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WO2025168946A1 - Immunogène pfrh5 - Google Patents

Immunogène pfrh5

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Publication number
WO2025168946A1
WO2025168946A1 PCT/GB2025/050240 GB2025050240W WO2025168946A1 WO 2025168946 A1 WO2025168946 A1 WO 2025168946A1 GB 2025050240 W GB2025050240 W GB 2025050240W WO 2025168946 A1 WO2025168946 A1 WO 2025168946A1
Authority
WO
WIPO (PCT)
Prior art keywords
pfrh5
polypeptide
seq
epitope
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2025/050240
Other languages
English (en)
Inventor
Matthew Kenneth HIGGINS
Thomas Edward HARRISON
Nawsad ALAM
Ivan CAMPEOTTO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxford University Innovation Ltd
Original Assignee
Oxford University Innovation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB2401825.1A external-priority patent/GB202401825D0/en
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Publication of WO2025168946A1 publication Critical patent/WO2025168946A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents

Definitions

  • 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.
  • the RTS,S/AS01 (MosquirixTM) 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).
  • GAA growth inhibitory activity
  • PfRH5 Reticulocyte-binding protein Homologue 5
  • WO 2012/114125 Reticulocyte-binding protein Homologue 5
  • 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.
  • 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.
  • 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.
  • PfRH5 Reticulocyte-binding protein Homologue 5
  • 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.
  • 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.
  • the present inventors have developed synthetic immunogens each comprising an epitope mimic for one of numerous growth inhibitory PfRH5 antibodies, including R5.034.
  • the epitope is presented on a helical scaffold, which comprises three ⁇ -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.
  • 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.
  • S aid 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.
  • S aid polypeptide, wherein (a) the consensus sequence of SEQ ID NO: 3 may correspond to a first portion of the epitope presented on the first ⁇ -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 ⁇ -helix of the scaffold.
  • S aid 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.
  • S aid polypeptide may comprise at least two cysteine residues positioned such that they form a disulphide bridge in the polypeptide.
  • S aid 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.
  • S aid 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 virus-like particle may be directly conjugated to the polypeptide according to the invention.
  • Said virus-like particle may be I53-50.
  • S aid 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.
  • GAA growth inhibitory activity
  • 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.
  • S aid 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.
  • T he 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 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.
  • T he 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.
  • GAA growth inhibitory activity
  • 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.
  • residues coloured dark grey are those which directly contact 9AD4 while the left-hand panel shows residues defined as forming the broader 9AD4 epitope.
  • 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.
  • the location of two disulphide bonds introduced to stabilise the synthetic immunogen are labelled CC1 and CC2.
  • Size exclusion chromatography traces obtained for the twelve designs.
  • 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.
  • FIG. 2 The epitope mimic binds growth-neutralising antibodies 9AD4, R5.016 and R5.034.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • “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 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.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the target molecule).
  • a "vector” or “construct” 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.
  • virus-like particle 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).
  • 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.
  • conjugate linker means a single bond or a group of atoms comprising at least one bond that connects a conjugate moiety to a polypeptide.
  • protein refers 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.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • 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.
  • protein and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing.
  • polynucleotides 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.
  • conservatively modified variants in a protein of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.
  • “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.
  • 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.
  • 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.
  • 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: A mino 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a “fragment” of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide.
  • 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).
  • a fragment as defined herein retains the same function as the full-length polynucleotide.
  • T he 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BMI body mass index
  • 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.
  • control and “reference population” are used interchangeably.
  • PfRH5 epitope The present invention provides a polypeptide comprising a PfRH5 epitope. T he 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.
  • 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.
  • the PfRH5 ⁇ NL 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 PfRH5 ⁇ NL 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.
  • T he 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • KSYNNNFCNTNKLNIWRTFQK (SEQ ID NO: 21, which is the neutralising epitope bin of the R5.004 antibody as described in WO2016/016651).
  • 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.
  • 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).
  • 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.
  • 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%.
  • RSA Relative Solvent Accessibility
  • the amino acids residues of the PfRH5 epitope as defined herein i.e.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • a PfRH5 epitope may specifically bind to one or more of R5.034, 9AD4, R5.016 and R5.004.
  • 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.
  • 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.
  • 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.
  • 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.
  • a PfRH5 epitope is grafted onto a scaffold, such that it is presented in an immunogenic conformation.
  • 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.
  • 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.
  • any two amino acid residues of the PfRH5 epitope may be separated by one or more amino acid residue from the scaffold.
  • 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.
  • 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.
  • 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
  • still another pair of amino acids of the PfRH5 epitope may be separated by 0 scaffold amino acids.
  • 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 ( ⁇ ) 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).
  • RMSD Root Mean Square Deviation
  • 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 ⁇ or less.
  • 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 ⁇ .
  • the nature of the scaffold amino acids separating any two amino acids of the PfRH5 epitope 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the consensus sequence of KxIAxxAFxKKIxEAxDKV (SEQ ID NO: 3) and the consensus sequence of KIxMDxKNYxTNLxEQ (SEQ ID NO: 4) are comprised in different ⁇ -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.
  • T he consensus sequence of SEQ ID NO: 3 may be presented on a first ⁇ -helix of the scaffold and the consensus sequence of SEQ ID NO: 4 may be presented on a second ⁇ - helix of the scaffold.
  • the consensus sequence of SEQ ID NO: 3 corresponds to a first portion of the PfRH5 epitope presented on the first ⁇ -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 ⁇ -helix of the 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.
  • a scaffold may be a molecular structure designed to display or present epitopes in a specific conformation.
  • the scaffold is a polypeptide.
  • the PfRH5 sequences and fragments described in US 2018/0193440 and WO 2020/074908 are not exogenous scaffolds according to the present invention.
  • 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.
  • the PfRH5 sequences of SEQ ID NOs: 1, 2 and 18 herein 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.
  • 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.
  • 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.
  • the invention relates to a scaffold which comprises at least two ⁇ -helices.
  • the term ⁇ -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 ⁇ - helices.
  • the scaffold comprises three ⁇ -helices.
  • a scaffold of the invention typically comprises two ⁇ -helices which comprise a PfRH5 epitope of the invention, as described herein. Said two ⁇ -helices may be resurfaced such that the PfRH5 epitope can be grafted onto them, allowing the successful presentation of the PfRH5 epitope.
  • the at least two ⁇ -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 ⁇ -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.
  • the RMSD of the three-dimensional arrangement of the at least two ⁇ -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 ⁇ or less.
  • the RMSD of the three-dimensional arrangement of the at least two ⁇ -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 ⁇ .
  • the invention relates to a scaffold wherein the at least two ⁇ -helices are sufficiently long that the scaffold fully contains a PfRH5 epitope of the invention.
  • an ⁇ -helix in a polypeptide of the invention is at least 15 amino acid residues in length.
  • an ⁇ -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 ⁇ -helix in a polypeptide of the invention may be independently selected. Alternatively, the length of each ⁇ -helix in a polypeptide of the invention may be the same.
  • a PfRH5 epitope of the invention is presented on two ⁇ -helices and a third ⁇ -helix is present to stabilise and/or multimerise the polypeptide as described herein
  • the lengths of each of the two ⁇ -helices presenting/comprising the PfRH5 epitope may be independently selected or may be the same.
  • the first ⁇ -helix and/or the second ⁇ -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.
  • the first ⁇ -helix may be 29 amino acids in length and the second ⁇ -helix may be 33 amino acids in length.
  • 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.
  • a trimerization helix of the invention may be incorporated into the structure of an immunogen, such as the polypeptides of the invention.
  • the immunogen may comprise at least two ⁇ -helices in addition to the trimerization helix.
  • each polypeptide monomer will typically comprise a trimerization helix which comprises the same zipper motif.
  • a ny suitable technique may be used to resurface the trimerization helix, examples of such techniques are well known in the art.
  • 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.
  • 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.
  • the present invention provides a polypeptide in which the PfRH5 epitope is grafted onto the scaffold.
  • 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.
  • Growth 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • T he polypeptide of the invention may comprise at least two cysteine residues positioned such that they form a disulphide bridge in the polypeptide.
  • 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.
  • 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.
  • T he 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.
  • 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.
  • the polypeptide of the invention may be dimerized, trimerized, tetramerized, pentamerized or hexamerized.
  • the polypeptide of the invention is trimerized.
  • 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.
  • 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 ⁇ g/ml, 2x10-4 ⁇ g/ml, 3x10-4 ⁇ g/ml, 4x10-4 ⁇ g/ml, 5x10-4 ⁇ g/ml, 6x10-4 ⁇ g/ml, 7x10-4 ⁇ g/ml, 8x10 -4 ⁇ g/ml, 9x10 -4 ⁇ g/ml, 1x10 -3 ⁇ g/ml, 2x10 -3 ⁇ g/ml, 3x10 -3 ⁇ g/ml, 4x10 -3 ⁇ g/ml, 5x10 -3 ⁇ g/ml, 6x10 -3 ⁇ g/ml
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the PfRH5 antibodies elicited by a polypeptide of the invention e.g. the GIA or binding affinity of antibodies induced by said polypeptide
  • 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.
  • W here 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.
  • 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.
  • 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.
  • the conjugate moiety is a multimeric scaffold.
  • suitable multimeric scaffolds include virus-like particle (VLP), multimeric proteins and multimeric synthetic polymers. I n some preferred embodiments, the conjugate moiety is a virus-like particle (VLP).
  • VLP virus-like particle
  • a virus-like particle 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.
  • 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.
  • the VLP may be a synthetic or designed VLP.
  • 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).
  • 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).
  • CnaB2 second immunoglobulin-like collagen adhesin domain
  • FbaB fibronectin-binding protein
  • the conjugate group may be cleavable from the polypeptide of the invention.
  • certain conjugate linkers may comprise one or more cleavable moieties.
  • a cleavable moiety is a cleavable bond.
  • 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.
  • the conjugate group is not cleavable from the polypeptide of the invention.
  • 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.
  • the virus-like particle is attached to the N-terminus of the polypeptide via a spy-tag.
  • the VLP is attached to the C-terminus of the polypeptide via a GS linker, such as a GS16 linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • a conjugate monomer of the invention may comprise or consist of SEQ ID NO: 63 as described herein.
  • 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.
  • the genetic strain of the blood-stage Plasmodium parasite is Plasmodium falciparum.
  • the growth inhibitory activity 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.
  • EC30 GIA value which is 100 ng/mL or less
  • 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.
  • 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.
  • 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.
  • nucleic acid refers 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.
  • 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).
  • 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.
  • 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)).
  • adenovirus of a human serotype such as AdHu5, a simian serotype such as ChAd63, ChAdOX1 or ChAdOX2, or another form
  • AAV adeno-associated virus
  • 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.
  • said viral vector is an AAV vector.
  • 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.
  • a vector of the invention may be capable of expression in vivo in a mammalian (and particularly a human) subject following immunisation.
  • 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 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.
  • 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.
  • 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.
  • Crystal structures can be determined at any appropriate resolution, such as 1.6 or 2.4 ⁇ .
  • antibody 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.
  • Ig immunoglobulin
  • L light
  • mutant, variant, or derivative antibody entities are known in the art, non-limiting embodiments of which are discussed below.
  • each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • VH heavy chain variable 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).
  • CDR complementarity determining regions
  • FR framework regions
  • 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).
  • the PfRH5 epitope binds to PfRH5 antibodies which are mAbs.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a 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.
  • Kabat numbering "Kabat definitions and “Kabat labelling” are used interchangeably herein.
  • Antibodies of the invention are not limited to a particular method of generation or production.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antibody of the invention has a GIA of at least 30% or more against Plasmodium parasites.
  • 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.
  • the antibody has a GIA of at least 50%, or more against Plasmodium parasites.
  • 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.
  • An antibody of the invention may have an effective concentration required for 30% GIA (EC30) GIA value which is 100 ng/mL or lower.
  • 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.
  • 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.
  • 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).
  • 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 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.
  • a composition, PfRH5 epitope, polypeptide, conjugate, antibody, nucleic acid, or vector e.g., a viral vector, RNA vaccine or DNA plasmid
  • 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.
  • 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.
  • a polypeptide may be used in combination with one or more additional Plasmodium antigens (e.g.
  • 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.
  • treatment or “treating” embraces therapeutic or preventative/prophylactic measures and includes post-infection therapy and amelioration of malaria.
  • 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.
  • compositions, 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 (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.
  • a subject e.g. a mammal such as a human or other primate
  • 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
  • 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.
  • 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.
  • 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 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.
  • additional antigens may be expressed in any suitable form.
  • expression may be as a virus like particle (VLP).
  • 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.
  • 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.
  • Such “boosting” may comprise the administration of a pox virus, such as MVA.
  • 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.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • 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.
  • the carrier is a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline.
  • the composition is in lyophilized form, in which case it may include a stabilizer, such as BSA.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 Full length PfRH5 amino acid sequence (3D7) including signal sequence
  • SEQ ID NO: 3 Consensus sequence of first portion of a preferred PfRH5 epitope as presented on a first ⁇ -helix of the scaffold
  • SEQ ID NO: 4 Consensus sequence of second portion of a preferred PfRH5 epitope as presented on a second ⁇ -helix of the scaffold
  • SEQ ID NO: 5 Exemplified immunogen 3 (PfRH5 epitope with intervening scaffold residues)
  • 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: 1 Full length PfRH5 amino acid sequence (3D7) including signal sequence 1 MIRIKKKLIL TIIYIHLFIL NRLSFENAIK KTKNQENNLT LLPIKSTEEE KDDIKNGKDI
  • 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 K NVNFLQYHF 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 ILDMSNE
  • 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 ⁇ -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).
  • residue positions in RH5- 34EM in Table 1 are also calculated based on the RH5-34EM sequence including an initial methionine.
  • 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.
  • 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).
  • the monoclonal antibody R5.034 was transiently expressed using Expi293FTM cells with the Expi293TM 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 HiTrapTM 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 HiTrapTM rProtein A column (Cytiva).
  • R5.016 bound with lower affinity to RH5-34EM than PfRH5, although still in the nanomolar range (115nM vs 2.4nM) ( Figure 2e).
  • 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 1.63 ⁇ resolution) and R5.034 (at 1.75 ⁇ resolution) were determined ( Figure 3, Table 3).
  • 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.
  • HbSAg hepatitis B surface protein
  • 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.
  • 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).

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Abstract

La présente invention concerne des polypeptides utilisés pour le traitement ou pour la prévention du paludisme.
PCT/GB2025/050240 2024-02-09 2025-02-07 Immunogène pfrh5 Pending WO2025168946A1 (fr)

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WO2012114125A2 (fr) 2011-02-25 2012-08-30 Isis Innovation Limited Traitement et prévention du paludisme
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WO2012046081A1 (fr) * 2010-10-08 2012-04-12 Genome Research Limited Vaccin contre le paludisme
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