codon encoding an initial methionine may be included in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) a codon encoding an initial methionine may excluded in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. A polypeptide of the invention may comprise a scaffold in which one or both of the at least two α-helices have been resurfaced such that the PfRH5 epitope is presented with the amino acid residues of the PfRH5 epitope have the same relative three-dimensional conformation as in PfRH5. The polypeptide of the invention may be multimerized. As described herein, a polypeptide of the invention which is to be multimerized may comprise a multimerization motif, typically within a third α-helix domain. A non-limiting example of a polypeptide of the invention comprising such a multimerization domain is found in SEQ ID NO: 57. A trimerized polypeptide of the invention may be formed by a trimer of three monomers comprising or consisting of a polypeptide or SEQ ID NO: 57, or a variant thereof as described herein. The term multimerized as described herein refers to the assembly of identical polypeptide units to form a larger, functional unit. For example, the polypeptide of the invention may be dimerized, trimerized, tetramerized, pentamerized or hexamerized. Preferably, the polypeptide of the invention is trimerized. For the avoidance of doubt, any and all disclosure herein in relation to polypeptides of the invention applies equally and without reservation to multimerized forms of the polypeptides, particularly trimerized forms. Wherein a polypeptide of the invention is multimerized, the monomers may all be the same polypeptide of the invention, or one or more of the polypeptides may be different. The use of the same polypeptide may be advantageous as it allows a greater immune stimulation
for a particular PfRH5 antigen of interest. The use of one or more different polypeptide may allow for an immune response to be stimulated against different PfRH5 antigens. As exemplified herein, the GIA of the PfRH5-specific antibodies induced by immunisation with a polypeptide of the invention is typically significantly greater than the GIA of PfRH5-specific antibodies induced by immunisation with PfRH5. The quality of the PfRH5 specific immune response (GIA as a factor of PfRH5-specific IgG) induced by polypeptides of the invention is typically significantly improved compared with that to PfRH5. Typically, the polypeptide of the invention may induce antibodies which have a GIA of at least 30% or more against Plasmodium parasites. For example, the polypeptide of the invention may induce antibodies which have a GIA of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more against Plasmodium parasites. Preferably the polypeptide of the invention may induce antibodies which have a GIA of at least 50%, or more against Plasmodium parasites. Preferably, the blood-stage Plasmodium parasite is Plasmodium falciparum. The polypeptide may induce antibodies that have a growth inhibitory activity (GIA) of at least 50% against a plurality of genetic strains of the blood-stage Plasmodium parasite. This is likely to be of importance in achieving protection against the variety of strains circulating in the natural environment. Accordingly, preferably, the polypeptide of the invention induces antibodies that have a GIA of at least 30%, at least 40%, 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. Preferably, the genetic strain of the blood- stage Plasmodium parasite is Plasmodium falciparum. The growth inhibitory activity (GIA) of PfRH5-specific antibodies may be measured at any appropriate concentration of the antibodies, for example the GIA may be measured at any appropriate concentration of the antibodies, for example the GIA may be measured at 1x10-4 μg/ml, 2x10-4 μg/ml, 3x10-4 μg/ml, 4x10-4 μg/ml, 5x10-4 μg/ml, 6x10-4 μg/ml, 7x10-4 μg/ml, 8x10-4 μg/ml, 9x10-4 μg/ml, 1x10-3 μg/ml, 2x10-3 μg/ml, 3x10-3 μg/ml, 4x10-3 μg/ml, 5x10-3 μg/ml, 6x10-3 μg/ml, 7x10-3 μg/ml, 8x10-3 μg/ml, 9x10-3 μg/ml, 0.01 μg/ml, 0.02 μg/ml, 0.03 μg/ml, 0.04 μg/ml, 0.05 μg/ml, 0.06 μg/ml, 0.07 μg/ml, 0.08 μg/ml, 0.09 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml, 0.4 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90 μg/ml, 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, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml of purified IgG antibody.
The growth inhibitory activity (GIA) of total IgG antibodies 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, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml or 10 mg/ml of purified IgG antibodies. Any appropriate technique may be used to determine the GIA. Exemplary techniques are described in the examples and conventional techniques are known in the art. A polypeptide of the invention may induce antibodies that have an effective concentration required for 30% GIA (EC30) GIA value which is 100 ng/mL or lower. For example, a polypeptide of the invention may induce antibodies that have an EC30 GIA value which is 100 ng/mL or less, 75 ng/mL or less, 50 ng/mL or less or 25 ng/mL or less. Any appropriate technique may be used to determine the EC30 GIA. Exemplary techniques are described in the Examples and conventional techniques are known in the art. A polypeptide of the invention may induce antibodies that have a growth inhibitory quality which is at least 100-fold greater than wild-type PfRH5. The term “quality” in relation to an antibody refers to eliciting a more specific induction of PfRH5 antibodies. The GIA of PfRH5-specific antibodies elicited by a polypeptide can be used as a measure of the of growth inhibitory quality. For example, a polypeptide of the invention may induce antibodies that have a growth inhibitory quality which is at least 100-fold, at least 200-fold, at least 300- fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold or at least 1000-fold greater than wild-type PfRH5. Preferably, a polypeptide of the invention may induce antibodies that have a growth inhibitory quality which is at least 500-fold greater than wild-type PfRH5. Any appropriate technique may be used to determine the growth inhibitory quality. Exemplary techniques are described in the Examples and conventional techniques are known in the art. It will be understood an antibody induced by a polypeptide of the invention may bind specifically to PfRH5. By specifically, it will be understood that the antibody binds to the molecule of interest, in this case the PfRH5 antigen and/or the epitope presented by the polypeptide of the invention. The polypeptide of the invention may induce PfRH5-specific antibodies which specifically bind to the PfRH5 epitope with a binding affinity in the range of 50 pM to 200 nM range. For example, the polypeptide of the invention may induce PfRH5-specific antibodies which specifically bind to the PfRH5 epitope with a binding affinity of 50 pM, 75 pM, 100 pM, 125 pM, 150 pM, 175 pM or 200 pM. The binding affinity of a PfRH5-specific antibody induced by a polypeptide of the invention may be quantified in any appropriate terms, e.g. dissociation constant (KD). KD
may be determined using any appropriate technique, such as surface plasmon resonance (SPR), which is used in the Examples herein. A multimerized (e.g. trimerized) form of a polypeptide of the invention may be associated with improved properties compared with the corresponding polypeptide in monomeric form. For example, a multimerized (e.g. trimerized) form of a polypeptide of the invention may elicit at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold or greater increase in the titre of PfRH5-specific antibodies elicited compared with the titre of PfRH5 specific antibodies elicited by a corresponding polypeptide in monomeric form. Alternatively or in addition, a multimerized (e.g. trimerized) form of a polypeptide of the invention may elicit at least a 1.5- fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold or greater decrease in the titre of scaffold- specific antibodies elicited compared with the titre of PfRH5 specific antibodies elicited by a corresponding polypeptide in monomeric form. In another example or in addition, scaffold residues which are completely covered in a multimerized (e.g. trimerized) form of a polypeptide of the invention may elicit no (or essentially no) antibody response when compared with the titre of scaffold-specific antibodies elicited by a corresponding polypeptide in monomeric form. Without being bound by theory, it is believed that the multimerized (e.g. trimerized) form of a polypeptide of the invention facilitates a more focused antibody response. Minimizing the antibody response against the rest of the polypeptide is advantageous as it reduces non-specific or off-target immune reactions. It will be understood that a polypeptide of the invention may need to be stable at elevated temperatures. A higher melting temperature makes the polypeptide more robust and resilient, allowing it to withstand different conditions without losing its structural and functional properties as well as being less prone to denaturation and degradation over time, leading to a longer half-life and improved shelf life. The polypeptide of the invention may have a melting temperature of greater than 75°C. For example, the polypeptide of the invention may have a melting temperature of greater than 75°C, great than 80°C, greater than 85°C, greater than 90°C, greater than 95°C or greater than 100°C. Any of the properties described herein of a polypeptide of the invention (e.g. the melt temperature of said polypeptide), or the PfRH5 antibodies elicited by a polypeptide of the invention (e.g. the GIA or binding affinity of antibodies induced by said polypeptide) may also be exhibited by a conjugate of the invention, or by a polypeptide encoded by a nucleic acid sequence or vector of the invention, or by the antibodies ultimately elicited from immunisation with said conjugate, nucleic acid or vector. Therefore, any and all disclosure regarding said properties given in the context of the polypeptides of the invention applies equally and without reservation to conjugates of the invention, nucleic acids and vectors of the invention, and to the antibodies ultimately elicited from immunisation with said conjugate, nucleic acid or vector.
Conjugates Typically, small immunogens (e.g. a polypeptide of the invention) are observed to induce lower immune responses compared with larger immunogens. Accordingly, conjugation of a polypeptide to a conjugate group may improve immune responses. The invention therefore provides a conjugate comprising a polypeptide of the invention and a conjugate group. A polypeptide of the invention may be covalently attached to one or more conjugate groups. A conjugate group may be attached to the N-terminus and/or the C-terminus of a polypeptide of the invention. A conjugate group may be attached to one or more non- terminal amino acid within the polypeptide. Covalent attachment of a conjugate group to a polypeptide of the invention may be interchangeably referred to herein as conjugation. Where conjugate groups are attached to both the N- and C-termini of the polypeptide different conjugate groups may be attached to the N-terminus and C-terminus, or the same conjugate group may be used for the N-terminus and the C-terminus. Where different conjugate groups are used, they may be selected independently. Where conjugate groups are attached to one or more non-terminal amino acid within the polypeptide, these may be the same as those present at the N-terminus or C-terminus (if present), or different thereto. When present, a conjugate group may preferably be attached to the N-terminus of a polypeptide of the invention. References herein to attachment of a conjugate group to a polypeptide of the invention refers to both attachment of said conjugate group to N-terminus and/or C-terminus, and/or to one or more non-terminal amino acid unless expressly stated to the contrary. A conjugate group may modify one or more properties of polypeptide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. A conjugate group may comprise or consist of a conjugate linker and/or a conjugate moiety. In some embodiments, the conjugate moiety is a multimeric scaffold. By way of a non-limiting example, suitable multimeric scaffolds include virus-like particle (VLP), multimeric proteins and multimeric synthetic polymers. In some preferred embodiments, the conjugate moiety is a virus-like particle (VLP). A virus-like particle (VLP) is a particle which resembles a virus but does not contain viral nucleic acid and is therefore non-infectious. VLPs commonly contain one or more virus capsid or envelope proteins which are capable of self-assembly to form the VLP. VLPs have been produced from components of a wide variety of virus families or can be synthetic and
designed using structure-guided methods (Noad and Roy (2003), Trends in Microbiology, 11:438-444; Grgacic et al., (2006), Methods, 40:60-65). Some VLPs have been approved as therapeutic vaccines, for example Engerix-B (for hepatitis B), Cervarix and Gardasil (for human papilloma viruses). Suitable VLPs are known in the art and can be readily selected by one of ordinary skilled without undue burden. By way of non-limiting example, suitable VLPs include Hepatitis B surface antigen (HBSAg), human papillomavirus (HPV) 18 L1 protein, HPV 16 L1 protein and/or Hepatitis E P239, preferably Hepatitis B surface antigen. In some preferred embodiments, the VLP may be a synthetic or designed VLP. By way of non-limiting example, suitable VLPs include I53-50 (as described in Wells et al. (2020). Cell vol.183,5 (2020): 1367-1382.e17, which is herein incorporated by reference). Preferably, a VLP used in a conjugate of the invention comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 60 and SEQ ID NO: 61. A conjugate group of the invention may be attached to polypeptide by any appropriate means. Conjugate moieties are typically attached to polypeptide through conjugate linkers. In certain conjugates, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an amino acid through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units. In certain embodiments, conjugate linkers comprise 1-30 linker-amino acids. A conjugate linker may be a protein coupling domain, such as that found in the spy- catcher/spy-tag system. SpyTag technology is a protein ligation method based on the SpyTag peptide and SpyCatcher protein (Zakeri et al. (2012). PNAS 109(12), 690-697, which is herein incorporated by reference), which are derived from the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin-binding protein (FbaB) of Streptococcus pyogenes (Spy). Suitable Spy-tag /Spy-catcher systems are known and commercially available in the art. The conjugate group may be cleavable from the polypeptide of the invention. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. Cleavage may be mediated by any suitable process, for example use of a protease or by chemical cleavage. Preferably the conjugate group is not cleavable from the polypeptide of the invention. Thus, certain conjugate linkers may not comprise any cleavable moieties. Preferably a conjugate of the invention comprises or consists of a polypeptide of the invention, which is conjugated to a VLP, particularly I53-50. The polypeptide may be conjugated to the VLP (e.g. I53-50) by any appropriate means. Even more preferably, a
conjugate of the invention comprises or consists of a polypeptide of the invention, which is directly conjugated to a VLP, particularly I53-50. In another embodiment, a conjugate of the invention comprises or consists of a polypeptide of the invention which is indirectly conjugated to a VLP (e.g. I53-50). By way of non-limiting example, a conjugate of the invention may comprise or consist of a polypeptide conjugated to a VLP (e.g. e.g. I53-50) using a spy-tag. In some embodiments, the virus-like particle is attached to the N-terminus of the polypeptide via a spy-tag. Preferably the VLP is attached to the C-terminus of the polypeptide via a GS linker, such as a GS16 linker. In some preferred embodiments, the invention provides a conjugate comprising or consisting of SEQ ID NO: 62. A conjugate of the invention may comprise or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 62. For example, the conjugate may comprise or consist of an amino acid sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to SEQ ID NO: 62. Preferably the conjugate may comprise or consists of an amino acid sequence having at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 62. A trimerized polypeptide of the invention may also be conjugated. In some embodiments, each monomer of a trimerized polypeptide to be conjugated may comprise or consist of SEQ ID NO: 57, as described herein. In a trimerized conjugate of the invention each polypeptide monomer may be conjugated to a monomer of the same conjugate group or to a different conjugate group, preferably to a monomer the same conjugate group. In other words, each monomer comprises or consists of the same polypeptide conjugated to the same conjugate group to form a conjugate monomer, and the trimeric form of the conjugate comprises three such conjugate monomers. In some embodiments, each polypeptide monomer may be conjugated to the same VLP, such as to an I53-50 sequence, particularly SEQ ID NO: 60 or 61 as described herein. Thus, by way of non-limiting example, a conjugate monomer of the invention may comprise or consist of SEQ ID NO: 63 as described herein. A trimeric conjugate may then comprise three copies of a conjugate monomer which comprises or consists of SEQ ID NO: 63. Typically, a conjugate of the invention may induce antibodies which have a GIA of at least 30% or more against Plasmodium parasites. For example, the conjugate of the invention may induce antibodies which have a GIA of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more against Plasmodium parasites. Preferably the polypeptide of the invention may induce antibodies which have a
GIA of at least 50%, or more against Plasmodium parasites. Preferably, the blood-stage Plasmodium parasite is Plasmodium falciparum. The conjugate may induce antibodies that have a growth inhibitory activity (GIA) of at least 50% against a plurality of genetic strains of the blood-stage Plasmodium parasite. This is likely to be of importance in achieving protection against the variety of strains circulating in the natural environment. Accordingly, preferably, the conjugate of the invention induces antibodies that have a GIA of at least 30%, at least 40%, 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. Preferably, the genetic strain of the blood-stage Plasmodium parasite is Plasmodium falciparum. The growth inhibitory activity (GIA) may be measured at any appropriate concentration of the antibodies as described herein. Any appropriate technique may be used to determine the GIA. Exemplary techniques are described in the examples and conventional techniques are known in the art. A conjugate of the invention may induce antibodies that have an effective concentration required for 30% GIA (EC30) GIA value which is 100 ng/mL or lower. For example, a conjugate of the invention may induce antibodies that have an EC30 GIA value which is 100 ng/mL or less, 75 ng/mL or less, 50 ng/mL or less or 25 ng/mL or less. Any appropriate technique may be used to determine the EC30 GIA. Exemplary techniques are described in the Examples and conventional techniques are known in the art. A conjugate of the invention may induce antibodies that have a growth inhibitory quality which is at least 100-fold greater than wild-type PfRH5. For example, a conjugate of the invention may induce antibodies that have a growth inhibitory quality which is at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600- fold, at least 700-fold, at least 800-fold, at least 900-fold or at least 1000-fold greater than wild-type PfRH5. Preferably, a conjugate of the invention may induce antibodies that have a growth inhibitory quality which is at least 500-fold greater than wild-type PfRH5. Any appropriate technique may be used to determine the growth inhibitory quality. Exemplary techniques are described in the Examples and conventional techniques are known in the art. It will be understood an antibody induced by a conjugate of the invention may bind specifically to PfRH5. By specifically, it will be understood that the antibody binds to the molecule of interest, in this case the PfRH5 antigen and/or the epitope presented by the conjugate of the invention. The conjugate of the invention may induce PfRH5-specific antibodies which specifically bind to the PfRH5 epitope with a binding affinity in the range of 50 pM to 200 nM range. For example, the conjugate of the invention may induce PfRH5-specific antibodies
which specifically bind to the PfRH5 epitope with a binding affinity of 50 pM, 75 pM, 100 pM, 125 pM, 150 pM, 175 pM or 200 pM. The binding affinity of a PfRH5-specific antibody induced by a conjugate of the invention may be quantified in any appropriate terms, e.g. dissociation constant (KD). KD may be determined using any appropriate technique, such as surface plasmon resonance (SPR), which is used in the Examples herein. It will be understood that a conjugate of the invention may need to be stable at elevated temperatures. A higher melting temperature makes the polypeptide more robust and resilient, allowing it to withstand different conditions without losing its structural and functional properties as well as being less prone to denaturation and degradation over time, leading to a longer half-life and improved shelf life. The polypeptide of the invention may have a melting temperature of greater than 75°C. For example, the polypeptide of the invention may have a melting temperature of greater than 75°C, great than 80°C, greater than 85°C, greater than 90°C, greater than 95°C or greater than 100°C. Isolated nucleotide sequences In other aspects, the invention provides an isolated nucleic acid sequence encoding a polypeptide or conjugate of the invention. The terms "nucleic acid", “nucleic acid sequence”, “nucleotide sequence” and “polynucleotide” are used herein interchangeably with the terms to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. The terms encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogues include, without limitation, phosphorothioates, phosphonamidites, methyl phosphonates, chiral-methyl phosphonates, 2-Omethyl ribonucleotides, peptide-nucleic acids (PNAs). In some embodiments, the nucleic acid may be an mRNA. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxy inosine residues (See: Batzer et al., Nucleic Acids Res 1991;25(19):5081; Ohtsuka et al., J Biol Chem 1985;260(5):2605-8; Rossolini et al., Mol Cell Probes 1994;8(2):91-8; the contents of each of which are herein incorporated by reference for this purpose).
In the case of a conjugate of the invention, the polypeptide of the invention may be encoded by a first nucleotide sequence and the conjugate group and/or conjugate moiety, particularly a VLP (e.g. I53-50) may be encoded by a second nucleotide sequence. Conjugation of the polypeptide and the conjugate group and/or conjugate moiety, particularly a VLP (e.g. I53-50) may then occur after the polypeptide and the conjugate group and/or conjugate moiety, particularly a VLP (e.g. I53-50) are expressed by the first and second nucleotide sequences respectively. The nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SOS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed.1987 Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York, the contents of which are herein incorporated by reference for this purpose. A nucleic acid of the disclosure can be, for example, DNA or RNA and may or may not contain intronic sequences. For example, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector. As discussed herein, for a nucleic acid sequence encoding an epitope, scaffold or polypeptide sequence of the invention: (a) a codon encoding an initial methionine may be included in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes; or (b) a codon encoding an initial methionine may excluded in the nucleotide sequence and (i) included for numbering purposes; or (ii) excluded for numbering purposes. Vectors and expression systems The present invention provides one or more vector or expression cassette encoding a polypeptide or conjugate of the invention. A vector or expression cassette of the invention typically comprises a nucleic acid of the invention operably linked to a promoter. Any suitable promoter may be used, conventional examples of which are known in the art. A conjugate of the invention may be expressed by: (i) one vector; or (ii) by two (or more) separate vectors or expression cassette. For example, for a conjugate of the invention, the polypeptide may be expressed by a first vector, and the conjugate group and/or conjugate moiety, particularly a VLP (e.g. I53-50), may be expressed by a second vector. The vector(s) may be present in the form of a formulation or composition.
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 some embodiments, 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 may be capable of expression in a mammalian cell, such as an immunised cell. Thus, a vector of the invention may be capable of expression in vivo in a mammalian (and particularly a human) subject following immunisation. Alternatively, or in addition, the vector may be capable of expression in a heterologous protein expression system. 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. By way of a non-limiting example, the vector is capable of expression in vivo. For example, the vector is capable of expression in a vaccinated mammal (e.g human.) The vector(s) may be a DNA vector, such as a DNA plasmid. A DNA vector(s) may be capable of expression in a mammalian cell expression system, such as an immunised cell. Thus, a DNA vector of the invention may be capable of expression in vivo in a mammalian (and particularly a human) subject following immunisation. The DNA vector may be suitable for expression in a bacterial and/or insect host cell or expression system, such as any of those exemplified herein (e.g. 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 an 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. A polypeptide or conjugate 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 also 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 polypeptides or conjugates 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. Alternatively, the polypeptides or conjugates of the invention may be mature forms from which the N-terminal signal peptide has been removed. A polypeptide or conjugate 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, a polypeptide or conjugate of the invention may be purified from heterologous expression systems without the need to use a purification tag. A polypeptide or conjugate of the invention may be expressed using any suitable systems. Polypeptides or conjugates of the invention may be expressed using conventional systems. A polypeptide or conjugate of the invention may be expressed in vivo. 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 (preferably CHO cells).
The invention also provides a host cell comprising a nucleic acid, expression cassette or vector of the invention which encodes for a polypeptide or conjugate of the invention. Preferably, the host cell may be an insect cell, optionally a Drosophila melanogaster cell, or a Pichia yeast cell, or an E. coli cell. Antibodies The present invention provides antibodies that bind specifically to the polypeptides or conjugates disclosed herein. Also provided are antibodies obtained following immunisation with the polypeptides or conjugates disclosed herein. Binding of an antibody of the invention to a polypeptide of the invention, and particularly the PfRH5 epitope comprised in said polypeptide may be determined using an appropriate technique. Those skilled in the art are able to choose a suitable mode of determining binding of the antibody or antigen-binding fragment thereof to an antigen (or epitope thereof) according to their preference and general knowledge, in light of the methods and disclosure herein. By way of non-limiting example, binding of an antibody of the invention to an epitope may be determined by X-ray crystallography or cry-EM. Suitable techniques are described in e.g., X-ray crystallography protocols (Harrison, T.E., Alam, N., Farrell, B., Quinkert, D., Lias, A.M., King, L.D., Draper, S.J., Campeotto, I.* and Higgins, M.K.* (2024) Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5. EMBO Molecular Medicine 162539-2559), NMR spectroscopy protocols (Valente, Ana P, and Mariana Manzano-Rendeiro. “Mapping conformational epitopes by NMR spectroscopy.” Current opinion in virology vol.49 (2021): 1- 6. doi:10.1016/j.coviro.2021.04.001) and cryo-EM protocols (Turner et al. “Protocol for analyzing antibody responses to glycoprotein antigens using electron-microscopy-based polyclonal epitope mapping.” STAR protocols vol.4,3 (2023): 102476. doi:10.1016/j.xpro.2023.102476), which are herein incorporated by reference, and which provide exemplary X-ray crystallography protocols for determining whether an antibody binds to an epitope according to the invention. Crystal structures can be determined at any appropriate resolution, such as 1.6 or 2.4 Å. The following definitions are generally applicable to all antibodies of the invention. 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 PfRH5 epitope binds to PfRH5 antibodies which are mAbs. Alternatively or in addition, the PfRH5 epitope may bind to PfRH5 antibodies which are pAbs. 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 “antibody” may also encompass an “antigen-binding fragment” of an antibody (or simply “binding fragment”). An “antigen binding fragment” or “binding fragment” 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 polypeptides 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 labelling" 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 (including full length antibodies or antigen-binding fragments thereof) of the invention may bind specifically to a PfRH5 epitope/polypeptide/conjugate of the invention. By specifically, it will be understood that the antibody binds to the molecule of interest, in this case the PfRH5 epitope/polypeptide/conjugate of the invention, with no significant cross-reactivity to any other molecule, particularly any other protein. For example, an antibody of the invention that is specific for a particular PfRH5
epitope/polypeptide/conjugate of the invention will show no significant cross-reactivity with other malarial invasion proteins. Cross-reactivity may be assessed by any suitable method. An antibody that is specific for the PfRH5 epitope/polypeptide/conjugate 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/polypeptide/conjugate. Preferably, the 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/polypeptide/conjugate. An antibody of the invention may specifically bind to the PfRH5 epitope/polypeptide/conjugate with a binding affinity in the range of 50 pM to 200 nM range. For example, an antibody of the invention may specifically bind to the PfRH5 epitope/polypeptide/conjugate with a binding affinity of 50 pM, 75 pM, 100 pM, 125 pM, 150 pM, 175 pM or 200 pM. The binding affinity of an antibody of the invention may be quantified in any appropriate terms, e.g. dissociation constant (KD). KD may be determined using any appropriate technique, such as surface plasmon resonance (SPR), which is used in the Examples herein. As described herein, a PfRH5 epitope/polypeptide/conjugate of the invention raise antibodies as described herein. The antibodies 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 antigens of the invention raise 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 a PfRH5 epitope/polypeptide/conjugate of the invention, or antibodies raised by said PfRH5 epitope/polypeptide/conjugate may be quantified using any appropriate technique and measured in any appropriate units. For example, effectiveness may be given in terms of their growth inhibitory activity (GIA), half maximal effective concentration (EC50), antibody titre stimulated (in terms of antibody units, AU) and/or EC50 in terms of AU (described herein in relation to antibodies of the invention) of the antibodies raised. The latter of these gives an indication of the quality of the antibody response stimulated by the PfRH5 epitope/polypeptide/conjugate of the invention. Any appropriate technique may be used to determine the GIA, EC50, AU or EC50/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 antigens or epitopes of the invention. Typically, an antibody of the invention has a GIA of at least 30% or more against Plasmodium parasites. For example, the polypeptide of the invention may induce antibodies
which have a GIA of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more against Plasmodium parasites. Preferably the antibody has a GIA of at least 50%, or more against Plasmodium parasites. Preferably, the blood-stage Plasmodium parasite is Plasmodium falciparum. The antibody may have a growth inhibitory activity (GIA) of at least 50% against a plurality of genetic strains of the blood-stage Plasmodium parasite. This is likely to be of importance in achieving protection against the variety of strains circulating in the natural environment. Accordingly, preferably, the antibody may have a GIA of at least 30%, at least 40%, 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. Preferably, the genetic strain of the blood-stage Plasmodium parasite is Plasmodium falciparum. 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, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml or 10 mg/ml of purified IgG antibody. Any appropriate technique may be used to determine the GIA. Exemplary techniques are described in the examples and conventional techniques are known in the art. An antibody of the invention may have an effective concentration required for 30% GIA (EC30) GIA value which is 100 ng/mL or lower. For example, an antibody may have an EC30 GIA value which is 100 ng/mL or less, 75 ng/mL or less, 50 ng/mL or less or 25 ng/mL or less. Any appropriate technique may be used to determine the EC30 GIA. Exemplary techniques are described in the Examples and conventional techniques are known in the art. An antibody may have a growth inhibitory quality which is at least 100-fold greater than an antibody raised against wild-type PfRH5. For example, an antibody may have a growth inhibitory quality which is at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900- fold or at least 1000-fold greater than an antibody raised against wild-type PfRH5, preferably at least 500-fold greater. Any appropriate technique may be used to determine the growth inhibitory quality. Exemplary techniques are described in the Examples and conventional techniques are known in the art. Therapeutic Indications The present invention also provides a method of stimulating or inducing an immune response in a subject comprising administering to the subject one or more composition, PfRH5
epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention (as described above). Thus, the invention provides a vaccine composition comprising one or more PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention. The method of stimulating or inducing an immune response in a subject may comprise administering one or more composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention to a subject. The invention provides a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention for use in therapy. For example, in some embodiments, the invention provides a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention for use in treating and/or preventing malaria. Preferably, the invention provides a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention for use in treating and/or preventing malaria. Particularly preferred is a polypeptide or conjugate of the invention for use in treating and/or preventing malaria. The invention also provides a method of treating and/or preventing malaria, said method comprising administering a therapeutically effective amount of a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention to a subject in need thereof. The invention also provides the use of a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention in the manufacture of a medicament for the prevention and/or treatment of malaria. In some embodiments, the treatment and/or prevention of malaria comprises (a) administering a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention to a subject; and (b) subsequently administering at least one dose of a full-length PfRH5 antigen which comprises or consists of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1, wherein preferably the full-length PfRH5 antigen is a thermostable form of PfRH5, optionally comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 17. Thus, the invention provides a composition comprising one or more composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector,
RNA vaccine or DNA plasmid) of the invention. A composition of the invention additionally optionally comprises one or more of a pharmaceutically acceptable excipient, diluent or carrier. In one embodiment, the one or more vectors are selected from a viral vector, RNA vaccine, or DNA plasmid. Preferably, a composition of the invention comprises a polypeptide or conjugate of the invention and a pharmaceutically acceptable excipient. 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. When used in a therapeutic application as described herein, a polypeptide of the invention may be provided in any appropriate form, e.g. as a (recombinant) protein, conjugate, vector, DNA plasmid, RNA vaccine or other form, as described herein. In addition, when used in a therapeutic application as described herein, a polypeptide may be used in combination with one or more additional Plasmodium antigens (e.g. Plasmodium merozoite antigens) which is independently provided in any appropriate form, e.g. as a (recombinant) protein, conjugate, vector, DNA plasmid, RNA vaccine or other form, as described herein. Any and all disclosure in relation to therapeutic applications of polypeptides and conjugates of the invention applies equally and without reservation to any combination of a polypeptide or conjugate of the invention and one or more additional Plasmodium antigens (e.g. Plasmodium merozoite antigens), regardless of the form of the polypeptide or conjugate and/or the one or more additional Plasmodium antigens (e.g. Plasmodium merozoite antigens). Thus, the polypeptide or conjugate of the invention may be in the form of a recombinant protein, conjugate, a virus-like particle, or a combination thereof as described herein. In addition, a polypeptide or conjugate of the invention may be used in combination with one or more further antigens selected from the group consisting of PfCyRPA, PfRH5, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181, PfCSS, PfPTRAMP and PfAARP, or a fragment thereof, for use in prevention or treatment of malaria. The present invention provides the use of a composition, antigen (e.g., RIPR antigen), fusion protein (e.g., RIPR-CyRPA fusion), antibody or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention either alone or in combination in the prevention or treatment of malaria. 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, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention 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, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) 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, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) 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 a PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention (described herein). Said compositions may further comprise one or more further components as described herein. By way of example, said vaccine composition may further comprise one or more additional malarial antigens (Plasmodium merozoite antigen) as described herein, and/or any further components as described herein. The one or more additional Plasmodium merozoite antigens which is independently provided in any appropriate form, e.g. as a (recombinant) protein, conjugate, vector, DNA plasmid, RNA vaccine or other form, as described herein.
A vaccine composition of the invention may further comprise one or more additional antigens selected from the group consisting of RIPR, PfCyRPA, PfRH5, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181, PfCSS, PfPTRAMP and PfAARP, or a fragment thereof. A vaccine composition of the invention may further comprise one or more vectors expressing one or more additional antigen selected from the group consisting of RIPR, PfCyRPA, PfRH5, PfP113, PfRhopH3, PfRAP2, PfAMA1, PfRON2, PfEBA175, PfRH1, PfRH2a, PfRH2b, PfRH4, PfRAP1, PfRAP3, PfMSRP5, PfRAMA, PfSERA9, PfEBA181, PfCSS, PfPTRAMP and PfAARP, or a fragment thereof. These additional antigens 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, as a conjugate. Examples of fusion proteins are hepatitis B surface antigen fusions (e.g. as in the RTSS 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 composition comprising a PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention may be combined with a composition comprising an additional antigen, 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). Thus, the composition comprising a PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention 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 comprising a PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention provides an antibody response (e.g. a neutralising antibody response) to Plasmodium parasitic infection. The composition comprising a PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA
vaccine or DNA plasmid) of the invention 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”. A PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) of the invention (as defined above) can be combined or administered in addition to a pharmaceutically acceptable carrier. Alternatively, or in addition the PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) 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 compositions 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 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 (e.g. a PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector (e.g., a viral vector, RNA vaccine or DNA plasmid) 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
composition 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’s adjuvant (CFA), Incomplete Freund’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, AS01, AS03 and AS04 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 polypeptide, conjugate, vector or 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.
Dosing Regimen As exemplified herein, the present inventors have shown that immunising with a polypeptide of the invention, followed by a subsequent immunisation with full-length PfRH5 gives rise to a higher quality immune response compared with immunising first with a full- length PfRH5, followed by a subsequent immunisation with a polypeptide of the invention. In other words, priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 gives a higher quality immune response compared with priming with a full-length PfRH5, followed by boosting with a polypeptide of the invention. Particularly preferred is priming with a polypeptide of the invention, followed by two boosts with full-length PfRH5. By higher quality immune response, it is mean that one or more properties of the antibodies (as described herein) elicited by priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 is improved compared with priming with a full- length PfRH5, followed by boosting with a polypeptide of the invention. For example, the GIA of the PfRH5-specific antibodies elicited by priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 may be greater compared with priming with a full-length PfRH5, followed by boosting with a polypeptide of the invention. The growth inhibitory quality of the antibodies elicited by priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 may be greater compared with priming with a full-length PfRH5, followed by boosting with a polypeptide of the invention. The binding affinity for PfRH5 of the antibodies elicited by priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 may be increased compared with priming with a full-length PfRH5, followed by boosting with a polypeptide of the invention. Priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 may elicit at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold or greater decrease in EC30 of PfRH5-specific antibodies elicited compared with the EC30 of PfRH5 specific antibodies elicited by priming with a full-length PfRH5, followed by boosting with a polypeptide of the invention. Alternatively or in addition, preferably in addition, priming with a polypeptide of the invention, followed by boosting with full-length PfRH5 may elicit at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold or greater increase in PfRH5-specific antibodies elicited compared with PfRH5-specific antibodies elicited by priming with a full-length PfRH5, followed by boosting with a polypeptide of the invention. This novel approach (priming with a specific antigen, followed by boosting with a full- length PfRH5) specifically induces antibodies which target the epitope region desired, as described herein. Accordingly, in some embodiments, the invention provides treatment and/or prevention of malaria , which comprises (a) administering the polypeptide, conjugate, nucleic acid, vector or composition to a patient; and (b) subsequently administering at least one
dose of a full-length PfRH5 antigen which comprises or consists of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1, optionally comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 17. This ordering of priming with a focused antigen (with a single or small number (e.g.5 or less epitopes) followed by boosting with an antigen containing broader epitope regions (e.g. a full-length protein or extracellular domain of a protein) is the opposite to the conventional approach, in which priming is carried out using an antigen containing broader epitope regions (e.g. a full-length protein or extracellular domain of a protein) followed by boosting with a focused antigen (with a single or small number (e.g.5 or less epitopes). Without being bound by theory, it is believed that priming with a focused antigen allows for antibodies to key epitopes (present in the focused antigen) to be generated without being masked by suboptimal epitopes present in the broader epitope region. Subsequent boosting with an antigen containing broader epitope regions then allows antibodies to the key epitope to be generated, together with antibodies to other epitopes within the boosting antigen, wherein some of these other antibodies may potentiate the activity of the antibodies raised against the key epitope. Accordingly, this dosing regimen is of broader applicability than PfRH5, although it is exemplified herein in that context. As such, the present invention also provides a method of treating or preventing a disease comprising one or more prime immunisations with a first, focused antigen (containing 5 or fewer epitopes, preferably 1 epitope), followed by one or more boost immunisations with a second, broader antigen (containing more epitopes than the first antigen, but typically including the epitope(s) of the first antigen). Preferably the first and second antigen are malarial antigens, particularly Plasmodium merozoite antigens such as those described herein. Priming may comprise 1, 2, 3, 4 or more separate immunisations with a first antigen (e.g. a polypeptide of the invention). Boosting may comprise 1, 2, 3, 4 or more separate immunisations with a second antigen (e.g. full-length PfRH5). In the context of the present invention, in some preferred embodiments, there may be one prime immunisation with a polypeptide of the invention (in whatever format, e.g. polypeptide, conjugate, vector, etc. as described herein) and two subsequent boost immunisations with full-length PfRH5 or a thermostable version thereof. Each prime administration may take place at an appropriate interval to be determined by a medical practitioner, such as at intervals of 1 day, 2 day, 3 days, 4 days, 5 days, 6 days, 1 week or more between each prime administration. Alternatively or in addition, each boost administration may take place at an appropriate interval to be determined by a medical
practitioner, such as at intervals of 1 day, 2 day, 3 days, 4 days, 5 days, 6 days, 1 week or more between each boost administration. The separation of the prime immunisation (or the last prime immunisation if there are more than 1) and the boost immunisation (or the first boost immunisation if there are more than 1) may be determined by a medical practitioner. For example, the prime immunisation (or the last prime immunisation if there are more than 1) and the boost immunisation (or the first boost immunisation if there are more than 1) may be separated by an interval of 1 day, 2 day, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, six months, or more. Typically, the prime immunisation (or the last prime immunisation if there are more than 1) and the boost immunisation (or the first boost immunisation if there are more than 1) may be separated by an interval of 28 days, 2 months, 3 months, 4 months, six months, or more. SEQUENCE HOMOLOGY 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 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 sequence from which the antigen of the invention is derived. Alternatively, the amino acid at a specified position within the 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 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 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 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). 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 & 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 __________________________________________ x 100 [length of the longer sequence plus the number of gaps Introduced into the longer sequence in order to align the two sequences] BLOSUM62 table A R N D C Q E G H I L K M F P S T 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 2 4 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 -211 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.
SEQUENCE INFORMATION Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs:, said residue/codon is optional. Where an initial Met amino acid residue or a corresponding initial codon is omitted in any of the following SEQ ID NOs:, said residue/codon may optionally be present. SEQ ID NO: 1: Full length PfRH5 amino acid sequence (3D7) including signal sequence SEQ ID NO: 2: PfRH5^NL SEQ ID NO: 3: Consensus sequence of first portion of a preferred PfRH5 epitope as presented on a first α-helix of the scaffold SEQ ID NO: 4: Consensus sequence of second portion of a preferred PfRH5 epitope as presented on a second α-helix of the scaffold SEQ ID NO: 5: Exemplified immunogen 3 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 6: Exemplified immunogen 3A (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 7: Exemplified immunogen 3B (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 8: Exemplified immunogen 3C (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 9: Exemplified immunogen 1 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 10: Exemplified immunogen 2 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 11: Exemplified immunogen 4 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 12: Exemplified immunogen 5 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 13: Exemplified immunogen 6 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 14: Exemplified immunogen 7 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 15: Exemplified immunogen 8 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 16: Exemplified immunogen 9 (PfRH5 epitope with intervening scaffold residues) SEQ ID NO: 17: Thermostable full-length PfRH5 amino acid sequence including signal sequence SEQ ID NO: 18: Thermostable PfRH5^NL SEQ ID NO: 19: Neutralising epitope bin of the R5.016 antibody SEQ ID NO: 20: Neutralising epitope of the R5.016 antibody SEQ ID NO: 21: Neutralising epitope bin of the R5.004 antibody SEQ ID NO: 22: Heavy chain CDR1 of the R5.034 antibody SEQ ID NO: 23: Heavy chain CDR2 of the R5.034 antibody SEQ ID NO: 24: Heavy chain CDR3 of the R5.034 antibody SEQ ID NO: 25: Light chain CDR1 of the R5.034 antibody SEQ ID NO: 26: Light chain CDR2 of the R5.034 antibody
SEQ ID NO: 27: Light chain CDR3 of the R5.034 antibody SEQ ID NO: 28: Heavy chain variable region of the R5.034 antibody SEQ ID NO: 29: Light chain variable region of the R5.034 antibody SEQ ID NO: 30: Heavy chain CDR1 of the 9AD4 antibody SEQ ID NO: 31: Heavy chain CDR2 of the 9AD4 antibody SEQ ID NO: 32: Heavy chain CDR3 of the 9AD4 antibody SEQ ID NO: 33: Light chain CDR1 of the 9AD4 antibody SEQ ID NO: 34: Light chain CDR2 of the 9AD4 antibody SEQ ID NO: 35: Light chain CDR3 of the 9AD4 antibody SEQ ID NO: 36: Heavy chain variable region of the 9AD4 antibody SEQ ID NO: 37: Light chain variable region of the 9AD4 antibody SEQ ID NO: 38: Heavy chain CDR1 of the R5.016 antibody SEQ ID NO: 39: Heavy chain CDR2 of the R5.016 antibody SEQ ID NO: 40: Heavy chain CDR3 of the R5.016 antibody SEQ ID NO: 41: Light chain CDR1 of the R5.016 antibody SEQ ID NO: 42: Light chain CDR2 of the R5.016 antibody SEQ ID NO: 43: Light chain CDR3 of the R5.016 antibody SEQ ID NO: 44: Heavy chain variable region of the R5.016 antibody SEQ ID NO: 45: Light chain variable region of the R5.016 antibody SEQ ID NO: 46: Heavy chain CDR1 of the R5.004 antibody SEQ ID NO: 47: Heavy chain CDR2 of the R5.004 antibody SEQ ID NO: 48: Heavy chain CDR3 of the R5.004 antibody SEQ ID NO: 49: Light chain CDR1 of the R5.004 antibody SEQ ID NO: 50: Light chain CDR2 of the R5.004 antibody SEQ ID NO: 51: Light chain CDR3 of the R5.004 antibody SEQ ID NO: 52: Heavy chain variable region of the R5.004 antibody SEQ ID NO: 53: Light chain variable region of the R5.004 antibody SEQ ID NO: 54: “Ungapped” consensus sequence of first portion of a preferred PfRH5 epitope as presented on a first α-helix of the scaffold SEQ ID NO: 55: “Ungapped” consensus sequence of second portion of a preferred PfRH5 epitope as presented on a second α-helix of the scaffold SEQ ID NO: 56: Exemplary repeating pattern of an isoleucine zipper. SEQ ID NO: 57: Exemplified immunogen sequence for trimerization (TB4) SEQ ID NO: 58: Further exemplified immunogen sequence (B4) SEQ ID NO: 59: Full length scaffold protein sequence using PDB 3LHP, chain S protein before resurfacing. SEQ ID NO: 60: Exemplary I53-50 sequence (I53-50A)
SEQ ID NO: 61: Exemplary I53-50 sequence (I53-50C>A) SEQ ID NO: 62: Exemplary conjugate sequence (B4-16GS-I53-50A-GSG-6HIS) SEQ ID NO: 63: Exemplary conjugate sequence for trimerization (TB4-16GS-I53-50A(C>A)- GSG-6HIS) SEQ ID NO: 64: Exemplary linker sequence for linking the heavy and light variable chains of R5.016 SEQ ID NO: 65: Exemplary linker sequence for linking the heavy and light variable chains of R5.034 SEQ ID NO: 66: Full length PfRH5 amino acid sequence (3D7) including signal sequence SEQ ID NO: 1: Full length PfRH5 amino acid sequence (3D7) including signal sequence 1 MIRIKKKLIL TIIYIHLFIL NRLSFENAIK KTKNQENNLT LLPIKSTEEE KDDIKNGKDI 61 KKEIDNDKEN IKTNNAKDHS TYIKSYLNTN VNDGLKYLFI PSHNSFIKKY SVFNQINDGM 121 LLNEKNDVKN NEDYKNVDYK NVNFLQYHFK ELSNYNIANS IDILQEKEGH LDFVIIPHYT 181 FLDYYKHLSY NSIYHKSSTY GKCIAVDAFI KKINEAYDKV KSKCNDIKND LIATIKKLEH 241 PYDINNKNDD SYRYDISEEI DDKSEETDDE TEEVEDSIQD TDSNHTPSNK KKNDLMNRTF 301 KKMMDEYNTK KKKLIKCIKN HENDFNKICM DMKNYGTNLF EQLSCYNNNF CNTNGIRYHY 361 DEYIHKLILS VKSKNLNKDL SDMTNILQQS ELLLTNLNKK MGSYIYIDTI KFIHKEMKHI 421 FNRIEYHTKI INDKTKIIQD KIKLNIWRTF 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 neutralising R5.004 antibody is shown in double underline. The (discontinuous) epitope for the neutralising R5.016 is shown in wavy underline. SEQ ID NO: 2: PfRH5^NL KNVNFLQYHF KELSNYNIAN SIDILQEKEG HLDFVIIPHY TFLDYYKHLS YNSIYHKSST 60 YGKCIAVDAF IKKINEAYDK VKSKCNDIKN DLIATIKKLE HPYDINNKNR AFKKMMDEYN 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 KKNKPLTQEP EA 342 Italicised and dotted underlined = additional residues that may be deleted (corresponding to residues 140-159 of the flexible N-terminal region of full-length PfRH5)
SEQ ID NO: 3: Consensus sequence of first portion of a preferred PfRH5 epitope as presented on a first α-helix of the scaffold KxIAxxAFxKKIxEAxDKV where x is any amino acid SEQ ID NO: 4: Consensus sequence of second portion of a preferred PfRH5 epitope as presented on a second α-helix of the scaffold KIxMDxKNYxTNLxEQ where x is any amino acid SEQ ID NO: 5: Exemplified immunogen 3 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVARKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYRKKKKIAIEAFLKKIEEAA DKVAREAKQRLDELEKKNQVDKEELEKAKEEVEKRARELRRRIREILERAKKWLDQ SEQ ID NO: 6: Exemplified immunogen 3A (PfRH5 epitope with intervening scaffold residues) GSGSYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYRKKKKIAIEAFLKKIEEAA DKVAREAKQRLDELEKKNQVDKEELEKCKEEVEKRARELRRRIREILERAKKWLDQ underlined residues are cysteine residues added to introduce disulphide bonds SEQ ID NO: 7: Exemplified immunogen 3B (PfRH5 epitope with intervening scaffold residues) GSGSYQDVARKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYCKKKKIAIEAFLKKIEEAA DKVAREAKQRLDELEKKNQVDKEELEKAKEEVEKRARELRRRIREILERAKKWCDQ underlined residues are cysteine residues added to introduce disulphide bonds SEQ ID NO: 8: Exemplified immunogen 3C (PfRH5 epitope with intervening scaffold residues) GSGSYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYCKKKKIAIEAFLKKIEEAA DKVAREAKQRLDELEKKNQVDKEELEKCKEEVEKRARELRRRIREILERAKKWCDQ underlined residues are cysteine residues added to introduce disulphide bonds SEQ ID NO: 9: Exemplified immunogen 1 (PfRH5 epitope with intervening scaffold residues)
GSGSYQDVLQKAEEKLRKIEMDAKNYRTNLEEQKDELSKTDDEKDKKKIAIEAFIKKIKEAAD KVAREAEEELRKLKDKNQVDSQELDKAEDKAKKKADELKDKIDNIEDDARKWLDQ SEQ ID NO: 10: Exemplified immunogen 2 (PfRH5 epitope with intervening scaffold residues) GSGSAEDVLKEAEEKLRKIAMDAKNYRTNLEEQKQELNKTDEERERKRIAIEAFIKKIEEAAD KVAREAKDKLDDLKKKNQVDEKKLEEVKQKVEREAREARRIIREAKDDAEKWLKQ SEQ ID NO: 11: Exemplified immunogen 4 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVAREAEEKLRKIEMDAKNYATNLEEQRDELSKTEEEIKKKKIAIEAFIKKIAEAADK VAREAEEELEKLKRKNQVDSKRLEDAKKRVKKLAEELKERIERIREKAEKWLKQ SEQ ID NO: 12: Exemplified immunogen 5 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVAERAERKLRKIEMDAKNYRTNLEEQKDELAKTEEEIKKKKIAIEAFIKKIKEAADK VAREADRELDELKKKNQVDSEELEKAKDKVRKWAEELRRRIDEAKKDAEKWLKQ SEQ ID NO: 13: Exemplified immunogen 6 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVKKEAEEKLDKIEMDAKNYRTNLEEQRQQLAKTEEEIKKKKIAIEAFIKKIEEAAD KVAREAEEKLDRLKKKNQVDEKKLEEAKDDVKDKADEVRKKIRDAKDDAEKWLKQ SEQ ID NO: 14: Exemplified immunogen 7 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVAREAKERLEKIEMDAKNYRTNLEEQKETLSKTEEEIKKKKIAIEAFIKKIEEAADK VAREAEERLRELEKKNQVDKNKLEKAEDEVKKKADEVRDKIRNARDDAEKWLKQ SEQ ID NO: 15: Exemplified immunogen 8 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVAREAKERLDKIEMDAKNYRTNLEEQKDELSKTEEEIKKKKIAIEAFIKKIKEAADK VAREAKKRLDELKKKNQVDSEKLDKAKEEVEKKARELKKKIEEIREDAEKWLKQ SEQ ID NO: 16: Exemplified immunogen 9 (PfRH5 epitope with intervening scaffold residues) GSGSYQDVAREAKEKLEKIEMDAKNYRTNLEEQRDQLAKTQEEIQKKKIAIEAFIKKIEEAAD KVAREADDKLDDLKKKNQVDSQELDKAKDEVRKKAEELKKKIREAREDAEKWLKQ
SEQ ID NO: 17: Thermostable full-length PfRH5 amino acid sequence including signal sequence 1 MIRIKKKLIL TIIYIHLFIL NRLSFENAIK KTKNQENNLT LLPIKSTEEE KDDIKNGKDI 61 KKEIDNDKEN IKTNNAKDHS TYIKSYLNTN VNDGLKYLFI PSHNSFIKKY SVFNQINDGM 121 LLNEKNDVKN NEDYKNVDYK NVNFLQYHFK ELSNYNLANS IDILQEKEGH LDFVIIPHYT 181 FLEYYKHLSY NSIYHKSSTY GKCIAVDAFI KKINETYDKV KSKCNDIKND LIKTIKKLEH 241 PYDINNKNDD SYRYDISEEI DDKSEETDDE TEEVEDSIQD TDSNHTPSNK KKNDLMNRTF 301 KKMFDEYNTK KNKFINCIKN HENDFNKICN DMKNYGTNLF EQLSCYNNNF CNTNGIRYHY 361 DEYIHKLILA VKSKNLNKDL NDMKNILQQS EKLLNNLEKK MGSYIYIDTI KFIHKEMKHI 421 FNRIEYHTKI INDKTKIIQD KIKLNIWRTF QKDELLKKIL DMSKEYALFI TSDHLRQMLY 481 NTFYSKEKHL NNIFHHLIYV LQMKLNDVPI KMEYFQTYKK NKPLTQ Signal sequence (amino acids 1 to 23) is in italics, flexible N-terminal (amino acids 1 to 139) and flexible loop (amino acids 248 to 296) regions are underlined Modified residues for thermostability are bold and double-underlined SEQ ID NO: 18: Thermostable PfRH5^NL KNVNFLQYHFKELSNYNLANSIDILQEKEGHLDFVIIPHYTFLEYYKHLSYNSIYHKSSTYGKYIAVD AFIKKINEAYDKVKSKCNDIKNDLIKTIKKLEHPYDINNKNRAFKKMFDEYNTKKNKFINCIKNHEND FNKICNDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILAVKSKNLNKDLNDMKNILQQSEK LLNNLEKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKKIL DMSKEYALFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKLNDVPIKMEYFQTYKKNKPLTQ Modified residues for thermostability are bold and double-underlined Italicised and dotted underlined = additional residues that may be deleted (corresponding to residues 140-159 of the flexible N-terminal region of full-length PfRH5) SEQ ID NO: 19: Neutralising epitope bin of the R5.016 antibody GKCIAVDAFIKKINETYDKKICMDMKNYGTNLFEQ SEQ ID NO: 20: Neutralising epitope of the R5.016 antibody GKCIAVDAFIKKINETYDKKICMDMKNY SEQ ID NO: 21: Neutralising epitope bin of the R5.004 antibody KSYNNNFCNTNKLNIWRTFQK SEQ ID NO: 22: Heavy chain CDR1 of the R5.034 antibody
GFTFNTYW SEQ ID NO: 23: Heavy chain CDR2 of the R5.034 antibody IQQDGSEK SEQ ID NO: 24: Heavy chain CDR3 of the R5.034 antibody ARDNPASAVAFDV SEQ ID NO: 25: Light chain CDR1 of the R5.034 antibody SSNIGNNA SEQ ID NO: 26: Light chain CDR2 of the R5.034 antibody FDD SEQ ID NO: 27: Light chain CDR3 of the R5.034 antibody AAWDDRLNGVV SEQ ID NO: 28: Heavy chain variable region of the R5.034 antibody EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMSWVRQAPGKGLEWVANIQQDGSEKD YLNSVRGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARDNPASAVAFDVWGQGAMVTVS S SEQ ID NO: 29: Light chain variable region of the R5.034 antibody QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPQLLIYYDDLLPSGVSD RFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGLVFGGGTKLTVL SEQ ID NO: 30: Heavy chain CDR1 of the 9AD4 antibody GFTFSDYG SEQ ID NO: 31: Heavy chain CDR2 of the 9AD4 antibody ISNMAYSI SEQ ID NO: 32: Heavy chain CDR3 of the 9AD4 antibody TRAIFDYAGYWYFDV SEQ ID NO: 33: Light chain CDR1 of the 9AD4 antibody ESVEYYGTSL
SEQ ID NO: 34: Light chain CDR2 of the 9AD4 antibody GAS SEQ ID NO: 35: Light chain CDR3 of the 9AD4 antibody QQSTKVPWT SEQ ID NO: 36: Heavy chain variable region of the 9AD4 antibody MGWSWIFLFLLSGTAGVHSEVKLVESGGGVVQPGGSRKLSCAASGFTFSDYGMAWVRQA PGKGPEWVTFISNMAYSIYYADTVTGRFTISRENAKNTLHLEMSSLRSEDTAMYYCTRAIFD YAGYWYFDVWGAGTTVTVS SEQ ID NO: 37: Light chain variable region of the 9AD4 antibody MVSTPQFLVFLLFWIPASRGDIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWFQQ KPGQPPRLLIHGASNVQSGVPARFSGSGSGTDFSLNIHPVEEDDFAMYFCQQSTKVPWTF GGGTKLEI SEQ ID NO: 38: Heavy chain CDR1 of the R5.016 antibody GYTFTSYG SEQ ID NO: 39: Heavy chain CDR2 of the R5.016 antibody ISGYDGNT SEQ ID NO: 40: Heavy chain CDR3 of the R5.016 antibody ARDGPQVGDFDWQVYYYYGMDV (S SEQ ID NO: 41: Light chain CDR1 of the R5.016 antibody QSINTW SEQ ID NO: 42: Light chain CDR2 of the R5.016 antibody KAS SEQ ID NO: 43: Light chain CDR3 of the R5.016 antibody QQYNSYLYT SEQ ID NO: 44: Heavy chain variable region of the R5.016 antibody
QVQLVQSGAEVKKPGASVRVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISGYDGNTN YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGPQVGDFDWQVYYYYGMDV WGQGTTVTVSS SEQ ID NO: 45: Light chain variable region of the R5.016 antibody AIRMTQSPSTLSASVGDRVTITCRASQSINTWLAWYQQKPGKAPNLLISKASSLESGVPSRF SGSGSGTEFTLTISSLQPDDFATYFCQQYNSYLYTFGQGTKVEIR SEQ ID NO: 46: Heavy chain CDR1 of the R5.004 antibody NYAIN SEQ ID NO: 47: Heavy chain CDR2 of the R5.004 antibody GIIPIFATTNYAQKFQG SEQ ID NO: 48: Heavy chain CDR3 of the R5.004 antibody DKHSWSYAFDI SEQ ID NO: 49: Light chain CDR1 of the R5.004 antibody SGSSSNIGSNTVN SEQ ID NO: 50: Light chain CDR2 of the R5.004 antibody SNNQRPS SEQ ID NO: 51: Light chain CDR3 of the R5.004 antibody AAWDDSLNGWV SEQ ID NO: 52: Heavy chain variable region of the R5.004 antibody EVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAINWVRQAPGQGLEWMGGIIPIFATTNYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARDKHSWSYAFDIWGQGTMVTVSS SEQ ID NO: 53: Light chain variable region of the R5.004 antibody QSVLTQPPSASGTPGLRVTISCSGSSSNIGSNTVNWYQHLPGTAPKLLIHSNNQRPSGVPD RFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQP SEQ ID NO: 54: “Ungapped” consensus sequence of first portion of a preferred PfRH5 epitope as presented on a first α-helix of the scaffold KIAAFKKIEADKV
SEQ ID NO: 55: “Ungapped” consensus sequence of second portion of a preferred PfRH5 epitope as presented on a second α-helix of the scaffold KIMDKNYTNLEQ SEQ ID NO: 56: Exemplary repeating pattern of an isoleucine zipper. IxxxIxxIxxxIxxIxxxIxxI SEQ ID NO: 57: Exemplified immunogen sequence for trimerization (TB4) SYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYRKKKKIAIEAFLKKIEEAADKVA REAKQRLDELEKKNQVDKEELEECKKEVEIRAKILRIEIKIILIEAKIWLID Isoleucine zipper residues are in bold and underlined. SEQ ID NO: 58: Further exemplified immunogen sequence (B4) SYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYRKKKKIAIEAFLKKIEEAADKVA REAKQRLDELEKKNQVDKEELEKCKEEVEKRARELRRRIREILERAKKWLDQ SEQ ID NO: 59: Full length scaffold protein sequence using PDB 3LHP, chain S protein before resurfacing HHHHHHGSISDIRKDAEVRMDKAVEAFKNKLDKFKAAVRKVFPTEERIKDWLKIVRGEAEQA RVAVRNVGRDANDKAAALGKDKEINWFDISQSLWDVQKLTDAAIKKIEAALADMEAWLTQG SEQ ID NO: 60: Exemplary I53-50 sequence (I53-50A) EKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKAL SVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTEL VKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGV GSALVKGTPDEVREKAKAFVEKIRGCTELE SEQ ID NO: 61: Exemplary I53-50 sequence (I53-50C>A) SGEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVI KALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPT ELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVG VGSALVKGTPDEVREKAKAFVEKIRGATELE SEQ ID NO: 62: Exemplary conjugate sequence (B4-16GS-I53-50A-GSG-6HIS)
SYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYRKKKKIAIEAFLKKIEEAADKVAREAKQ RLDELEKKNQVDKEELEKCKEEVEKRARELRRRIREILERAKKWLDQGGSGGSGGSGGSGGSGEKAAK AEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGA IIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLF PGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEK IRGCTELEGSGHHHHHH Bold and underlined residues = the B4 PfRH5 polypeptide of SEQ ID NO: 58 Italics = the GS16 linker Double underlined = the I50-53A VLP of SEQ ID NO: 60 Dotted underlined = GSG-6His tag SEQ ID NO: 63: Exemplary conjugate sequence for trimerization (TB4-16GS-I53-50A(C>A)- GSG-6HIS) SYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEEYRKKKKIAIEAFLKKIEEAADKVAREAKQ RLDELEKKNQVDKEELEECKKEVEIRAKILRIEIKIILIEAKIWLIDGGSGGSGGSGGSGGSGEKAAK AEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGA IIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLF PGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEK IRGATELEGSGHHHHHH Bold and underlined residues = the TB4 PfRH5 polypeptide of SEQ ID NO: 57 Italics = the GS16 linker Double underlined = the I50-53(C>A) VLP of SEQ ID NO: 61 Dotted underlined = GSG-6His tag SEQ ID NO: 64: Exemplary linker sequence for linking the heavy and light variable chains of R5.016 GGSSRSSSSGGGGSGGGG SEQ ID NO: 65: Exemplary linker sequence for linking the heavy and light variable chains of R5.034 GGGGSGGGGSGGGGS SEQ ID NO: 66: Full length PfRH5 amino acid sequence (3D7) including signal sequence 1 MIRIKKKLIL TIIYIHLFIL NRLSFENAIK KTKNQENNLT LLPIKSTEEE KDDIKNGKDI 61 KKEIDNDKEN IKTNNAKDHS TYIKSYLNTN VNDGLKYLFI PSHNSFIKKY SVFNQINDGM 121 LLNEKNDVKN NEDYKNVDYK NVNFLQYHFK ELSNYNIANS IDILQEKEGH LDFVIIPHYT 181 FLDYYKHLSY NSIYHKSSTY GKCIAVDAFI KKINETYDKV KSKCNDIKND LIATIKKLEH
241 PYDINNKNDD SYRYDISEEI DDKSEETDDE TEEVEDSIQD TDSNHTPSNK KKNDLMNRTF 301 KKMMDEYNTK KKKLIKCIKN HENDFNKICM DMKNYGTNLF EQLSCYNNNF CNTNGIRYHY 361 DEYIHKLILS VKSKNLNKDL SDMTNILQQS ELLLTNLNKK MGSYIYIDTI KFIHKEMKHI 421 FNRIEYHTKI INDKTKIIQD KIKLNIWRTF 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 neutralising R5.004 antibody is shown in double underline. The (discontinuous) epitope for the neutralising R5.016 is shown in wavy underline. EXAMPLES The invention is now described with reference to the Examples below. These are not limiting on the scope of the invention, and a person skilled in the art would be appreciate that suitable equivalents could be used within the scope of the present invention. Thus, the Examples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination. Example 1: Structure-guided design of a focused vaccine immunogen based on PfRH5 The aim was to design and produce a focused vaccine immunogen based on PfRH5. Methods Design of the epitope mimics The epitope mimics were designed using the Rosetta software suite. The region of PfRH5 containing the epitope was manually aligned with the scaffold structure (PDB: 3LHP) in Coot, allowing a composite model to be generated in which residues 202-220 and 327- 342 from PfRH5 were transplanted to the new epitope mimic. The resultant model was subjected to ab initio modelling using the energy minimisation function deployed in Rosetta. The folding process was carried out 10,000-times, scoring the output for stability. Next, the Rosetta package was used to perform computational site saturation mutagenesis for all residues in the synthetic immunogen, other than those specified as invariant due to their role in forming the epitope. This process generated 500 designs, which were scored for their Rosetta score as well as their root-mean-square-deviation from the desired design. Of the 500 designs, 447 were in a single cluster of closely related Rosetta scores and showed a Ca
RMSD of <1Å from the original design. The best 100 designs were then analysed by evolutionary trace analysis in Jalview, allowing selection of nine designs which best represented sequence diversity for the designed proteins. Expression and purification of epitope mimics The epitope mimics were each expressed with an N-terminal tag consisting of His6- tag – thrombin cleavage site – Spy tag – TEV cleavage site. This allowed expression of proteins which could be purified using metal ion affinity and could be cleaved to either remove all tags, or to leave a spy tag at the N-terminus to allow conjugation to virus-like particles which display the spy-catcher protein. The genes were inserted into the pEt15b vector (Novegen) and were transformed into Shuffle T7 express competent cells (New England Biolabs). Protein expression was induced when cells reached an optical density at 600nm of 0.6 by addition of IPTG to a final concentration of 0.5mM and cells were harvested after a further 4 hours at 37 °C. Cells were lysed by sonication and proteins were purified using Ni-NTA affinity chromatography (Qiagen). Analytical size exclusion chromatography Analytical size exclusion filtration was performed with a Superdex 75 Increase 10/300 column (Cytiva) in 20 mM Hepes (pH 7.5) and 150 mM NaCl. Results The epitope for neutralising antibody 9AD4 is contained entirely within two approximately anti-parallel α-helices which form one side of PfRH5, close to, but not overlapping the basigin binding site, with PfRH5 residues 202, 205, 209, 212, 213, 331, 334, 335, 338, 339, 341 and 342 directly contacting 9AD410 (Figure 1a). It was investigated whether a region of PfRH5 containing this epitope (Figure 1b) could be recapitulated on a synthetic immunogen built from a molecular architecture consisting of three α-helices, two of which are re-surfaced to present the 9AD4 epitope (Figure 1c). A three-helical bundle from the E. coli ribosome recycling factor was identified which had the correct helical topology to allow epitope grafting (PDB 3LHP, chain S). While this scaffold protein contains nearly anti-parallel helices, the equivalent helices forming the PfRH5 epitope diverge in their separation across the epitope, requiring redesign of the immunogen core to stabilise the conformation of these splayed helices. An in-silico model of the immunogen was constructed after grafting residues 19-34 and 48-66 from PfRH5 onto the helical bundle (Figure 1c). Rosetta-based design was used to improve folding of this immunogen. Through this process, the 25 residues which form the
epitope surface and make direct contains with 9AD4 were fixed as invariant, but all other residues were allowed to diversify (Table 1). Table 1: Interactions list of PfRH5 or RH5-34EM with 9AD4, R5.016 and R5.034. The left-hand column shows the PfRH5 residue number. The second column shows the equivalent residue in the RH5-34EM immunogen, with residues shown with an asterisk being those which have their identity retained from PfRH5. The remaining columns show the interactions observed in crystal structures of antibody and either PfRH5 or RH5-34EM, with residues which make interactions depicted by a tick and those which do not by a cross. In Table 1 the residue positions in PfRH5 are correspond to those in SEQ ID NO: 1, which includes an initial methionine (Met) residue. Similarly, the residue positions in RH5- 34EM in Table 1 are also calculated based on the RH5-34EM sequence including an initial methionine. In the immunogen sequences herein (i.e. SEQ ID NOs: 6-16), the initial methionine has been omitted, to reflect standard co-translational cleavage during protein synthesis. The position of the residues for RH5-34EM in column 2 of Table 1 are therefore 1 greater than the position of the corresponding residues in SEQ ID NOs: 6-16. By way of illustration, when present in the exemplified immunogens of SEQ ID NOs: 6-16: K46 in Table 1 corresponds to K45 in the immunogens; A62 in Table 1 corresponds to A61 in the immunogens; and N30 in Table 1 corresponds to N29 in the immunogens. For the avoidance of doubt, it is equally valid to number the residues including or excluding the initial methionine. When excluding the initial methionine the first residue after the methionine would be numbered as residue 1. When including the initial methionine, this is numbered as residue 1. Both methods of numbering are consistent with the invention and can be used interchangeably, depending on the context or specific requirement. Residue in Residue in 9AD4 R5.016 R5.034 PfRH5 RH5- PfRH5 PfRH5 RH5- PfRH5 RH5- 34EM 34EM 34EM Y200 K46 X X ^ X X G201 K47 X ^ ^ X X K202 K48* ^ ^ ^ X X Y203 K49 X X X X X I204 I50* X ^ ^ X X A205 A51* ^ ^ ^ X X D207 E53 X ^ X X X A208 A54* X ^ ^ ^ ^
F209 F55* ^ ^ ^ ^ ^ K211 K57* X ^ ^ X K212 K58* ^ ^ ^ ^ ^ I213 I59* ^ X X X X E215 E61* X ^ ^ X X A216 A62* X X X X X D218 D64* X X X X X K219 K65* X ^ X ^ ^ V220 V66* X X X X X N323 E15 X X X ^ X K327 K19* X ^ ^ ^ ^ I328 I20* X X X X X M330 M22* X X X ^ ^ D331 D23* ^ ^ X ^ ^ K333 K25* X X X X X N334 N26* ^ X X Y335 Y27* ^ ^ ^ X X T337 T29* X X X X X N338 N30* ^ X ^ ^ ^ L339 L31* ^ ^ X X E341 E33* ^ X X X X Q342 Q34* ^ X X X ^ S344 A35 ^ X X X X C345 N36 X X ^ X X This process generated a set of 500 designs, which were scored for their stability, as estimated through the Rosetta-score, and their predicted root-mean-squared-deviation from the starting model. Of these, 447 showed broadly equivalent stability and similarity scores. The diversity of the top 100 designs was assessed through evolutionary trace analysis, with one design selected as representative of each branch in the resultant evolutionary tree. These nine designs (Table 2) showed average pairwise sequence identifies from 63% - 84%, with variance found in 53 of the 118 positions on the immunogen. Table 2: Sequences of designed immunogens Protein sequences for the designed immunogens. The residues in bold and underlined in 3A, 3B and 3C are the cysteine residues added to introduce disulphide bonds.
Immunogen Sequence SEQ ID NO 1 GSGSYQDVLQKAEEKLRKIEMDAKNYRTNLEEQKDELSKTDD 9 EKDKKKIAIEAFIKKIKEAADKVAREAEEELRKLKDKNQVDSQE LDKAEDKAKKKADELKDKIDNIEDDARKWLDQ 2 GSGSAEDVLKEAEEKLRKIAMDAKNYRTNLEEQKQELNKTDE 10 ERERKRIAIEAFIKKIEEAADKVAREAKDKLDDLKKKNQVDEKK LEEVKQKVEREAREARRIIREAKDDAEKWLKQ 3 GSGSYQDVARKAKEKLDKIEMDAKNYETNLKEQANNADKTEE 5 YRKKKKIAIEAFLKKIEEAADKVAREAKQRLDELEKKNQVDKEE LEKAKEEVEKRARELRRRIREILERAKKWLDQ 4 GSGSYQDVAREAEEKLRKIEMDAKNYATNLEEQRDELSKTEE 11 EIKKKKIAIEAFIKKIAEAADKVAREAEEELEKLKRKNQVDSKRL EDAKKRVKKLAEELKERIERIREKAEKWLKQ 5 GSGSYQDVAERAERKLRKIEMDAKNYRTNLEEQKDELAKTEE 12 EIKKKKIAIEAFIKKIKEAADKVAREADRELDELKKKNQVDSEEL EKAKDKVRKWAEELRRRIDEAKKDAEKWLKQ 6 GSGSYQDVKKEAEEKLDKIEMDAKNYRTNLEEQRQQLAKTEE 13 EIKKKKIAIEAFIKKIEEAADKVAREAEEKLDRLKKKNQVDEKKL EEAKDDVKDKADEVRKKIRDAKDDAEKWLKQ 7 GSGSYQDVAREAKERLEKIEMDAKNYRTNLEEQKETLSKTEE 14 EIKKKKIAIEAFIKKIEEAADKVAREAEERLRELEKKNQVDKNKL EKAEDEVKKKADEVRDKIRNARDDAEKWLKQ 8 GSGSYQDVAREAKERLDKIEMDAKNYRTNLEEQKDELSKTEE 15 EIKKKKIAIEAFIKKIKEAADKVAREAKKRLDELKKKNQVDSEKL DKAKEEVEKKARELKKKIEEIREDAEKWLKQ 9 GSGSYQDVAREAKEKLEKIEMDAKNYRTNLEEQRDQLAKTQE 16 EIQKKKIAIEAFIKKIEEAADKVAREADDKLDDLKKKNQVDSQEL DKAKDEVRKKAEELKKKIREAREDAEKWLKQ 3A GSGSYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEE 6 YRKKKKIAIEAFLKKIEEAADKVAREAKQRLDELEKKNQVDKEE LEKCKEEVEKRARELRRRIREILERAKKWLDQ 3B GSGSYQDVARKAKEKLDKIEMDAKNYETNLKEQANNADKTEE 7 YCKKKKIAIEAFLKKIEEAADKVAREAKQRLDELEKKNQVDKEE LEKAKEEVEKRARELRRRIREILERAKKWCDQ
3C GSGSYQDVCRKAKEKLDKIEMDAKNYETNLKEQANNADKTEE 8 YCKKKKIAIEAFLKKIEEAADKVAREAKQRLDELEKKNQVDKEE LEKCKEEVEKRARELRRRIREILERAKKWCDQ The nine designs were each expressed in E. coli (Figure 6a), generating various profiles on a size exclusion column, with some indicative of a correctly folded protein of the predicted size (Figure 1e). Purified designs were assessed for correct folding by measuring their secondary structure using circular dichroism, with all nine designs showing the expected high α-helical content (Figure 6b). To test whether the epitope was correctly folded, surface plasmon resonance was used, flowing the designs over immobilised 9AD4 antibody and measuring the kinetics of binding and dissociation (Figure 1f, Figure 7). The designs were further defined by introducing a disulphide bond to stabilise the correct relative packing of the helices. Design 3 from Table 2 was used as the starting point, which showed a symmetrical size exclusion chromatography profile, which bound 9AD4 with a high affinity and slow off rate, and which gave the closest predicted root-mean-square- deviation to the starting epitope configuration during the design process. Two sites were identified in which residues from two neighbouring helices were correctly spaced to allow disulphide formation, CC1 and CC2 (Figure 1d), and three variants were produced (3A-C), each containing one or two disulphides (Figure 6). Once again, these were expressed in E. coli, generating symmetric profiles on a size exclusion column (Figure 1e). Circular dichroism was used to show that they adopt the correct fold (Figure 6b) and surface plasmon resonance was used to test their binding to 9AD4 (Figure 1f, Figure 7). In each case, these yielded similar results to design 3, with a highly helical secondary structure content and correct assembly of the epitope as measured by surface plasmon resonance. Conclusion Using this approach, a focused vaccine immunogen based on PfRH5 was successfully designed and produced. Example 2: Human monoclonal antibodies with high potency target the 9AD4 epitope The monoclonal antibodies R5.034 and R5.016 are growth-neutralising human antibodies which target PfRH5. Further studies were conducted to determine whether the synthetic immunogen generated in Example 1 can also mimic the epitope for these antibodies. Methods Expression and purification of Fab and scFv fragments
The monoclonal antibody R5.034 was transiently expressed using Expi293F™ cells with the Expi293™ Expression System Kit (Thermo Fisher). Culture supernatants were harvested and passed through a 0.45 mm filter. Antibody was purified using a pre-packed 1 ml HiTrap™ Protein G HP column (Cytiva). Fab fragments were prepared by cleavage with immobilised Papain (20341, Thermo Scientific) overnight at 37 °C, and Fc and Fab fragments were separated using a pre-packed 1 ml HiTrap™ rProtein A column (Cytiva). The unbound fraction containing the Fab was retained and exchanged into PBS. Single chain variable fragments of R5.016 and R5.034 were constructed by linking the heavy and light variable chains using linkers of 18- and 15-residues respectively (GGSSRSSSSGGGGSGGGG for R5.016 (SEQ ID NO: 64) and GGGGSGGGGSGGGGS for R5.034 (SEQ ID NO: 65)). These were cloned into the pHLsec vector and were expressed transiently in FreeStyle™ 293-F cells (Thermo Fisher) in FreeStyle™ F17 Expression Medium supplemented with L-glutamine and 1x MEM non-essential amino acids (Gibco). Cultures were harvested after 5 days (R5.016) or 6 days (R5.034) and supernatants were adjusted to pH 8.0 and 0.45 μm filtered before incubating with Ni-NTA resin equilibrated in 25 mM Tris pH 8.0, 150 mM NaCl (TBS). After washing the resin with 10 column volumes of TBS, followed by 20 column volumes of 25 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole, bound proteins were eluted with TBS with 500 mM imidazole. To isolate monomeric scFv, eluted proteins were further purified by gel filtration on a Superdex 7510/300 column (R5.016) or a Superdex 200 Increase 10/300 column (R5.034) into 20 mM HEPES pH 7.5, 150 mM NaCl. Expression of PfRH5ΔNL RH5ΔNL (a construct of RH5 encompassing residues K140-K247 and N297-Q526, thereby lacking its flexible N-terminus and internal loop, with substitutions T216A and T299A, to remove potential glycosylation sites, and C203Y, of the 7G8 Plasmodium falciparum strain, and a C-terminal His-tag) was expressed and secreted from a stable S2 cell line (ExpreS2ion Biotechnologies) in EX-CELL® 420 Serum Free Medium (Sigma Aldrich). After 3-4 days, the culture supernatant was harvested and adjusted to pH 8 with Tris, centrifuged at 9,000 g for 15 mins and 0.45 μm filtered, then incubated with Ni Sepharose™ excel resin (Cytiva) for 2 hours. Beads were washed with 5 column volumes of TBS, and 20 column volumes of wash buffer (25 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole), then bound proteins eluted with elution buffer (TBS + 500 mM imidazole). Eluted proteins were diluted 1:1 in ConA binding buffer (20 mM Tris pH 7.5, 500 mM NaCl, 1 mM MnCl2, 1 mM CaCl2), then incubated with ConA Sepharose 4B resin (Cytiva) overnight at 4°C, after which the unbound fraction containing RH5ΔNL was recovered. RH5ΔNL was
further purified by gel filtration using an S200 Increase 10/300 column into SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol). Results The structure of the R5.016, which had previously been determined, bound to PfRH5 and it was found that 13/15 of the residues on PfRH5 contacted by R5.016 are retained in the epitope mimic designs of the invention (Table 1). To determine the degree to which the epitope for R5.034 is recapitulated in the epitope mimic, the crystal structure of the Fab fragment of R5.034 bound to a version of PfRH5 lacking the flexible N-terminus and the flexible central loop (PfRH5ΔNL) was determined at 2.4Å resolution (Figure 2a, Table 3). It was found that R5.034 binds to an epitope which overlaps those of both 9AD4 and R5.016. However, R5.034 has a more compact binding site than R5.016, further from the tip of the PfRH5 diamond. The R5.034 epitope is recapitulated in the synthetic designed immunogen with 9/10 of the PfRH5 residues contacted by R5.034 also found in the immunogen (Figure 2b, Table 1). Conclusion These studies therefore support the design of immunogens which recapitulate the epitope for R5.016 and R5.034, as well as that for 9AD4, as a potential route to inducing the most effective PfRH5-targeting growth-neutralising antibodies during human vaccination and indicates that the epitope mimics designed to recapitulate the 9AD4 epitope will also be effective for R5.016 and R5.034.
Table 3: Crystallographic statistics RH5-34EM: PfRH5:R5.034 RH5-34EM: R5.016 R5.034 Data collection Space group P212121 P212121 P61 Cell dimensions a, b, c (Å) 64.281, 114.01, 82.75, 82.99, 111.83, 111.83, 217.09 118.75 91.57 ^^^^^^^ (^) 90, 90, 90 90, 90, 90 90, 90, 120 Wavelength 0.9795 Å 0.9786 Å 0.9999 Å Resolution (Å) 217.10 – 1.63 82.99-2.40 55.92-1.75 (1.66 – 1.63) (2.49-2.40) (1.78-1.75) Total Observations 1306445 (64462) 420026 (44946) 655283 (30833) Total Unique 199219 (9819) 32741 (3402) 65543 (3252) Rpim (%) 3.3 (88.9) 3.6 (46.4) 4.4 (119.7) CC1/2 1.00 (0.52) 0.999 (0.911) 0.998 (0.598) I/^(I) 12.0 (1.3) 15.2 (2.0) 9.6 (0.9) Completeness (%) 100.0 (100.0) 100.0 (100.0) 100.0 (99.9) Multiplicity 6.6 (6.6) 12.8 (13.2) 10.0 (9.5) Wilson B factor 25.0 55.49 25.22 Refinement Reflections 199083 32667 65500 Rwork / Rfree (%) 19.10/20.59 25.74/30.46 19.86/21.84 Average B factor 36.0 72.0 44.0 Number of residues Protein 1392 708 667 Glycerol 4 0 0 Water 1232 65 419 R.m.s deviations Bond lengths (Å) 0.008 0.006 0.009 Bond angles (^) 0.92 0.87 0.95 Ramachandran plot Favored (%) 99.4 96.4 97.5 Allowed (%) 0.6 3.6 2.5 Outliers (%) 0 0 0
Example 3: Structural and biophysical assessment of the epitope mimic Structural and biophysical studies were completed to test the hypothesis that the epitope mimic replicates the epitope for 9AD4, as well as those for human neutralising antibodies R5.016 and R5.034. Methods Circular dichroism Circular dichroism experiments were conducted using a Jasco J815 spectrophotometer. Proteins were buffer exchanged into 20mM sodium phosphate pH 7.5, 20mM NaF using PD-10 columns (Cytiva) and adjusted to a final concentration of 0.1mg/ml. For each sample, 10 scans were performed at 20°C from 260nm to 190nm wavelength, with measurements taken every 0.5mm. A baseline determined using buffer alone was subtracted. For thermal melt experiments, spectra were taken from 260nm to 190nm wavelength at 2°C intervals from 20°C to 90°C with measurements taken every 0.2nm. A baseline measurement for buffer alone at 20°C was subtracted from all spectra. Surface plasmon resonance Surface plasmon resonance experiments were carried out using a Biacore T200 instrument (Cytiva) with a buffer of 20 mM Hepes pH 7.5, 150 mM NaCl, 0.005% Tween-20. Recombinant Protein A/G (Pierce) was coupled to a CM5 chip by amine coupling. Antibody was captured onto the chip through binding to Protein A/G, and the epitope mimics were then flowed over at 30 μL/min with a contact time of 240 seconds and dissociation time of 400 seconds. After each cycle, the surface was regenerated using 10 mM using glycine pH 2. Two-fold dilution series from 500 nM to 3.9 nM were studied for each sample. Data were analysed using the BiaEvaluation software. Crystallisation and structure determination To determine the structure of RH5-34EM bound to R5.016 scFv, components were combined at a 1:1 molar ratio, incubated for 1 hour and complex was purified by size- exclusion chromatography using a Superdex 7510/300 column in 20 mM Hepes pH 7.5, 150 mM NaCl. Crystals were obtained at 10 mg/ml in sitting drops by vapour diffusion using a well solution of 20% PEG8000, 0.1 M HEPES pH 7.0 after mixing 100 nl protein and 100 nl reservoir solution. They were cryoprotected through transfer into drops of well solution supplemented with 25% glycerol prior to cryocooling for data collection in liquid nitrogen. Data were collected at Diamond Light Source at the I04 beamline and were indexed using DIALS (v3.0)33 and scaled using AIMLESS (v0.73)34 giving a dataset at a resolution of 1.63
Å. The structure was solved using molecular replacement using Phaser MR35 (v2.8.3), with the VH and VL domains of the scFv (PDB: 4U0R)10 as search models. The model was built and refined using cycles of COOT31 (v0.8.9.2) and BUSTER36 (v2.10). To determine the structure of RH5-34EM bound to R5.034 scFv, components were mixed at approximately equimolar ratio and complex was isolated by gel filtration using an S75 Increase 10/300 in 25 mM Tris pH 7.4, 150 mM NaCl. Crystals were obtained at 15 mg/mL in sitting drops by vapour diffusion at 18 °C in Morpheus A1 (0.1 M Buffer System 1 pH 6.5, 0.06 M Divalents, 30% v/v Precipitant Mix 1) after mixing 100 nl protein and 100 nl reservoir solution. Data were collected at Diamond Light Source on beamline I24 (wavelength 0.9999 Å). Diffraction data were processed using xia2-3dii37 to 1.75 Å. Molecular replacement was performed in PHASER35 using the RH5-34EM and scFv scaffold from the RH4-34EM:R5.016 structure, determined here, as search models. The structural model was further built and refined using COOT31 (0.9.3) and BUSTER36 (2.10.4). Two copies of the RH5-34EM:scFv complex are present in the unit cell. To determine the structure of PfRH5 bound to R5.034, RH5ΔNL was treated with 1 ug/ml Endoproteinase Glu-C (Sigma) overnight at room temperature, then mixed with the R5.034 Fab fragment at slight molar excess. The complex was isolated by gel filtration using an S200 Increase 10/300 column into 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol. Crystals were obtained at 12.35 mg/mL in sitting drops by vapour diffusion at 18 °C in JCSG+ A9 (0.2 M ammonium chloride, 20% w/v PEG 3350) containing Silver Bullets C10 (0.16% w/v β-Cyclodextrin, 0.16% w/v D-(+)-Cellobiose, 0.16% w/v D-(+)-Maltotriose, 0.16% w/v D-(+)-Melezitose hydrate, 0.16% w/v D-(+)-Raffinose pentahydrate, 0.16% w/v Stachyose hydrate, 0.02 M HEPES sodium pH 6.8) after mixing 100 nl protein, 100 nl reservoir solution and 50 nl additives. Data were acquired at the Synchrotron SOLEIL on beamline PROXIMA-1 (wavelength 0.9786 Å). Diffraction data were processed using DIALS33 and AIMLESS34 in the CCP4 suite to 2.4 Å. Molecular replacement was performed using PHASER35 using a crystal structure of PfRH5 (PDB: 6RCU), the scFv of R5.034 (determined here) and the constant domain of a Fab fragment (PDB: 4LLD) as search models. The model was further built using COOT31 (0.9.3) and refined using BUSTER36 (2.10.4) then PHENIX38 (1.20.1-4487). The unit cell contains one copy each of PfRH5 and R5.034 Fab fragment. The heavy chain constant domain is less well resolved than the rest of the complex. Results Circular dichroism analysis at increasing temperatures and show that secondary structure content is retained up to 90°C (Figure 2c), compared with a melting temperature of ~65°C for the previous thermally stabilised PfRH5_HS1 design.
Surface plasmon resonance (SPR) measured the binding kinetics of 9AD4, R5.016 and R5.034 for the RH5-34EM and for PfRH5. RH5-34EM bound to 9AD4 with an affinity of 3.4nM compared with 1.6nM for PfRH5, albeit with a faster on-rate and faster-off rate (Figure 2d). R5.016 bound with lower affinity to RH5-34EM than PfRH5, although still in the nanomolar range (115nM vs 2.4nM) (Figure 2e). However, R5.034 showed a very similar high affinity and binding kinetics for both RH5-34EM and PfRH5 (73pM vs 94pM) (Figure 2f). Therefore, RH5-34EM effectively mimics the epitope of the most effective growth neutralising antibody, R5.034 and substantially mimics that of R5.016. Crystal structures of the epitope mimic bound to scFv fragments of human monoclonal antibodies R5.016 (at 1.63Å resolution) and R5.034 (at 1.75Å resolution) were determined (Figure 3, Table 3). Comparison of these structure with those of PfRH5 bound to R5.016 and R5.034 showed significant similarity, both in the fold of the epitope mimic and in the interactions made with antibodies. Of the 15 interactions formed between PfRH5 and R5.016, 13 were retained with the epitope mimic. In the case of R5.034, of the 10 interactions made with PfRH5, 9 were also observed with the epitope mimic (Figure 3, Table 1). These structural alignments also showed the greater match between the R5.034 epitopes on RH5-34EM and PfRH5 than between the R5.016, with R5.034 binding closer to the centre of the RH5-34EM immunogen, while R5.016 binds more towards the end of the immunogen, where the structural similarity to PfRH5 starts to diverge (Figure 3c). Conclusion These structural and biophysical studies confirm that the inventors have designed a small, highly stable synthetic immunogen which recapitulates the epitopes for neutralising antibodies R5.034, R5.016 and 9AD4 on a small stable scaffold, referred to going forward as RH5-34EM as it most effectively mimics the R5.034 epitope of RH5. Example 4: RH5-34EM generates a high-quality immune response The immunogenicity of RH5-34EM in comparison to that of PfRH5 was assessed. Methods Cohorts of six rats were immunised with either three doses of RH5-34EM or three doses of PfRH5, both formulated with the adjuvant matrix M (Figure 4a). As small immunogens are likely to induce lower responses, RH5-34EM was conjugated, through a spy-tag at the N-terminus, to virus-like particles consisting of the hepatitis B surface protein (HbSAg) fused to a spy-catcher28. Fourteen days after the final dose, sera were harvested, total IgG were purified, and the IgG were analysed for antibody responses against RH5- 34EM and PfRH5 using ELISA.
Immunisation of rats Six 8-10-week-old female Wistar rats per cohort were injected intramuscularly (IM) with antigens equimolar to 2 µl of soluble RH5.1 formulated in 25 µg of MatrixM adjuvant (Novavax). Blood samples were taken on days -2, 28, 56 for serum preparation and rats were terminally bled on day 70. Each rat was analysed individually by ELISA and GIA. ELISA measurements Nunc Maxisorp plates (Thermo Fisher Scientific) were coated overnight (4°C, >16 h) with 50 µl/well of RH5.1 or Bundle-4 at 2 μg/mL diluted in Dulbecco’s PBS (DPBS). The coated plates were washed in 6-times with wash buffer (PBS with 0.05 % Tween 20; PBST) and blocked with 200 μL/well of RT Starting Block T20 (Thermo Fisher Scientific) for 1 h. Blocked plates were washed 6x in DPBS/T and dilutions of the reference serum, internal control and test serum prepared in Starting block T20 were added 50µL/well and left for 2h at RT. After incubation with serum, the plates were washed 6x in DPBS/T and 50μL of goat anti-rat secondary antibody (1:1000 prepared in Starting Block T20) was pipetted per well and left for 1h at RT. Plates were washed 6-times with DPBS/T and 100 μL development buffer (p- nitrophenyl phosphate substrate diluted in diethanolamine buffer) was added per well and developed according to internal controls (average OD = 1.0, approx. range 0.8 – 1.2). All serum samples were tested in triplicate against each coating antigen. The test serum samples were diluted to achieve OD 405nm reading in the linear part of the standard curve. Growth-inhibitory activity measurement Erythrocytes from O+ individuals for 3D7 Plasmodium falciparum culture and growth inhibition assays (GIA) were obtained via the NHS Blood and Transplant service. On day 1 of the assay, 20 ml of 3D7 Plasmodium falciparum culture in complete medium (incomplete medium [RPMI, 1% L-glutamine, 0.005% hypoxanthine, 25 mM HEPES], 10% heat- inactivated filtered pooled human serum from O+ individuals, 10 μg/ml gentamycin) at 2% haematocrit were sorbitol-synchronized. On day 2, 20 μl of synchronised parasites in 2x complete medium, 0.4% parasitaemia, 2% haematocrit were added to the twelve wells containing 20 μl 10 mM EDTA or incomplete medium or to the triplicate wells containing rat serum-purified IgG dilutions, 20 μl positive controls (40 μg/ml 2AC7, 30 μg/ml R5.016, 5 μg/ml R5.034), or 20 μl of a negative control monoclonal antibody (500 μg/ml EBL040) in incomplete medium. A parallel tracker Plasmodium falciparum culture (0.2% final parasitaemia) was also prepared. The assay plates and the tracker culture were then placed in a modular incubator and cultured at 37 °C for 2 days. On day 4, the assay plates were washed twice with cold PBS, and erythrocytes were resuspended by shaking.
The inhibition of Plasmodium falciparum growth was measured using the lactate dehydrogenase (LDH) assay, with 120 μl LDH substrate containing 3-acetylpyridine adenine dinucleotide (50 μg/ml), diaphorase (1 U/ml), and nitro blue tetrazolium (0.2 mg/ml) added to each well. The assay plates were developed until reaching an optical density of 0.4-0.6 in the wells containing parasitized erythrocytes and read at 650 nm. Percent inhibition of Plasmodium falciparum growth was calculated using the following formula: ^^^^^^ − ^650 ^^^^ℎ^^^^^^^ ^^^^)
^^^^^^^ − ^650 ^^^^ℎ^^^^^^^ ^^^^)
Results Immunisation with RH5-34EM required two injections to induce PfRH5-specific antibodies, with little increase in titre after a third injection (Figure 4b). However, immunisation with PfRH5 resulted in around a thousand-fold higher titre of PfRH5-specific antibodies after three immunisations. When studying RH5-34EM-specific antibodies, it was found that immunisation with RH5-34EM generated high titres after just one immunisation (Figure 4c). In contrast, immunisation with PfRH5 required three immunisations to induce RH5-34EM-specific antibodies and at approximately a thousand-fold lower titre than those induced through immunisation with RH5-34EM. As the only region shared between PfRH5 and RH5-34EM is the 9AD4/R5.016/R5.034 epitope, it was expected that RH5-34EM- specific antibodies induced by PfRH5 and PfRH5-specific antibodies induced by RH5-34EM will bind to this shared epitope. It is therefore noteworthy that these two groups showed a similar ELISA reactivity after three doses, suggesting that RH5-34EM immunisation induces an equivalent titre against this epitope to that resulting from immunisation with PfRH5 (Figure 4d). The quality of the induced antibodies in a growth-inhibition assay was then assessed. Total IgG were purified from rat sera on day 70 and the growth inhibitory activity (GIA) was assessed for a dilution series of these antibodies (Figure 4e,f). The antibody concentration required for 30% GIA (EC30) was around two-fold lower for sera from PfRH5-immunised rats than for sera from those immunised with RH5-34EM, albeit this difference was not statistically significant. In addition, to assess the quality of the antibody responses, the growth-inhibitory activity was also assessed after calibration for the concentration of PfRH5- specific antibodies, rather than total IgG. ELISA was used to measure the concentration of RH5-specific antibodies in both sera, allowing conversion of these data to reveal the effect of PfRH5-specific IgG on growth (Figure 4g,h). In this case, the PfRH5-specific antibodies from RH5-34EM-immunised rats showed an EC30 approximately one thousand-fold lower than those from PfRH5-immunised rats.
Conclusion When growth-inhibitory activity was considered as a factor of total IgG, immunisation with PfRH5 was around two-fold more effective than RH5-34EM, suggesting that PfRH5 immunisation generates a larger response, as the result of antibodies targeting multiple neutralising epitopes on PfRH5. However, the growth-inhibitory quality of the PfRH5-specific antibodies induced through immunisation with RH5-34EM was around one thousand-fold greater that of the PfRH5-specific antibodies induced through immunisation with PfRH5, suggesting that the RH5-34EM immunogen gives rise to a significantly higher quality immune response than immunisation with PfRH5. Example 5: Priming with a focused immunogen followed by a PfRH5 boost generates the most growth-neutralising antibody response As immunisation with RH5-34EM generated a high-quality, lower-quantity response, while immunisation with PfRH5 generated a lower-quality, higher-quantity response, we next aimed to determine whether different prime-boost regimes, in which RH5-34EM and PfRH5 are provided in different sequences, could generate a high-quality, high-quantity response. Methods Rats were immunised, using the same regime as Figure 4a, with each of the six different remaining sequences possible for the PfRH5 and RH5-34EM immunogens (Figure 5, Figure 8). ELISA measurements were used to assess the PfRH5-specific and RH5-34EM-specific responses resulting from these immunisation regimes and were largely as predicted. ELISA measurements Nunc Maxisorp plates (Thermo Fisher Scientific) were coated overnight (4°C, >16 h) with 50 µl/well of RH5.1 or Bundle-4 at 2 μg/mL diluted in Dulbecco’s PBS (DPBS). The coated plates were washed in 6-times with wash buffer (PBS with 0.05 % Tween 20; PBST) and blocked with 200 μL/well of RT Starting Block T20 (Thermo Fisher Scientific) for 1 h. Blocked plates were washed 6x in DPBS/T and dilutions of the reference serum, internal control and test serum prepared in Starting block T20 were added 50µL/well and left for 2h at RT. After incubation with serum, the plates were washed 6x in DPBS/T and 50μL of goat anti-rat secondary antibody (1:1000 prepared in Starting Block T20) was pipetted per well and left for 1h at RT. Plates were washed 6-times with DPBS/T and 100 μL development buffer (p- nitrophenyl phosphate substrate diluted in diethanolamine buffer) was added per well and developed according to internal controls (average OD = 1.0, approx. range 0.8 – 1.2). All
serum samples were tested in triplicate against each coating antigen. The test serum samples were diluted to achieve OD 405nm reading in the linear part of the standard curve. Growth-inhibitory activity measurement Erythrocytes from O+ individuals for 3D7 Plasmodium falciparum culture and growth inhibition assays (GIA) were obtained via the NHS Blood and Transplant service. On day 1 of the assay, 20 ml of 3D7 Plasmodium falciparum culture in complete medium (incomplete medium [RPMI, 1% L-glutamine, 0.005% hypoxanthine, 25 mM HEPES], 10% heat- inactivated filtered pooled human serum from O+ individuals, 10 μg/ml gentamycin) at 2% haematocrit were sorbitol-synchronized. On day 2, 20 μl of synchronised parasites in 2x complete medium, 0.4% parasitaemia, 2% haematocrit were added to the twelve wells containing 20 μl 10 mM EDTA or incomplete medium or to the triplicate wells containing rat serum-purified IgG dilutions, 20 μl positive controls (40 μg/ml 2AC7, 30 μg/ml R5.016, 5 μg/ml R5.034), or 20 μl of a negative control monoclonal antibody (500 μg/ml EBL040) in incomplete medium. A parallel tracker Plasmodium falciparum culture (0.2% final parasitaemia) was also prepared. The assay plates and the tracker culture were then placed in a modular incubator and cultured at 37 °C for 2 days. On day 4, the assay plates were washed twice with cold PBS, and erythrocytes were resuspended by shaking. The inhibition of Plasmodium falciparum growth was measured using the lactate dehydrogenase (LDH) assay, with 120 μl LDH substrate containing 3-acetylpyridine adenine dinucleotide (50 μg/ml), diaphorase (1 U/ml), and nitro blue tetrazolium (0.2 mg/ml) added to each well. The assay plates were developed until reaching an optical density of 0.4-0.6 in the wells containing parasitized erythrocytes and read at 650 nm. Percent inhibition of Plasmodium falciparum growth was calculated using the following formula: ^^^^^^ − ^650 ^^^^ℎ^^^^^^^ ^^^^)
^^^^^^^ − ^650 ^^^^ℎ^^^^^^^ ^^^^)
Results ELISA reactivity against RH5-34EM scaled with the number of doses of RH5-34EM, with two or three doses generating equivalent reactivity, one dose of RH5-34EM generating an intermediate reactivity and only PfRH5 immunisation generating the lowest reactivity (Figure 5a). Apart from the PfRH5-PfRH5-RH5-34EM regimen, the order in which doses were provided did not affect overall reactivity against RH5-34EM. A similar outcome was seen when assessing PfRH5-reactivity (Figure 5b). Here similar reactivity resulted from two or three doses of PfRH5, provided in any order. One dose of PfRH5, provided at any of the three immunisation time points resulted in an intermediate PfRH5-reactivity, while the lowest
was generated by three doses of RH5-34EM. Again, no significant difference was observed depending on the point at which PfRH5 was provided in the immunisation sequence. The quality of the response by considering PfRH5-specific IgG, was investigated (Figure 5c). Three doses of RH5-34EM generated PfRH5-specific IgG with the lowest EC30. Next most effective was two doses of RH5-34EM and one dose of PfRH5. These IgG had a significantly (p=0.0303 to P=0.0043) higher EC30 than those induced through three RH5- 34EM doses. In addition, the position within the immunisation sequence of the PfRH5 dose had a significant effect, with priming with RH5-34EM and boosting with PfRH5 generating a higher quality response. Finally, two or three doses of PfRH5 generated the highest EC30, with no significant difference between these four cohorts. Therefore, while RH5-34EM alone generates the highest quality PfRH5-specific response, two doses of RH5-34EM followed by one of PfRH5 has a four-fold weaker EC30, with a focused immunogen prime, followed by a broader PfRH5 boost giving higher quality IgG than other dosing regimens. Finally, the growth-inhibitory activity from total IgG was assessed (Figure 5d). The lowest mean EC30 resulted from one dose of RH5-34EM followed by two doses of PfRH5. The next lowest involved a dose of PfRH5 followed by RH5-34EM followed by PfRH5. These two regimens both resulted in approximately two-fold lower EC30 for growth inhibition than three doses of PfRH5, despite the ~375-fold lower titre of PfRH5-specific antibodies in these samples. Conclusion The leading dosing regime in terms of growth-inhibitory activity from total IgG is one dose of a focused RH5-34EM immunogen, followed by two of the full PfRH5 immunogen. Example 6: Trimerization of RH5-34EM To improve RH5-34EM, a rationally designed new version of the immunogen was creates in which it is trimerized. The design aim was to increase the immunogenicity specific for the epitope of interest through better presentation. Design of the trimerized RH5-34EM RH5-34EM trimerization was carried out using the Rosetta software suite. The design rationale was that RH5-34EM can be trimerized through the redesign of third helix, which does not contain the epitope, to form a new trimerization interface. To achieve this, we selected an isoleucine-rich zipper scaffold. These scaffolds assemble, through interactions between involving an alpha helix, to form a trimeric arrangement involving three parallel alpha-helices, one contributed by each monomer (PDB ID 1GCM). The helix from this scaffold was used to replace the C-terminal helix of RH5-34EM using the Rosetta MotifGraft
protocol. A set of seven grafted designs were obtained and were manually inspected using PyMOL to choose a single candidate that can assemble into a trimer without any steric clash. The trimer candidate was redesigned using Rosetta FastRelax to incorporate the Ile zipper residues as well as mutations to stabilize the inter-chain interface. A set of 100 designs were analysed and a consensus design was selected was subsequently successfully validated to form a trimer. Expression and purification of trimerized RH5-34EM The trimerized RH5-34EM epitope mimic was expressed with an N-terminal tag consisting of His6-tag – thrombin cleavage site – Spy tag – TEV cleavage site. This allowed expression of proteins which could be purified using metal ion affinity and could be cleaved to either remove all tags, or to leave a spy tag at the N-terminus to allow conjugation to virus-like particles which display the spy-catcher protein. The genes were inserted into the pEt15b vector (Novegen) and were transformed into Shuffle T7 express competent cells (New England Biolabs). Protein expression was induced when cells reached an optical density at 600nm of 0.6 by addition of IPTG to a final concentration of 0.5mM and cells were harvested after a further 4 hours at 37 °C. Cells were lysed by sonication and proteins were purified using Ni-NTA affinity chromatography (Qiagen). Crystallisation and structure determination To determine the structure of trimerized RH5-34EM bound to R5.016 scFv, components were combined at a 1:1 molar ratio, incubated for 1 hour and complex was purified by size-exclusion chromatography using a Superdex 20010/300 column in 20 mM Hepes pH 7.5, 150 mM NaCl. Crystals were obtained at 8 mg/ml in sitting drops by vapour diffusion using a well solution of 0.1 M MES, pH 6, 0.2 M Potassium acetate, 15 % v/v Pentaerythritol ethoxylate (15/4 EO/OH) after mixing 100 nl protein and 100 nl reservoir solution. They were cryoprotected through transfer into drops of well solution supplemented with 25% glycerol prior to cryocooling for data collection in liquid nitrogen. Data were collected at Diamond Light Source at the I04 beamline and were indexed using DIALS (v3.0)33 and scaled using AIMLESS (v0.73)34 giving a dataset at a resolution of 2.0 Å. The structure was solved using molecular replacement using Phaser MR35 (v2.8.3), with the VH and VL domains of the R5.016 scFv and RH5-34EM as search models . Coupling RH5-34EM, TB4 on HepB surface antigen VLP The immunogen coupling to the Hepatitis B surface antigen (HBsAg) virus-like particle (VLP) was performed using the SpyTag-SpyCatcher system. RH5-34EM and TB4 were buffer-exchanged into SpyBuffer (40 mM Na₂HPO₄, 200 mM NaCitrate, pH 7.5) and
then combined with HBsAg (in PBS) at a 3:1 molar ratio. The reaction volume was doubled by adding SpyBuffer and incubated overnight at room temperature. The reaction mixture was then processed using size-exclusion chromatography. Immunisation of rats Six 8-10-week-old female Wistar rats per cohort were injected intramuscularly (IM) with antigens equimolar to 2 µl of soluble RH5.1 formulated in 25 µg of MatrixM adjuvant (Novavax). Blood samples were taken on days -2, 28, 56 for serum preparation and rats were terminally bled on day 70. Each rat was analysed individually by ELISA and GIA, as described in example 4. Results Initial pre-clinical experiments in rats showed that TB4 induces an approximately 2.5- fold higher PfRH5-specific response compared to RH5-34EM, while lowering scaffold- specific response by 4-fold (Figure 9c). Also, the EC50 for growth-inhibitory antibodies is approximately 4-fold lower for IgG purified from rats immunised with TB4 than those immunised with RH5-34EM. Therefore, the new immunogen is a further improvement over RH5-34EM.