Stabilised protein

Abstract

The present disclosure relates to synthetic Plasmodium reticulocyte binding protein 2b (mRBP2b) antigens that have improved properties such as thermal stability, as well as the use of these antigens to detect a Plasmodium infection in a diagnostic device or for use in a vaccine.

Description

[0001] STABILISED PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to a synthetic Plasmodium reticulocyte binding protein 2b (mRBP2b) antigen, as well as the use of these synthetic antigens to detect a Plasmodium infection or in a vaccine.

[0004] BACKGROUND OF THE INVENTION

[0005] According to recent reports (The Lancet, 2024), malaria is still rampant, and some 249 million cases and 608 000 deaths were reported globally in 2022 (WHO, 2023). Malaria is marked by severe fever and pain, and can be fatal. The symptoms are caused by Plasmodium parasites that infect and destroy red blood cells in the subject.

[0006] There are multiple species of parasite that cause malaria in humans, the main being; Plasmodium vivax, P. falciparum, P. malariae, P. ovale and P. knowlesi. Transmission from humans to mosquitoes can only occur when the sexual stages of the parasite (gametocytes) are circulating in the blood. Liver-stage infection with hypnozoites is completely undetectable and asymptomatic, and upon relapses, constitutes a significant transmission reservoir. P. falciparum and P. knowlesi do not have this ability. P. malariae can cause recurrent infections but it remains unclear if these infections persist in the bloodstream, the liver or another organ.

[0007] Parasite species differ in their preference to the type of red blood cells they can invade. Of the five major species, the P. vivax parasite is the most evasive. It can “hide” in the liver of a subject and remain dormant - and thus the subject is asymptomatic - before remerging to cause renewed blood-stage infections and malarial symptoms. This ability to hide from the immune system in the liver for prolonged periods makes P. vivax particularly difficult to detect and treat.

[0008] P. vivax is confined to reticulocytes by specific recognition of the red blood cell type from parasite proteins expressed at the apical pole of the merozoite. These expressed proteins are named reticulocyte binding proteins (PvRBP) (Galinski et al., 1992) and given their crucial implication in the invasion process, PvRBP proteins are considered to be good vaccine candidates.

[0009] Diagnosis of subjects with P. vivax infections is of paramount importance to reduce or even eliminate transmission in a population. Such diagnosis would also significantly help individual subjects to receive proper treatment, including those that have only silent liver-stage infections. Given the high degree of population mobility today, particularly in response to natural disasters or human conflicts, accurate and rapid diagnosis of all P. vivax infections has become even more important to controlling the disease. In addition, as transmission in countries decreases (as each population approaches elimination of the disease), population-level surveillance becomes increasingly important.

[0010] Current diagnostics to detect Plasmodium infection in programmatic settings rely on either the use of rapid diagnostic tests (detecting parasite antigens in the blood) or microscopy of Giemsa-stained blood-smears. Both these methods will only detect a current blood-stage infection, and hence will miss all asymptomatic hypnozoite infections. These methods are also insensitive to the common low parasitaemic infections (at the blood-stage) of P. vivax. In the context of P. vivax elimination, the overwhelming majority of infectious P. vivax episodes that are caused by relapsing hypnozoite infections (Robinson et al., 2015) reiterates the need to innovatively target this reservoir of disease. The ability to identify individuals who are infected with hypnozoites would be revolutionary in P. vivax control, however the sparsity of infection throughout the liver and the inaccessibility of this organ makes this exceptionally challenging.

[0011] Identifying antigen-specific antibodies associated with P. vivax as a result of exposure or infection is key to malaria diagnostics. Some proteins have been very well studied and characterized for diagnostic purposes. For example, merozoite surface protein 1, MSP1, (US6958235, WO9208795), MSP3 (US7488489) and rhoptry associated membrane antigen (RAMA) (EP0372019) to name a few.

[0012] Other researchers have focused efforts on vaccine antigens based on apical membrane antigen 1 (PfAMAl). However, antibodies against Pf A A 1 appear only to be effective at an extremely high concentration. In addition, PfAMAl induces strainspecific antibodies which are not effective against genetically diverse strains of the Plasmodium parasite (Goodman and Draper, 2010). Research at Oxford University, in collaboration with Yeda Research and Development, focused on reticulocytebinding protein homologue 5 (PfRh5) as a potential antigen vaccine candidate against P. falciparum (WO2012114125).

[0013] Meta analysis of cohort studies derived from mostly Brazilian data (Cutts et al., 2014) showed that antibody responses to the P. vivax proteins, circumsporozoite protein (PvCSP,) PvMSP-119, PvMSP-9RIRII, and PvAMAl correlated with protection from clinical disease. However, there were considerable heterogeneity in observed estimates. Evaluating data from Thailand and Brazil, researchers at the Walter Eliza Hall Institute showed that antibodies for a range of parasitic proteins correlated with infection time frames (Longley et al., 2020; W02018130871). One of antigens in the study, PvRBP2b, was identified as being a significant marker and its inclusion in a multiplex array significantly influenced specificity and selectivity.

[0014] These proteins or their fragments would make good vaccine candidates. The only currently approved vaccines are based on the CSP protein and act by blocking infection of P. falciparum in the liver (vaccines: RTS,S and R21). RTS,S has been administered to nearly 2 million children in pilot studies in Ghana, Kenya, and Malawi, resulting in a 13% reduction in child mortality, and Phase 3 results for R21 / Matrix-M, (The Lancet, 2024 , showed 75 % efficacy in children aged 5-36 months. However, its supply is limited and getting it to those in need in a timely manner is still challenging.

[0015] Alternative vaccine development has been hampered by the requirement for potentially reactogenic chemical adjuvants in addition to the antigen to induce sufficient antibody responses in human subjects. Some of the Plasmodium antigens suffer from limited stability at high temperatures and poor expression levels in various expression systems. Stability is important in the context of use of these antigens in hot and underdeveloped countries where transportation requires deployment of cold chain systems. Thus, there is an ongoing need for alternative, more stable, Plasmodium antigens, for use in surveillance tools and vaccines that will retain efficacy when stored at elevated temperatures.

[0016] SUMMARY

[0017] In an aspect, the present disclosure provides a synthetic antigen which comprises at least one amino acid substitution when compared to a wild type P. vivax reticulocyte binding protein 2b (PvRBP2b) having a sequence of amino acids provided as SEQ ID NO: 1, wherein the at least one amino acid substitution is at a position corresponding to the following amino acid number of SEQ ID NO: 1 : 243, 247, 248, 264, 269, 270, 276, 278, 280, 283, 291, 305, 309, 313, 317, 319, 322, 324, 337, 339, 347, 352, 354, 356, 357, 361, 367, 369, 371, 372, 377, 378, 382, 393, 398, 402, 404, 405, 406, 407, 409, 413, 417, 418, 428, 431, 436, 439, 444, 459, 463 or 467.

[0018] In an embodiment, the synthetic antigen has a higher expression level in cell culture when compared to a synthetic antigen having the amino acid sequence of SEQ ID N0:2.

[0019] In an embodiment, the synthetic antigen has higher thermostability when compared to an antigen having the amino acid sequence of SEQ ID NO:2.

[0020] In an embodiment, the synthetic antigen has a higher affinity to an anti- Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody when compared to an antigen having the amino acid sequence of SEQ ID NO:2. In an embodiment, the synthetic antigen is at least 302 amino acids in length. In an embodiment, the synthetic antigen is 302 amino acids in length.

[0021] Also provided is a polynucleotide encoding the synthetic antigen of the disclosure.

[0022] In an aspect, the present disclosure provides a vector comprising the polynucleotide of the disclosure.

[0023] In an aspect, the present disclosure provides a host cell comprising the polynucleotide of the disclosure and / or the vector of the disclosure.

[0024] In an aspect, the present disclosure provides a method of producing a synthetic antigen of the disclosure, the method comprising expressing in a cell or cell free expression system the polynucleotide of the disclosure.

[0025] In an aspect, the present disclosure provides a method of producing the cell of the disclosure, the method comprising the step of introducing the polynucleotide of the disclosure and / or the vector of the disclosure, into a cell.

[0026] In an aspect, the present disclosure provides a composition comprising one or more of the synthetic antigen of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, or the cell of the disclosure, and a suitable carrier.

[0027] In an embodiment, the composition is a vaccine composition.

[0028] In an embodiment, the vaccine composition comprises an adjuvant.

[0029] In an embodiment, the composition comprises the synthetic antigen of the disclosure.

[0030] Also provided is a solid support comprising the synthetic antigen of the disclosure.

[0031] In an embodiment, the solid support is a dipstick, test strip, or micro-well plate.

[0032] In an embodiment, the test strip is a lateral flow test strip.

[0033] In an aspect, the present disclosure provides a diagnostic device comprising the solid support of the disclosure.

[0034] In an aspect, the present disclosure provides a kit comprising one or more of the synthetic antigen of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the cell of the disclosure, the composition of the disclosure, the solid support of the disclosure or the diagnostic device of the disclosure.

[0035] In an aspect, the present disclosure provides a method of determining whether a subject has, or has had, Plasmodium sp. infection, the method comprising determining the presence or absence of an antibody which binds the synthetic antigen of the disclosure in a sample obtained from the subject, wherein the presence of the antibody is indicative of the subject having, or previously having, a. Plasmodium sp. infection. In an aspect, the present disclosure provides a method of treating a Plasmodium sp. infection in a subject, the method comprising administering an anti- Plasmodium sp. agent to the subject if they have been determined to have a Plasmodium sp. infection using the method of the disclosure.

[0036] In an aspect, the present disclosure provides a method of raising an immune response in a subject, the method comprising administering to the subject one or more of the synthetic antigen of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the cell of the disclosure, or the composition of the disclosure.

[0037] In an aspect, the present disclosure provides a method of treating or preventing a Plasmodium sp. infection in a subject, the method comprising administering to the subject one or more of the synthetic antigen of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the cell of the disclosure, or the composition of the disclosure.

[0038] In an embodiment, the above methods treat or prevent at least on symptom of malaria.

[0039] In an embodiment, the Plasmodium sp. is Plasmodium vivax.

[0040] Also provided is the use of the synthetic antigen of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the cell of the disclosure, or the composition of the disclosure in the manufacture of a medicament for raising an immune response.

[0041] Further provided is the use of the synthetic antigen of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the cell of the disclosure, or the composition of the disclosure in the manufacture of a medicament for treating or preventing Plasmodium sp. infection in a subject.

[0042] In an aspect, the present disclosure provides a method selecting a synthetic antigen which binds an &nA-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody, the method comprising i) producing a synthetic antigen of the disclosure, ii) determining if the synthetic antigen binds the anti-RBP2b antibody, and iii) selecting the synthetic antigen if it binds the anti-RBP2b antibody.

[0043] In an embodiment, the method further comprises expressing the synthetic antigen in a cell and determining if more synthetic antigen is produced when compared to a corresponding wild type antigen, such as synthetic antigen having the amino acid sequence of SEQ ID NO:2, produced under the same conditions. In an embodiment, the method further comprises detecting the thermostability of the synthetic antigen, and step iii) comprises selecting the synthetic antigen if it has a higher thermostability when compared to a corresponding wild type antigen such as antigen having the amino acid sequence of SEQ ID NO:2.

[0044] In an embodiment, the method further comprises detecting the affinity of the antigen-antibody binding, and step iii) comprises selecting the synthetic antigen if it has a higher affinity when compared to a corresponding wild type antigen such as antigen having the amino acid sequence of SEQ ID NO:2. Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0045] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0046] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0047] The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0048] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0049] FIGURE 1 - Purification yields and biophysical characterization of parental PvRBP2b 169-470 and variants (A) Coomassie-stained SDS-PAGE gel of Ni-NTA affinity purified parental PvRBP2bi69-47o and nine variants in reducing conditions. (B) Final yields (mg / L bacterial culture) for parental and stabilized variants after Ni-NTA affinity, ion exchange and size exclusion purification steps with fold change increase relative to parental yields shown on top of the corresponding bar graphs. The dotted line separates the two independent replicates for protein purification.

[0050] FIGURE 2 - Structural comparison of parental PvRBP2bi69-47o with stabilized variants WHT2483 and 2484. (A) Crystal structures of WHT2483 and WHT2484 are overlayed with parental PvRBP2bi69-47o with RMSD values provided. (B) Mutated residues are shown as spheres mapped onto ribbon representations of the parental PvRBP2b 169-470 structure. Representative mutations that demonstrate stabilising effects are highlighted on the tertiary structure compared with parental PvRBP2b 169-470. (C) Binding footprints of human antibodies against parental PvRBP2b 169-470. Parental PvRBP2b 169-470 is shown in surface representation (white) and coloured regions denote residues involved in antibody binding, with light chain interactions in a lighter shade and heavy chain interactions in a darker shade for each antibody (D) Total antibody bound surface for the eight human monoclonal antibodies is shown on parental PvRBP2b 169-470 surface (white).

[0051] FIGURE 3 - Receiver operator characteristic (ROC) curves demonstrating the sensitivity and specificity of classifying recent P. vivax exposure in the prior 9 months when using IgG antibodies against a panel of 8 P. vivctx synthetic antigens. The reference utilises RBP2b P25, this is then swapped for either RBP2b P87 WT or one of the variants. The results excluding all RBP2bs demonstrate the importance of this synthetic antigen for the classification. The classification was performed using IgG antibody responses in the Brazil, Thai, Solomon Island and malaria-naive cohorts, in a Random Forest classifier where the truth was the monthly PCR results from the prior year.

[0052] KEY TO THE SEQUENCE LISTING

[0053] SEQ ID NO: 1 - Full length P. vivax RBP2b (Wild type - WT).

[0054] SEQ ID NO: 2 - Antigenic fragment of P. vivax RBP2b (amino acid numbers 169 to 470 of SEQ ID: 1).

[0055] SEQ ID NO: 3 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2483. SEQ ID NO: 4 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2482. SEQ ID NO: 5 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2484. SEQ ID NO: 6 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2476. SEQ ID NO: 7 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2477. SEQ ID NO: 8 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2478. SEQ ID NO: 9 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2479. SEQ ID NO: 10 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2480. SEQ ID NO: 11 - Synthetic antigenic fragment of P. vivax RBP2b - WHT2481.

[0056] SEQ ID NO: 12 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2483.

[0057] SEQ ID NO: 13 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2482.

[0058] SEQ ID NO: 14 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2484. SEQ ID NO: 15 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2476.

[0059] SEQ ID NO: 16 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2477.

[0060] SEQ ID NO: 17 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2478.

[0061] SEQ ID NO: 18 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2479.

[0062] SEQ ID NO: 19 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2480.

[0063] SEQ ID NO: 20 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2481.

[0064] SEQ ID NO: 21 - Open reading frame encoding antigenic fragment of P. vivax RBP2b - WHT2483 - with N-terminal extension including a start Met and a Hexa His.

[0065] SEQ ID NO: 22 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2482 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 23 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2484 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 24 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2476 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 25 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2477 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 26 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2478 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 27 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2479 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 28 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2480 - with N-terminal extension including a start Met and a Hexa His. SEQ ID NO: 29 - Open reading frame encoding synthetic antigenic fragment of P. vivax RBP2b - WHT2481 - with N-terminal extension including a start Met and a Hexa His.

[0066] DETAILED DESCRIPTION OF THE DISCLOSURE

[0067] General Techniques and Definitions

[0068] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, malaria diagnostics, malaria vaccines, protein chemistry, and biochemistry).

[0069] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0070] As used herein, the term about, unless stated to the contrary, refers to + / - 10%, or more preferably + / - 5%, more preferably + / - 1%, of the designated value.

[0071] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0072] As used herein “antigen” refers to a substance, typically a protein, capable of stimulating an immune response in a subject.

[0073] As used herein, “raising an immune response” or similar terms refers to the immune system of the subject identifying an antigen of the disclosure as foreign resulting in the formation of antibodies (from B lymphocytes) and / or lymphocytes (such as T lymphocytes) capable of reacting with the antigen.

[0074] As used herein, the phrase “at least one amino acid substitution is at a position corresponding to the following amino acid number of SEQ ID NO” or variations thereof refers to the relative position of the amino acid compared to surrounding amino acids. In this regard, in some embodiments a polypeptide of the disclosure may have deletional or substitutional mutation, or be a fragment, which alters the relative positioning of the amino acid when aligned against, for instance, SEQ ID NO: 1. As the skilled person would appreciate, when the synthetic antigen is a fragment of a full length protein the “corresponding” amino acid can be determined by an alignment. As an example, amino acid number 74 of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 corresponds to amino acid number 214 for SEQ ID NO:1.

[0075] As used herein, “thermal stability”, “thermostability”, or variations thereof refers to the ability of the synthetic antigen to maintain antigenic activity at a higher temperature and / or for an extended period of time when compared to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2. Standard techniques are known in the art, including but not limited to, circular dichroism, differential scanning calorimetry and surface plasmon resonance, and can be used to quantify thermal stability of a synthetic antigen of the disclosure. In an embodiment, a synthetic antigen of the disclosure has a higher thermal stability of at least 2°C, at least 3 °C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 25°C, or more, when compared to a suitable control antigen such as a antigen consisting of an amino acid sequence provided as SEQ ID NO:2.

[0076] In an embodiment, a synthetic antigen of the disclosure has a higher thermal stability of at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 25°C, or more, when compared to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2 as measured by dynamic light scattering.

[0077] As used herein, the term “TonSet” refers to the temperature of the onset of melting (the temperature at which the synthetic antigen begins to unfold). In an embodiment, Tonset is determined using the Tycho NT.6 (NanoTemper Technologies) (see, for instance, Example 1).

[0078] As used herein, the term “T ” refers to the inflection temperature, the temperature of the unfolding transition in the 350 nm / 330 nm ratio fluorescence signal. Unfolding could result in denaturation of the antigen. In an embodiment, Ti is determined using the Tycho NT.6 (NanoTemper Technologies) software (1.2.0.750) (see, for instance, Example 1).

[0079] As used herein, the phrase “higher expression level in cell culture”, “more antigen is produced” or similar phrases refer to more synthetic antigen of the disclosure being produced ( greater synthetic antigen yield) when compared to a suitable control antigen, such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2, expressed under identical conditions such as those described in Example Un an embodiment, the synthetic antigen is expressed in E. coli.

[0080] In an embodiment, the synthetic antigen is expressed in a mammalian cell.

[0081] As used herein, “affinity” refers to the strength of binding between a synthetic antigen of the disclosure and an anh-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody. In an embodiment, a synthetic antigen of the disclosure has a higher affinity to an a.ni\-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody when compared to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2.

[0082] In an alternative embodiment, a synthetic antigen of the disclosure has an affinity to an \ -Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody that is comparable to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2. In an alternative embodiment the synthetic antigen of the disclosure maintains recognition to an mh-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody that is comparable to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2.

[0083] In an embodiment, the naturally occurring r\ -Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody is selected from 237235, 241242, 243244, 251249, 253245, 258259, 260261, 273264 and 283284 as described in Chan et al. (2021)

[0084] In a preferred embodiment, the naturally occurring &ni\-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody is selected from 241242, 253245, 258259, and 273264 as described in Chan et al. (2021)

[0085] Reference to "subject" or "subjects" includes a subject susceptible to a Plasmodium sp. infection, or at risk of exposure to a Plasmodium sp. The subject may be infected or uninfected and may be symptomless or in need of treatment. For example, the subject can be a mammal. Exemplary subject’s include but are not limited to human, primate, livestock (e.g. sheep, cow, chicken, horse, donkey, pig), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animal (e.g. fox, deer), zoo animals (e.g. lion, tiger, bear), reservoir animals (e.g. bats, camels, pangolin). In an embodiment, the subject is a mammal. In one embodiment, the subject is human. In an embodiment, the human is a fetus, infant, child, early adult or an adult. As used herein, the term "prevention" or “prophylaxis” refers to reducing the likelihood of contracting or developing Plasmodium sp. infection or a symptom thereof such as a symptom of malaria. Prevention need not be complete and does not imply that a subject will not eventually contract or develop the infection or a symptom thereof.

[0086] The terms “treating” or “treatment” as used herein, refer to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject), or indirect treatment, effected, by at least one party, (e.g., a medical doctor, a nurse, a pharmacist, or a pharmaceutical sales representative) by providing instructions, in any form, that (i) instruct a subject to self-treat according to a method of the disclosure (e.g., self-administer a therapeutic, such as a vaccine of the disclosure) or (ii) instruct a third party to treat a subject according to a method of the disclosure. In an embodiment, the subject has malaria. Also encompassed within the meaning of the term “treating” is administering a therapeutic at a sufficiently early phase of disease to prevent or slow its progression.

[0087] Reticulocyte binding protein 2b (RBP2b) and modified versions thereof

[0088] RBP2b binds to transferrin receptor 1 (TfRl) to mediate a critical invasion pathway for entry at least some Plasmodium sp. into reticulocytes . Mouse monoclonal antibodies raised against PvRBP2b abolished the PvRBP2b binding to reticulocytes and inhibit P. vivax entry into reticulocytes. (Gruszczyk et al, Science (2018), Gruszczyk et al., Nature 2018.)

[0089] As used herein, PvRBP2b refers to naturally occurring, unmodifed, RBP2b from P. vivax (such as having an amino acid sequence provided as SEQ ID NO: 1), or an antigenic fragment thereof (such as consisting of an amino acid sequence provided as SEQ ID NO: 2).

[0090] As used herein, unless stated otherwise, RBP2b or “unmodified RBP2b” refers to a naturally occurring RBP2b from Plasmodium sp., or an antigenic fragment thereof.

[0091] As used herein, a synthetic protein which is an antigen refers to a man made variant of RBP2b or an antigenic fragment thereof, wherein the resultant, synthetic protein, mRBP2b, which comprises at least one amino acid substitution when compared to a wild type P. vivax reticulocyte binding protein 2b (PvRBP2b) having a sequence of amino acids provided as SEQ ID NO: 1, wherein the at least one amino acid substitution is at a position corresponding to the following amino acid number of SEQ ID NO: 1 : 243, 247, 248, 264, 269, 270, 276, 278, 280, 283, 291, 305, 309, 313, 317, 319, 322, 324, 337, 339, 347, 352, 354, 356, 357, 361, 367, 369, 371, 372, 377, 378, 382, 393, 398, 402, 404, 405, 406, 407, 409, 413, 417, 418, 428, 431, 436, 439, 444, 459, 463 or 467. In an embodiment in which one or more amino acid position is substituted relative to the corresponding unmodified RBP2b antigen, the substitution may be a conservative substitution or a non-conservative substitution. A conservative substitution is defined as substitution by an amino acid pertaining to the same physiochemical group to the amino acid present in the unmodified RBP2b antigen. 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 unmodified RBP2b antigen. In more detail, amino acids are, in principle, divided into different physiochemical groups. Aspartate and glutamate belong to the negatively-charged amino acids. Histidine, arginine and lysine belong to the positively-charged amino acids. Asparagine, glutamine, serine, threonine, cysteine and tyrosine belong to the polar amino acids. Glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine and tryptophan belong to the non-polar amino acids. Aromatic side groups are to be found among the amino acids histidine, phenylalanine, tyrosine and tryptophan. Thus, as a non-limiting example, a conservative substation may involve the substitution of a non-polar amino acid by another non-polar amino acid, such as substituting leucine with isoleucine. As another non-limiting example, a non-conservative substitution may involve the substation of a non-polar amino acid (e.g. leucine) with a negatively-charged amino acid (e.g. aspartate), a positively-charged amino acid (e.g. arginine), or a polar amino acid (e.g. asparagine).

[0092] At least one amino acid substitution as described herein may be a conservative amino acid substitution, such as substituting one negatively charged amino acid for another. Other conservative amino acid substitutions encompassed by the present disclosure include substituting a hydrophobic amino acid by another hydrophobic amino acid, substituting a positively-charged amino acid by another positively-charged amino acid, or substituting a polar amino acid by another polar amino acid.

[0093] At least one amino acid substitution as described herein may be a non- conservative amino acid substitution, such as substituting a polar amino acid with a hydrophobic amino acid. Other non-conservative amino acid substitutions encompassed by the present disclosure include, but are not limited to, substituting a hydrophobic amino acid by a positively-charged amino acid, substituting a hydrophobic amino acid by a negatively-charged amino acid, substituting a hydrophobic amino acid by a polar amino acid , substituting a polar amino acid by a positively- or negatively-charged amino acid, or by a hydrophobic amino acid, substituting a positively- or negatively-charged amino acid by a polar or hydrophobic amino acid, or by substituting a positively-charged amino acid by a negatively-charged amino acid, or vice versa. A synthetic antigen of the disclosure may comprise at least 1, at least 2, at least 3, at least 4, 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 30, or at least 35, of the listed amino acid substitutions, or any combination thereof.

[0094] A synthetic antigen of the disclosure may comprise at least 17 of the listed amino acid substitutions, or any combination thereof.

[0095] In an embodiment, the synthetic antigen has a substitution at one or more or all of position(s): 243, 247, 248, 270, 309, 322, 324, 337, 347, 354, 357, 361, 367, 369, 371, 372, 377, 378, 398, 402, 405, 406, 418, 431, 436, or 439.

[0096] In an embodiment, the synthetic antigen has a substitution at one or more or all of position(s): 243, 247, 248, 270, 309, 322, 324, 337, 347, 354, 357, 361, 367, 369, 371, 372, 377, 378, 398, 402, 405, 406, 418, 431, 436, or 439, the substitutions resulting in a synthetic antigen with higher thermal stability when compared to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2 as measured by dynamic light scattering.

[0097] In an embodiment, the synthetic antigen has a substitution at one or more or all of position(s): 243, 247, 322, 337, 347, 354, 357, 367, 377, 378, 398, 406, 418, 431 or 436.

[0098] In an embodiment, the synthetic antigen has a substitution at one or more or all of position(s): 243, 247, 322, 337, 347, 354, 357, 367, 377, 378, 398, 406, 418, 431 or 436, the substitutions resulting in a synthetic antigen with higher thermal stability when compared to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2 as measured by dynamic light scattering.

[0099] In an embodiment, the synthetic antigen has a substitution at all of position(s): 243, 247, 270, 322, 337, 347, 354, 357, 367, 369, 371, 377, 378, 398, 406, 418, 431, 436 and 439.

[0100] In an embodiment, the synthetic antigen has a substitution at all of position(s): 243, 247, 270, 322, 337, 347, 354, 357, 367, 369, 371, 377, 378, 398, 406, 418, 431, 436 and 439, the substitutions resulting in a synthetic antigen with higher thermal stability when compared to a suitable control antigen such as an antigen consisting of an amino acid sequence provided as SEQ ID NO:2 as measured by dynamic light scattering. In an embodiment, the synthetic antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, identical to the amino acid sequence provided as SEQ ID NO:3.

[0101] In an embodiment, the synthetic antigen comprises or consists of the amino acid sequence provided as SEQ ID NO:3.

[0102] In an embodiment, the synthetic antigen has a substitution at all of position(s): 243, 247, 270, 322, 337, 347, 354, 357, 361, 367, 377, 378, 398, 406, 418, 431 and 436.

[0103] In an embodiment, the synthetic antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, identical to the amino acid sequence provided as SEQ ID NO:4.

[0104] In an embodiment, the synthetic antigen comprises or consists of the amino acid sequence provided as SEQ ID NO:4.

[0105] In an embodiment, the synthetic antigen has a substitution at all of position(s): 243, 247, 248, 270, 309, 322, 324, 337, 347, 354, 357, 361, 367, 369, 371, 372, 377, 378, 398, 402, 405, 406, 418, 431, 436 and 439.

[0106] In an embodiment, the synthetic antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, identical to the amino acid sequence provided as SEQ ID NO: 5.

[0107] In an embodiment, the synthetic s antigen comprises or consists of the amino acid sequence provided as SEQ ID NO: 5.

[0108] In an embodiment, the amino acid at position 243 is leucine.

[0109] In an embodiment, the amino acid at position 247 is leucine.

[0110] In an embodiment, the amino acid at position 248 is leucine.

[0111] In an embodiment, the amino acid at position 264 is asparagine.

[0112] In an embodiment, the amino acid at position 269 is glutamic acid.

[0113] In an embodiment, the amino acid at position 270 is leucine.

[0114] In an embodiment, the amino acid at position 276 is threonine.

[0115] In an embodiment, the amino acid at position 278 is leucine.

[0116] In an embodiment, the amino acid at position 280 is glutamic acid.

[0117] In an embodiment, the amino acid at position 283 is asparagine.

[0118] In an embodiment, the amino acid at position 291 is glutamic acid.

[0119] In an embodiment, the amino acid at position 305 is lysine.

[0120] In an embodiment, the amino acid at position 309 is arginine.

[0121] In an embodiment, the amino acid at position 313 is threonine. In an embodiment, the amino acid at position 317 is serine. In an embodiment, the amino acid at position 319 is lysine. In an embodiment, the amino acid at position 322 is tryptophan. In an embodiment, the amino acid at position 324 is phenylalanine. In an embodiment, the amino acid at position 337 is leucine. In an embodiment, the amino acid at position 339 is aspartic acid. In an embodiment, the amino acid at position 347 is phenylalanine. In an embodiment, the amino acid at position 352 is asparagine. In an embodiment, the amino acid at position 354 is tyrosine or phenylalanine. In an embodiment, the amino acid at position 354 is tyrosine. In an embodiment, the amino acid at position 356 is asparagine. In an embodiment, the amino acid at position 357 is leucine. In an embodiment, the amino acid at position 361 is tyrosine. In an embodiment, the amino acid at position 367 is aspartic acid. In an embodiment, the amino acid at position 369 is isoleucine or aspartic acid. In an embodiment, the amino acid at position 369 is isoleucine. In an embodiment, the amino acid at position 371 is leucine. In an embodiment, the amino acid at position 372 is glutamine. In an embodiment, the amino acid at position 377 is valine. In an embodiment, the amino acid at position 378 is leucine. In an embodiment, the amino acid at position 382 is lysine. In an embodiment, the amino acid at position 393 is glutamic acid or arginine. In an embodiment, the amino acid at position 398 is alanine. In an embodiment, the amino acid at position 402 is isoleucine. In an embodiment, the amino acid at position 404 is aspartic acid. In an embodiment, the amino acid at position 405 is isoleucine. In an embodiment, the amino acid at position 406 is alanine. In an embodiment, the amino acid at position 407 is asparagine. In an embodiment, the amino acid at position 409 is glutamic acid. In an embodiment, the amino acid at position 413 is tyrosine. In an embodiment, the amino acid at position 417 is aspartic acid. In an embodiment, the amino acid at position 418 is glutamic acid or threonine In an embodiment, the amino acid at position 418 is glutamic acid. In an embodiment, the amino acid at position 428 is tyrosine. In an embodiment, the amino acid at position 431 is alanine.

[0122] In an embodiment, the amino acid at position 436 is alanine or serine. In an embodiment, the amino acid at position 436 is alanine.

[0123] In an embodiment, the amino acid at position 439 is glutamine or asparagine.

[0124] In an embodiment, the amino acid at position 439 is glutamine.

[0125] In an embodiment, the amino acid at position 444 is glutamic acid or arginine.

[0126] In an embodiment, the amino acid at position 459 is serine.

[0127] In an embodiment, the amino acid at position 463 is serine.

[0128] In an embodiment, the amino acid at position 467 is lysine.

[0129] In an embodiment, the amino acid position is a position corresponding to the amino acid sequence provided as SEQ ID NO: 1.

[0130] In an embodiment, the synthetics antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 11, or which is at least 80% identical to one or more or all of SEQ ID NO’ s 3 to 11.

[0131] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 5, or which is at least 80% identical to one or more or all of SEQ ID NO’s 3 to 5.

[0132] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 11, or which is at least 90% identical to one or more or all of SEQ ID NO’ s 3 to 11.

[0133] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 5, or which is at least 90% identical to one or more or all of SEQ ID NO’s 3 to 5.

[0134] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 11, or which is at least 95% identical to one or more or all of SEQ ID NO’ s 3 to 11.

[0135] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 5, or which is at least 95% identical to one or more or all of SEQ ID NO’s 3 to 5.

[0136] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 11, or which is at least 99% identical to one or more or all of SEQ ID NO’ s 3 to 11.

[0137] In an embodiment, the synthetic antigen of the disclosure comprises a sequence of amino acids provided as any one of SEQ ID NO’s 3 to 5, or which is at least 99% identical to one or more or all of SEQ ID NO’s 3 to 5.

[0138] In an embodiment, the synthetic antigen has an N-terminal methionine residue.

[0139] In an embodiment, the synthetic antigen of the disclosure is a synthetic antigenic fragment of full length RBP2b. In an embodiment, the synthetic antigen is 302 amino acids in length. In an embodiment, the fragment corresponds to amino acid numbers 169 to 470 of SEQ ID NO:1. Examples of such synthetic antigens of the disclosure include, but are not limited to, those consisting of an amino acid sequence provided as SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0140] In an embodiment, the synthetic antigen is at least 302, at least 350, or at least 400 amino acids in length.

[0141] In an embodiment, the synthetic antigen of the disclosure lacks the native RBP2b N-terminal signal sequence.

[0142] In an embodiment, the synthetic antigen of the disclosure has a TOnset of at least 40°C, between 40°C and 60°C, between 50°C and 60°C, between 55°C and 60°C, or between 55 °C and 59.4°C.

[0143] In an embodiment, the synthetic antigen of the disclosure has a Ti of at least 65°C, between 65°C and 80°C, between 70°C and 80°C, or between 70°C and 75°C.

[0144] In an embodiment, the synthetic antigen of the disclosure has at least a 5 fold, at least a 8 fold, between a 5 and 20 fold, between an 8 and 20 fold, or between an 8 and 16 fold, higher protein yield when compared to SEQ ID NO:2 expressed under identical conditions.

[0145] In an embodiment, the synthetic antigen has a Ka(x 105T's'1) for an anti- Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody which is less than 12, less than 10, between 4 and 12, between 4 and 10, or between 5 and 10.

[0146] In an embodiment, the synthetic antigen has a KD (nM) for an mh-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody which is less than 0.1, less than 0.01, less than 0.005, or less than 0.001. In an embodiment, the antibody is 237235. In an embodiment, the antibody is 241242. In an embodiment, the antibody is 251249. In an embodiment, the antibody is 253245.

[0147] In an embodiment, the synthetic antigen has a Kd (x 10'5s'1) for an anti- Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody which is less than 10, less than 5, less than 1, less than 0.1, or less than 0.01. In an embodiment, the antibody is 237235. In an embodiment, the antibody is 241242.

[0148] The synthetic antigens of the disclosure may additionally comprise an N- or C- terminal tag, for example to assist in recombinant production and / or purification. Any N- or C-terminal tag may be used, including conventional tags known in the art. Suitable tags sequences include C-terminal hexa-histidine tags and the "C-tag" (the four amino acids EPEA at the C-terminus), which are commonly used in the art to aid purification from heterologous expression systems, e.g. insect cells, mammalian cells, bacteria, or yeast. Other examples of suitable tags include GST and MBP tags, or any other conventional tag which may be used to facilitate increased expression of a synthetic antigen. In other embodiments, the synthetic antigens of the disclosure are purified from heterologous expression systems without the need to use a purification tag.

[0149] Immunogenicity of the synthetic antigens of the disclosure can also be assessed using multiplexed antibody assays based on Luminex xMAP beads. Here, antigens are bound to uniquely colour coded beads that can be detected by lasers or LED. This enables multiplexing. Antigen coupled beads are mixed with monoclonal antibody or polyclonal human plasma / sera then binding revealed through addition of a fluorescent anti-human (or other species) IgG detector antibody. Plates are run on a Luminex instrument, such as MAGPIX, Bioplex-200, Intelliflex. The fluorescent signal is quantified by a 2nd laser or LED.

[0150] The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. In an embodiment, the query sequence is atleast 301 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 301 amino acids. In an embodiment, the GAP analysis aligns two sequences over their entire length.

[0151] Amino acid sequence variants of the synthetic antigens of the present disclosure can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present disclosure, or by in vitro synthesis of the desired synthetic antigen. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired characteristics.

[0152] Polynucleotides

[0153] As used herein, a "polynucleotide" or "nucleic acid" or "nucleic acid molecule" means a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA, and cDNA. The term "polynucleotide" is used interchangeably herein with the term "nucleic acid". Preferred polynucleotides of the disclosure encode a synthetic antigen of the disclosure.

[0154] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 29, or which is at least 80% identical to one or more or all of SEQ ID NO’s 12 to 29. In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 80% identical to one or more or all of SEQ ID NO’s 12 to 20.

[0155] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 80% identical to one or more or all of SEQ ID NO’s 12 to 14 and 21 to 23.

[0156] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 80% identical to one or more or all of SEQ ID NO’s 12 to 14.

[0157] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 29, or which is at least 90% identical to one or more or all of SEQ ID NO’s 12 to 29.

[0158] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 90% identical to one or more or all of SEQ ID NO’s 12 to 20.

[0159] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 90% identical to one or more or all of SEQ ID NO’s 12 to 14 and 21 to 23.

[0160] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 90% identical to one or more or all of SEQ ID NO’s 12 to 14.

[0161] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 29, or which is at least 95% identical to one or more or all of SEQ ID NO’s 12 to 29.

[0162] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 95% identical to one or more or all of SEQ ID NO’s 12 to 20.

[0163] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 95% identical to one or more or all of SEQ ID NO’s 12 to 14 and 21 to 23.

[0164] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 95% identical to one or more or all of SEQ ID NO’s 12 to 14.

[0165] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 29, or which is at least 99% identical to one or more or all of SEQ ID NO’s 12 to 29. In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 99% identical to one or more or all of SEQ ID NO’s 12 to 20.

[0166] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 99% identical to one or more or all of SEQ ID NO’s 12 to 14 and 21 to 23.

[0167] In an embodiment, a polynucleotide of the disclosure comprises a sequence of nucleotides provided as any one of SEQ ID NO’s 12 to 20, or which is at least 99% identical to one or more or all of SEQ ID NO’s 12 to 14.

[0168] The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 903 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 903 nucleotides. In an embodiment, the GAP analysis aligns two sequences over their entire length.

[0169] With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

[0170] In a further embodiment, the present disclosure relates to polynucleotides which are substantially identical to those specifically described herein. As used herein, with reference to a polynucleotide the term "substantially identical" means the substitution of one or a few (for example 2, 3, or 4) nucleotides whilst maintaining at least one activity of the native protein encoded by the polynucleotide. In addition, this term includes the addition or deletion of nucleotides which results in the increase or decrease in size of the encoded protein by one or a few (for example 2, 3, or 4) amino acids whilst maintaining at least one activity of the protein encoded by the polynucleotide. In particular, this includes polynucleotides which encode the same polypeptide or amino acid sequence but which vary in nucleotide sequence by redundancy of the genetic code. Vectors, Host Cells and Expression Systems

[0171] The present disclosure includes use of vectors for manipulation or transfer of genetic constructs. By “vector” or "chimeric vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably is double-stranded DNA and contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or capable of integration into the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a cell, is integrated into the genome of the recipient cell and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene or other gene that can be used for selection of suitable transformants. Examples of such genes are well known to those of skill in the art.

[0172] The vector(s) may be a viral vector. Such a viral vector may be an adenovirus (of a human serotype such as AdHu5, a simian serotype such as ChAd63, ChAdOXl or ChAdOX2, or another form), an adeno-associated virus (AAV), or poxvirus vector (such as a modified vaccinia Ankara (MV A)). ChAdOXl and ChAdOX2 are disclosed in WO2012 / 172277. ChAdOX2 is a BAC-derived and E4 modified AdC68-based viral vector. Preferably said viral vector is an AAV vector.

[0173] Viral vectors are usually non-replicating or replication impaired vectors, which means that the viral vector cannot replicate to any significant extent in normal cells (e.g. normal human cells), as measured by conventional means - e.g. via measuring DNA synthesis and / or viral titre. Non-replicating or replication impaired vectors may have become so naturally (i.e. they have been isolated as such from nature) or artificially (e.g. by breeding in vitro or by genetic manipulation). There will generally be at least one celltype in which the replication-impaired viral vector can be grown - for example, modified vaccinia Ankara (MV A) can be grown in CEF cells. In one embodiment, the vector is selected from a human or simian adenovirus or a poxvirus vector.

[0174] Typically, the viral vector is incapable of causing a significant infection in an animal subject, typically in a mammalian subject such as a human or other primate.

[0175] The vector(s) may be a DNA vector, such as a DNA plasmid. The DNA vector(s) is typically capable of expression in a mammalian cell expression system, such as an immunised cell. The vector may be suitable for expression in a bacterial and / or insect host cell or expression system, such as any of those exemplified herein. A non-limiting example of a suitable expression vector is a pET15b vector, which may be optionally modified to encode an N-terminal tag, such as a hexa-histidine tag and / or a protease cleavage site, such as a TEV protease cleavage site.

[0176] The vector(s) may be an RNA vector, such as a self-amplifying RNA vaccine The vector(s) may be a phage vector, such as an AAV / phage hybrid vector, synthetic antigen

[0177] The synthetic antigen of the disclosure may include a leader sequence, for example to assist in recombinant production and / or secretion. Any suitable leader sequence may be used, including conventional leader sequences known in the art. Suitable leader sequences include Bip leader sequences, which are commonly used in the art to aid secretion from insect cells and human tissue plasminogen activator leader sequence (tPA), which is routinely used in viral and DNA based vaccines and for protein vaccines to aid secretion from mammalian cell expression platforms. By way of a nonlimiting example, a synthetic antigen of the disclosure may include the secretory signal from bovine tissue plasminogen activator, or may include another signal to direct the subcellular trafficking of the epitopes / antigens and fusion proteins. Alternatively, the synthetic antigen of the disclosure may be the mature form in which the N-terminal signal peptide has been removed.

[0178] In an embodiment, a polynucleotide of the disclosure is operably linked to a promoter. Operably linking a promoter or enhancer element to a transcribable polynucleotide means placing the transcribable polynucleotide (e.g., protein-encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription of that polynucleotide. In the construction of heterologous promoter / structural gene combinations, it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide which is approximately the same as the distance between that promoter and the protein coding region it controls in its natural setting; i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer etc) with respect to a transcribable polynucleotide to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived.

[0179] Another embodiment of the present disclosure includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present disclosure, or progeny cells thereof. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, particle bombardment / biolistics, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. In an embodiment, gene editing is used to transform the target cell using, for example, targeting nucleases such as TALEN, Cpfl or Cas9-CRISPR or engineered nucleases derived therefrom.

[0180] . Host cells for expression of Plasmodium antigens are well known in the art. The host cell can be any suitable cell such as a Drosophila melanogaster cell, or a Pichia yeast cell, an Escherichia coli cell or a mammalian cell.

[0181] Synthetic antigens of the disclosure may be expressed using conventional systems. Such a system may be a prokaryotic or a eukaryotic system. Examples of such systems are well-known in the art. Non-limiting examples of suitable host systems include Escherichia coli, Saccharomyces cerevisiae, Pichia pasloris, non-lytic insect cell expression systems such as Schneider 2 (S2) and Schneider 3 (S3) cells from Drosophila melanogaster and Sf9 and Sf21 cells from Spodoptera frugiperda, and mammalian expression systems such as CHO cells and human embryonic kidney (HEK / HEK293) cells.

[0182] Preferably the expression system used is one that does not result in the synthetic antigen being glycosylated.

[0183] Typically using an expression system as defined herein and standard purification techniques known in the art, it is possible to obtain a purified synthetic antigen of the disclosure at a concentration of at least 2 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 11 mg / mL, at least 12 mg / mL, at least 13 mg / mL, at least 14 mg / mL, at least 15 mg / mL, at least 16 mg / mL, at least 17 mg / mL, at least 18 mg / mL, at least 19 mg / mL, at least 20 mg / mL, at least 21 mg / mL, at least 22 mg / mL, at least 23 mg / mL, at least 24 mg / mL, or more, or between about 10 mg / mL to about 25 mg / mL or between about 10 mg / mL to about 30 mg / mL.

[0184] Typically a synthetic antigen of the disclosure will be expressed at least 2 fold, at least 3 fold, at least4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 -fold, at least 9 fold, at least 10 fold, at least 15 fold, or more, or between about 7 fold and about 17 fold, or between about 8 fold and about 16 fold, relative to the expression level of the corresponding unmodified RBP2b synthetic antigen in any given expression system, including each of those individualised herein. In an embodiment, the synthetic antigen of the disclosure will be expressed at least 8 fold relative to the corresponding unmodified RBP2b synthetic antigen in any given expression system, including each of those individualised herein. In an embodiment, the synthetic antigen of the disclosure will be expressed at least 10 fold relative to the corresponding unmodified RBP2b synthetic antigen in any given expression system, including each of those individualised herein. In an embodiment, the synthetic antigen of the disclosure will be expressed at least 16 fold relative to the corresponding unmodified RBP2b synthetic antigen in any given expression system, including each of those individualised herein. In an embodiment, the synthetic antigen of the disclosure will be expressed between about 7 fold and about 17 fold relative to the corresponding unmodified RBP2b synthetic antigen in any given expression system, including each of those individualised herein. In an embodiment, the synthetic antigen of the disclosure will be expressed between about 8 fold and about 16 fold relative to the corresponding unmodified RBP2b antigen in any given expression system, including each of those individualised herein.

[0185] Pharmaceutical Compositions

[0186] The term "vaccine" is herein used interchangeably with the terms "therapeutic / prophylactic composition", "formulation" or "medicament".

[0187] The vaccine of the disclosure (as defined above) can be combined or administered in addition to a pharmaceutically acceptable carrier. Alternatively or in addition the vaccine of the disclosure can further be combined with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and / or antimicrobial compound.

[0188] Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0189] Administration of immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (e.g. vaccines) is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. Accordingly, immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (e.g. vaccines) of the disclosure are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.

[0190] The active immunogenic ingredients (such as a synthetic RBP2b synthetic antigen of the disclosure) are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants which enhance the effectiveness of the vaccine.

[0191] Generally, the carrier is a pharmaceutically-acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate- buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as BSA. In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomers al or sodium azide, to facilitate long term storage.

[0192] Methods of Use

[0193] The synthetic antigen provided herein is useful in a method of determining whether a subject has, or has had, a Plasmodium sp. infection.

[0194] Detection methodologies utilizing antigens are known in the art and include, e.g., radioimmunoassay (RIA), magnetic immunoassay (MIA), immunocytochemical (ICC) assays, immunohistochemical (IHC) assays, immunofluorescent assays, ELISA, EIA, ELISPOT, enzyme multiplied immunoassay, radiobinding assay, Western blotting, immunoprecipitation, dot blots, flow cytometry, real-time immunoquantitative PCR, protein microarrays, and the like. For use as a diagnostic or surveillance tool, it is preferable that a synthetic antigen of the disclosure is used with multiple malaria antigens in a multiplex diagnostic test. This is typically an immunobased assay and could take the form of a bead based (such as the Luminex described above) or lateral flow based. For a bead based test, typically the synthetic antigen is covalently coupled to magnetic microspheres (BioRad Laboratories, USA) harnessing functional groups of the magnetic microspheres such as carboxyl groups activated with N-hydroxysulfosuccinimide (Sigma, USA) and N-(3- Dimethylaminopropyl)-N-ethylcarbodiimide (Sigma, USA).

[0195] Examples of such additional malaria antigens include, but are not limited to, PfAMAl, PfEBA175, PfRHl, PfRH2a, PfRH2b or PfRH4, PfCyRPA, PfRIPR, PfP113 or PfAARP, or a fragment thereof. These could also be synthetic variants or fragments of any or all of these malaria antigens.

[0196] A biological sample is, in various aspects, obtained from a human (or other mammalian subject), for example, by collecting a bodily fluid sample or swabbing a body orifice. The sample may be collected by, e.g., a health care work or self-sampling. The biological sample may be blood, interstitial fluid, plasma, serum, urine, cerebral spinal fluid, sweat, saliva, or other clinically relevant sample, which may be processed (if desired) to remove various components naturally found in the sample other than antibodies. Pre-treatment of the sample can involve filtration, precipitation, dilution, distillation, concentration, inactivation of interfering components, and the addition of reagents. In some embodiments, the sample is an undiluted sample, i.e., the sample is obtained from the biological source and directly tested without any pre-dilution of the sample.

[0197] In an aspect, the present disclosure provides a method of treating a Plasmodium sp. infection in a subject, the method comprising administering an anti- Plasmodium sp. agent to the subject if they have been determined to have a Plasmodium sp. infection using the method of the disclosure.

[0198] In an aspect, the present disclosure provides a method of raising an immune response in a subject, the method comprising administering to the subject one or more of the synthetic antigens of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, or the composition of the disclosure.

[0199] In an aspect, the present disclosure provides a method of treating or preventing a Plasmodium sp. infection in a subject, the method comprising administering to the subject one or more of the synthetic antigens of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, or the composition of the disclosure.

[0200] A method of the disclosure may include the administration, of either the same composition or a different composition, of a different Plasmodium sp. antigen. Examples of such additional malaria antigens include, but are not limited to, PfAMAl, PfEBA175, PfRHl, PfRH2a, PfRH2b or PfRH4, PfCyRPA, PfRIPR, PfP113 or PfAARP, or a synthetic variant or a fragment thereof.

[0201] When administered to a subject (e.g. a mammal such as a human or other primate) that already has malaria, or is showing symptoms associated with Plasmodium parasite infection, a synthetic antigen of the disclosure can cure, delay, reduce the severity of, or ameliorate one or more symptoms, and / or prolong the survival of a subject beyond that expected in the absence of such treatment.

[0202] Alternatively, a synthetic antigen of the disclosure 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.

[0203] Solid Supports and Kits

[0204] The disclosure provides a solid support comprising a synthetic antigen of the disclosure. Any solid support suitable for biological applications is appropriate for use in the context of the disclosure. For example, solid supports include, but are not limited to, a tube, a dish, a flask, a bag, a plate (e.g., a micro-well or microtiter plate), a test strip, a membrane, a filter, a bead (including micro- or nano-particle), a dipstick, a fiber, and the like.

[0205] In exemplary embodiments, the solid support is made of a polymer. In exemplary aspects, the solid support comprises agarose, cellulose, dextran, polyacrylamide, latex, or controlled pore glass. In exemplary aspects, the solid support is composed of polyvinyl difluoride (PVDF), nitrocellulose, nylon 66, protran nitrocellulose, or paper. In exemplary aspects, the solid support comprises a membrane, which is selected from Immobilon®, Protran®, QuickDraw®, Westran®, Whatman® or Hybond® membranes (Sigma-Aldrich, St. Louis, MO). Optionally, the solid support comprises pre-aliquoted amounts of the synthetic antigen. The synthetic antigen may be adhered to the solid support using any suitable method so long as the synthetic antigen retains ability to bind an anti-RBP2b antibody.

[0206] The solid support is useful in a variety of contexts, including in methods of detecting or quantifying an anti-RBP2b antibody in a sample. If desired, the solid support may be configured in a lateral flow assay format and / or in an immunochromatographic assay format. Lateral flow assay systems are known in the art (see, for example, Grant et al., 2016 and Cross et al., 2016).

[0207] An exemplary solid support is arranged as follows. For purposes of illustration, the test strip comprises a porous matrix (e.g., a membrane) which allows movement of a fluid sample along the length of the test strip, the matrix optionally adhered to a more rigid backing to facilitate handling. In the exemplary embodiment, the solid support comprises a sample application zone. The sample application zone is a region of the test strip upon which a sample (e.g., a biological sample or other type of sample in which an anti-RBP2b antibody may be present) is applied. Adjacent to the sample application zone is a test zone adjacent. The test zone comprises the synthetic antigen described herein. The synthetic antigen is immobilized on the solid support in the test zone in such a matter that the synthetic antigens bind their target when exposed to the sample, thereby creating a zone with antibody-antigen complexes when anti-RBP2b antibodies are present in the sample. Finally, the solid support optionally comprises a wick, which facilitates movement of the sample (or components thereof) into or along the test strip.

[0208] Merely to illustrate the operation of this exemplary embodiment, a sample is applied to the sample application zone, which is optionally impregnated with buffer or surfactant or other reagent which promotes maintenance of anti-RBP2b antibodies or distribution of the sample along the solid support. The sample migrates to the test zone which comprises the synthetic antigen Anti-RBP2b antibodies in the sample bind the synthetic antigen . Binding of the anti-RBP2b antibodies to the synthetic antigen (the complex) is identified by detecting binding of the anti-human IgG secondary antibody conjugated to a suitable marker such as colloidal gold.

[0209] The lateral flow device may be partnered with a reader system to enable quantification and inclusion of machine-learning algorithms.

[0210] Also provided herein are kits comprising any one or more of the synthetic antigens described herein, optionally adhered to a solid support. Kits generally comprise one or more synthetic antigens of the disclosure in or on a suitable container or support alongside instructions for use, and optionally further comprising reagents for use in connection with the synthetic antigens.

[0211] EXAMPLES

[0212] 1 - Materials and Methods

[0213] Design

[0214] The procedure for designing more stable and expressible variants of PvRBP2b followed the PROSS design strategy except the following steps were observed:

[0215] 1) The parental PvRBP2b crystallographic structures were analysed to identify regions that interact with human antibodies. Surfaces involved in such interactions were disabled from design. 2) PROSS starts by generating a multiple sequence alignment (MSA) of homologs (>= 35% sequence identity) of the target protein (PvRBP2b) and generating a positionspecific scoring matrix (PSSM) that serves as a statistical model for mutation at each position of the target. Typically, dozens of unique homologs are available for this analysis.

[0216] In this disclosure the inventors sought to design stabilized synthetic variants of PvRBP2b, under stringent conditions of minimally perturbing the solvent-accessible surfaces to maintain its antigenicity profile. Furthermore, for some of the designs, due to limited diversity of natural PvRBP2b homologs, another Al-based design method, called ProteinMPNN2 (Dauparas et al., 2022) was thus used to generate a statistical model of mutations at each position of PvRBP2b. Briefly, starting from the PvRBP2b structure (PDB entry: 5W53), backbone coordinates were used as inputs to initialise ProteinMPNN, allowing the network to build a backbone-specific mutation model. Log probabilities describing the likelihood of an amino acid at a given position were extracted from the model. Probabilities were averaged over 50 initialisations of MPNN and rounded to the closest integer. The resulting matrix of dimension (Lx 20) with L the length of the protein of interest was treated exactly as a PSSM is treated in the PROSS workflow.

[0217] 3) The structure-based energy filtering of mutations and design steps were performed exactly as in PROSS. Three variants were analysed visually, and point mutations deemed risky for stability and folding were eliminated. The final preferred variants comprised 17, 19, and 26, mutations relative to wild type PvRBP2b (see Tables 1 and 2).

[0218] Other considerations include introducing mutations in solvent-exposed positions because those are less likely to compromise protein foldability. In the case of PvRBP2b, structural analyses indicated that >48% of the solvent-accessible surface is targeted by naturally acquired human antibodies from previously infected individuals..

[0219] Table 1. Point mutations following design (and following expression). Table 2. Amino acid changes compared to Wild Type (WT).

[0220] Arrows refer to either an increase in the change or a decrease in the type of AA from the WT

[0221] PvRBP2b 169-470 and synthetic variants cloning and sequencing

[0222] The sequence of PvRBP2b from P. vivctx strain Salvador I was obtained from PlasmoDB Database (www.plasmodb.org; accession number: PVX 094255, 2,806 amino acids). Synthetic DNA was codon-optimized for expression in E. coli (Life Technologies). The nucleotide sequence encoding amino acids 169 to 470 of PvRBP2b was cloned into pPROEX HTb vector which included sequences for a N-terminal 6xHis- tag followed by a TEV cleavage site. This sequence refers to the “wildtype” PvRBP2bt69- 470. Restriction enzyme cloning was used to clone the synthetic DNA fragments (obtained from Twist Biosciences) of PvRBP2bi69-47o variants into pPROEX HTb vector.

[0223] Expression and purification of PvRBP2bi69-47o synthetic variants

[0224] Parental PvRBP2bi69-47o was expressed using E. coli strain SHuffle® T7 (New England Biolabs) and Terrific Broth (TB) supplemented with 100 pg / mL of carbenicillin. Flasks containing 1 L of medium were incubated in a Multitron shaker (Infers HT) at 37 °C at 180 rpm. At ODeoo of around 1.0, IPTG (Astral) was added to the final concentration of 1.0 mM and protein expression was allowed to continue for 20 hours at 16 °C. Cells were harvested by centrifugation at 6,000 x g, resuspended in freezing buffer containing 50 mM Tris HC1 pH 7.5, 500 mM NaCl, 10 % (v / v) glycerol supplemented with complete EDTA-free protease inhibitor cocktail (Roche) and stored at -80 °C until further processing.

[0225] For the purification, cell pellet was thawed on ice and resuspended in the freezing buffer supplemented with 0.5 mg / mL of DNase and 1.0 mg / mL of lysozyme (Sigma- Aldrich). Cells were lysed using sonicator Sonopuls UW 3200 (Bandelin) equipped with VS 70 T probe. The obtained crude cell extract was clarified by centrifugation at 30,000 x g for 45 minutes at 4°C. The supernatant was loaded onto the 5 mL HisTrap excel column (GE Healthcare) pre-equilibrated with the freezing buffer. Unbound material was removed using 10 column volumes of wash buffer: 20 mM Tris HC1 pH 7.5, 500 mM NaCl and 10 mM imidazole. The bound protein was eluted from the column using the same buffer but containing 300 mM imidazole. Eluted fractions were pooled and dialyzed overnight into the dialysis buffer containing 20 mM Tris pH 7.5 and 100 mM NaCl. The resulting protein sample was applied on the 5 mL HiTrap SP HP cation exchange chromatography column (GE Healthcare) pre-equilibrated with the dialysis buffer. Unbound material was removed using 10 column volumes of the buffer. Protein was eluted from the column using a gradient of 20 mM Tris pH 7.5 and 1.0 M NaCl.

[0226] Collected fractions were analyzed on SDS PAGE and fractions of interest were concentrated using an Amicon Ultra-4 lO kDa molecular weight cut-off concentrator (Millipore) and loaded onto S75 Superdex 16 / 600 size exclusion column (GE Healthcare) pre-equilibrated with 20 mM HEPES pH 7.5 and 150 mM NaCl. The monodisperse peak fractions containing protein were pooled and concentrated using the same concentrator, flash-frozen in liquid nitrogen and stored at -80°C. Expression and purification of WHT2482, WHT2483 and WHT2484 synthetic antigen variants were performed in a similar manner as described above.

[0227] Thermal stability assays

[0228] Parental PvRBP2bi 69-470 and synthetic variants were diluted to 1 mg / mL and transferred to an Aurora 384 well plate for dynamic light scattering measurements in the DynaPro plate reader III (Wyatt Technology). All samples were centrifuged at 17,000 x g for 5 min at 4 °C to sediment any precipitate. The plate was sealed, and measurements were performed over a temperature ramp of 25 to 80 °C at a ramp rate of 0.1 °C / min, with 4 s acquisition time averaged over 5 acquisitions. The onset model was fit to the data to obtain TOnset values for all constructs. TonSet values are the temperature at which the protein begins to unfold. Data from at least two different batches of protein in triplicate were evaluated using Dynamics software v8.0.0.89. Thermal shift assays were performed using the Tycho NT.6 (NanoTemper Technologies). Parental PvRBP2bi69-47o and variants were measured at 10 pM in storage buffer. 10 uL of each sample was transferred into a capillary and measured from 35 to 95 °C using a Tycho NT.6 (Nanotemper). The inflection temperatures of each protein were calculated by the Tycho NT.6 software (1.2.0.750). Technical triplicates were measured in three independent experiments. Data were analysed using GraphPad Prism.

[0229] The inflection temperatures of each protein were calculated by the Tycho NT.6 software (1.2.0.750). Technical triplicates were measured in three independent experiments. Data were analysed using GraphPad Prism.

[0230] Antibody expression and purification

[0231] Recombinant human monoclonal antibodies (mAbs) were expressed in Expi293 HEK cells (Life Technologies) maintained in suspension at 37 °C and 8% CO2. Cells were transfected at a density of 3 * 106with equal amounts of heavy and light-chain paired plasmids using polyethyleneimine (PEI, Sigma- Aldrich) at a ratio of 1 :03 of the total amount of plasmid to PEI.

[0232] One day after transfection, valproic acid was added to cultures to a final concentration of 0.025 M.

[0233] Seven days after transfection, the supernatant was collected by centrifugation and filtered through a 0.22 pm filter. Human mAbs were purified by loading the supernatant onto a 1 mL Protein A HP HiTrap column (GE Healthcare). Columns were equilibrated and washed using Dulbecco’s phosphate-buffered saline (DPBS). Human mAbs were eluted using 0.1 mM citric acid pH 3.00 and neutralized with 1 M Tris-HCl pH 9.0. A second purification step was performed by loading Protein A eluate on a Hiload 16 / 600 Superdex 200 pg gel filtration column (GE Healthcare), which was pre-equilibrated with DPBS. Human mAbs were concentrated using Amicon Ultra-04 5 kDa (Millipore). Antibody concentration was determined by absorbance measurement at 280 nm using a Nanodrop and purity was determined using SDS-PAGE.

[0234] Bio-layer interferometry

[0235] Antibody affinities were measured using an Octet RED96 instrument. Assays were performed at 25 °C in solid black 96-well plates agitated at 1000 rpm. The kinetic buffer was composed of PBS 0.1% BSA, 0.05% TWEEN. A 60 s biosensor baseline step was applied before human mAbs were loaded onto anti-human IgG Fc capture sensor tips (AHC) by submerging sensor tips in 5 pg / mL human mAb until a response of 0.5 nm then washed in a kinetic buffer for 60 s. Association measurements were performed using a two-fold concentration gradient of PvRBP2bi69-47o and synthetic variants from 0.63-10 nM for 200 s and dissociation was measured in a kinetic buffer for 300 s. Sensor tips were regenerated using a cycle of 5 s in 100 mM glycine pH 1.5 and 5 s in kinetic buffer repeated five times. Baseline drift was corrected by subtracting the average shift of a human mAb loaded sensor not incubated with PvRBP2b 169-470 and synthetic variants, and an unloaded sensor incubated with PvRBP2bi69-47o and synthetic variants. Curve fitting analysis was performed with Octet Data Analysis 10.0 software using a global fit 1 : 1 model to determine KD values and kinetic parameters. Curves that could not be reliably fitted were excluded from further analysis.

[0236] Crystallography methods

[0237] Crystallization trials were undertaken at 20 °C using 96 well sitting drop vapour diffusion plates (Greiner). Crystals were obtained for WHT2483 from a solution containing 5 % (v / v) MPD, 10 % (w / v) PEG 6000 and 0.1 M HEPES pH 7.5 at 10 mg / mL. Crystals were obtained for WHT2484 using 10 % (v / v) Propan-2-ol, 10 % (w / v) PEG MME 5000, 0.1 M Na Cacodylate pH 6 at 15 mg / mL. Crystals were frozen in liquid nitrogen at 100 K following cryoprotection with 15 to 20 % glycerol in reservoir solution. Datasets were collected to 2.4 A (WHT2483) and 1.9 A (WHT2484) using the MX2 beamline at the Australian Synchrotron (Melbourne, Victoria). Data were recorded using an Eiger 16M detector (Dectris) and processed using the XDS package (Kabsch, et al 2010)). Molecular replacement was undertaken using Phaser (McCoy et al., 2007). A search model was generated using AlphaFold2 models for WHT2483 and WHT2484 respectively (Jumper et al 2021).

[0238] The WHT2483 structure was solved in space group P2i2i2i with two copies in the asymmetric unit while WHT2484 was solved in space group P4i2i2 and had a single copy in the asymmetric unit. Refinement through iterative rounds of model building in COOT (52) and refinement in Phenix vl.19.2 generated models with an RObs / Rfree = 19.52 / 24.53 for WHT2483 and 18.88 / 22.17 for WHT2484 (Adams, et al 2010). Structural models were deposited in the PDB under PDB ID 9DZC and 9DZD respectively. Figures were prepared using the PyMOL Molecular Graphics System (The PyMOL Molecular Graphics System, Version 3.0 Schrodinger, LLC). Antibody measurements

[0239] IgG levels were measured using a multiplexed bead-based assay. Briefly, 2.5 x io6COOH magnetic microspheres (also called beads) (Luminex Corp) were incubated for 20 min at room temperature in 100 mM monobasic sodium phosphate (pH 6.2); 50 mg / mL sulfo-NHS (A-hydroxysulfosuccinimide sodium salt) was added to convert the protein’s carboxyl groups to amine-reactive NHS esters and 50 mg / mL of EDC [A-(3-dimethylaminopropyl)-7V'-ethylcarbodiimide hydrochloride] used to crosslink P. vivax proteins to the microspheres, with incubation at 4 °C overnight.

[0240] 50 pL of protein-conjugated microspheres in buffer were added to a 96-well black plate (500 microspheres per well) and incubated with 50 pL of test plasma at a 1 / 100 dilution for 30 min at room temperature on a plate shaker. All dilutions were made in phosphate buffered saline containing 1% bovine serum albumin and 0.05% (v / v) Tween- 20 (denoted as PBT), and all samples were run singularly. Following the incubation, the plate was washed three times with 100 pl of PBT using an automatic plate washer. The washed microspheres were incubated for 15 min with 1 / 100 detector antibody, PE- conjugated anti-human IgG Fc (1 mg / mL, Jackson ImmunoResearch), at room temperature on a plate shaker. The microspheres were then washed and assayed on a MAGPIX instrument and the results expressed as the median fluorescent intensity (MFI). MFI measurements were converted to arbitrary relative antibody units (RAU) using a 2- fold 10-point serial dilution of a positive control plasma pool, by running a 5-paramater logistic function as previously described (Mazhari PLoS One 2020).

[0241] Example 2 - Protein expression.

[0242] Wildtype PvRBP2bi69-47o and nine synthetic variants of the present disclosure were expressed in E. coli with Ni-NTA affinity chromatography as the first purification step. SDS-Page showed expression of each of the synthetic variants, as evidence by a band corresponding to the expected molecular weight of the antigens.

[0243] SDS-PAGE analyses also showed that three PvRBP2bi69-47o synthetic variants (WHT2482, WHT2483 and WHT2484) had increased yields (stronger band on the SDS PAGE gel) relative to the other synthetic variants (Figure 1 A) and compared to Wildtype PvRBP2b 169-470 (WT). For these three variants, two additional purification steps using cation exchange and size exclusion chromatography were then adopted. After the three- step purification process, WHT2482, WHT2483 and WHT2484 showed increased protein yields up to 8, 16 and 10-fold respectively compared to wildtype PvRBP2bi69-47o from a 4L expression (Figure IB). Example 3 - Thermostability

[0244] The three synthetic variants with increased expression levels were also assessed for their thermal stability. This was done by dynamic light scattering (DLS). Dynamic light scattering measurements showed that the three synthetic variants / antigens displayed significantly higher thermal stability with TonSet values of 54 to 59 °C compared to 45 °C for the parental protein (Table 3), translating to an improvement of 9 to 14 °C. thermal stability measurements were also conducted using a label-free differential scanning fluorimeter and it is observed that the synthetic variants / antigens showed higher thermal stability as described by the inflection temperature (T0 by 8 to 10 °C compared to parental PvRBP2bi69-47o (Table 4).

[0245] Table 3. DLS measurements of parental and three synthetic variants / antigens showing the hydrodynamic radius (Rh) and unfolding onset temperatures (TOnset) from two independent replicates.

[0246] Table 4. Inflection temperatures (T0 of parental PvRBP2bi69-47o and the synthetic variants using label free thermal shift analysis (Tycho NT.6, Nanotemper).

[0247] Til and Ti2 refer to two different inflection points upon unfolding of the protein, as it is possible the protein unfolds in a bimodal fashion. Example 4 - Affinity

[0248] Bio-layer interferometry was used to determine the binding kinetics and affinities of the interaction between human monoclonal antibodies (mAbs) (Chan et al., 2021) and the three synthetic variants that had higher expression yields and greater stability compared to WT, namely, WHT2482, WHT2483 and WHT2484 (Table 5). The panel of nine human mAbs bind each of these synthetic antigens, WHT2482, WHT2483 and WHT2484, with high affinity in the low nanomolar range. While the affinities were not uniformly as high compared to wildtype PvRBP2bi69-47o, it was clear that the three synthetic variants retained human antibody recognition across the panel.

[0249] Table 5. Affinity results Example 5 - Crystallography

[0250] The crystal structures of WHT2483 and WHT2484 were determined to 2.4 A and 1.9 A resolution (Figure 2A and B). An overlay of the structural coordinates of PvRBP2b 169-470 (PDB ID 5W53, Gruszczyk, et al., 2018) with WHT2483 and WHT2484 show very similar overall architectures with RMSD of 0.750 A and 0.879 A respectively. The greatest divergence in the structural backbones occurs at the C-terminal portions of the a5 and a7 helices with up to 1.7 A difference in position at His 464 in the a7 helix (Figure 2 A, B).

[0251] Comparison of the binding footprints of the human antibody panel with the mutations introduced in WHT2483 and WHT2484 reveals that the binding surfaces are largely conserved, with few of the introduced mutations being within the antibody bound surface of parental PvRBP2bi69-47o (Figure 2C). There are three introduced mutations which fall within the antibody binding footprints, these are Lys248Leu and Met324Phe which are present in WHT2484, as well as Gln378Leu which is present in both WHT2483 and WHT2484 (Figure 2C). These mutations fall within the binding footprints of human mAbs 237235, 283284 and 241242 respectively. Despite the presence of these mutations, these human mAbs still bound with nanomolar affinities to the stabilized variants (Table 3). Recognition may be retained for Lys248 and Met324 as the contacts are solely mediated by Van-Der-Waals interactions with the sidechains, which could either be retained by the residue swap or are long range (> 5.0 A). The interaction of 241242 with Gln378 is mediated through the peptide backbone which would remain unchanged in the stabilized designs.

[0252]

[0253] PvRBP2b can be produced in recombinant microbial systems resulting in a well- folded and functional protein. Nevertheless, its expression yields and stability are low, limiting its development as a serological marker for diagnostic tests, particularly for use in malaria endemic countries, which are the main targets for elimination efforts. The preferred product characteristics for a diagnostic test for risk of P. vivax relapse, call for a test shelf-life of >18 months at >35°C and 90% relative humidity. Several computational and experimental approaches have been developed to address marginal protein stability and low expressibility. Typically, stabilizing, mutations are introduced in solvent-exposed positions because those are less likely to compromise protein foldability. In the case of PvRBP2b, however, structural analyses indicated that >48% of the solvent-accessible surface is targeted by naturally acquired human antibodies from previously infected individuals. To serve as a useful serological marker, the designed, synthetic antigen must exhibit nearly the same antigenic profile as parental PvRBP2b, dictating that much of the surface should be free of mutations, and that the natural protein backbone conformation must be carefully maintained. Therefore, the design strategy sought to mostly introduce core mutations without, however, perturbing the backbone structure. Introducing many stabilizing core mutations while mostly maintaining the protein surface demands very high accuracy in design.

[0254] Three of the synthetic antigen variants of this disclosure, which had been assessed as being highly stable and with good expression levels compared to WT, were tested in a diagnostic device in combination with several other P. vivax antigens to assess their improved specificity and selectivity compared to WT PvRBP2bi69-47o. The additional different malaria antigens (from P. vivax protein constructs) were as follows: EBP (KMZ83376.1), sl6 (PVX_000930), Pv-fam-a (PVX_096995), MSP5 (PVX_003770), RBP2b.P25 (PVX_094255), MSP1-19 (PVX_099980), RBP2a.P9 (PVX_121920), MSP8 (PVX_097625) and PTEX150 (PVX_084720), wherein the 6 / 7 digit numbers refer to PlasmoDB gene ID (plasmodb.org) except in the case of EBP wherein the code refers to an accession number in ENA (https: / / www.ebi.ac.uk / ena / browser / home). These proteins mentioned above have been previously described in WO2018130871.

[0255] Proteins were expressed and purified using hexa-histidine tags using mammalian (HEK293E), Escherichia coli or insect cell expression systems. Cohort samples

[0256] Malaria-endemic samples were utilised from three existing year-long longitudinal cohorts as previously described (Longley et al., 2020). Briefly, yearlong cohort studies were conducted in Thailand (Kanchanaburi and Ratchaburi provinces), Brazil (Manaus) and the Solomon Islands (Ngella) across 2013-2014. Each site enrolled between 928 and 1,274 individuals with blood samples taken every month for qPCR detection of P. vivax infections and plasma stored for antibody measurements. This enabled total IgG antibody responses measured at the final visit of the yearlong cohorts to be related to the time since prior P. vivax infection. IgG antibody levels against WT RBP2b and the variants were highly correlated (r=0.97, p<2.2e-16).

[0257] The inventors also utilised four panels of malaria-naive negative control plasma samples as previously described (Longley et al., 2020). Briefly, this included 102 individuals from the Volunteer Blood Donor Registry in Melbourne, Australia; 100 individuals from the Australian Red Cross, Melbourne, Australia; 72 individuals from the Thai Red Cross, Bangkok, Thailand; and 96 individuals from the Rio de Janeiro State Blood Bank, Rio de Janeiro, Brazil.

[0258] Classification of recent P. vivax infection

[0259] IgG antibody levels measured in the endemic country samples and controls were used to train machine-learning algorithms to classify individuals as recently exposed within the prior 9-months or not, with the truth being the PCR-detected P. vivax bloodstage infections from the monthly sampling during the prior year of the cohort. The ability to correctly classify recent exposure is presented as receiver operating characteristic (ROC) curves (Figure 3), that show the sensitivity and specificity of the classification. In this case we have used a Random Forest classified. The classification utilises the IgG antibody data from a panel of 8 antigens, that includes either RBP2b P25 as the reference, or replaces this with P87 and each of the synthetic variants. To demonstrate the important of including a RBP2b in the classification algorithm, a top 8 panel excluding any version of RBP2b is also shown.

[0260] A summary of this can be measured as the area under the curve (AUC) and is used in Table 6 to compare the different antigen combinations in a quantitative manner. Table 6. Diagnostic results. Classification performance, as measured by AUC, when utilizing IgG antibody responses to different combinations of 8 malaria relevant antigens. The reference is using RBP2b P25 as in the Longley et al. (2020). RBP2b P25 is then swapped for P87 WT or one of the synthetic antigens, and a combination with no RBP2b is also included.

[0261] For a good diagnostic the AUC is preferably >0.8. These results show that the new stabilized, synthetic antigens performed above that cut off and were not much less compared to the WT fragment (RBP2b P87).

[0262] In summary, the current data shows the results from testing with three of the designed synthetic c antigens bearing core mutations relative to PvRBP2b. Each expressed up to 11 mg / L, and had 14°C higher thermal tolerance than the parental protein. The synthetic antigens retained binding to naturally acquired human monoclonal antibodies with nanomolar affinities, suggesting that the immunologically competent surfaces were retained as was confirmed by crystallographic analyses. Using longitudinal observational cohorts from malaria endemic regions of Thailand, Brazil and the Solomon Islands, diagnostic tests showed that antibody responses against the antigens are highly correlated with those against the parental protein and can classify individuals as recently infected with P. vivax.

[0263] Testing of the other variants of the current disclosure would involve the routine steps herein described. These steps would involve cloning the nucleotide sequences of SEQ ID 21 to SEQ ID 29 encoding amino acids SEQ ID NO 6 to SEQ ID 11 respectively, into a vector. The vector is then added to the to an E. coli strain which subsequently expresses the required protein. Cells can then be harvested and the antigens purified by affinity chromatography. Measurement of activity can be performed by Bio-layer interferometry, whilst thermal stability can be measured by DLS.

[0264] Example 7- Expression in mammalian cells.

[0265] Transfection of 30 mL Expi293 cell culture was performed essentially as described in the Expi293 Expression System User Guide (Thermo Fisher Scientific) with the addition of 0.5% v / v lupin peptone (Cell Biosciences) and IX Antibiotic- Antimycotic (Thermo Fisher Scientific) to the cell culture. Cell culture medium containing the secreted protein was harvested 6 days after transfection by centrifugation, filtered and kept at 4°C until use.

[0266] Media containing the secreted protein was batch bound with 1 mL Complete His-tag purification resin. Following incubation, the resin was washed with wash buffer (DPBS, 5 mM Imidazole). Bound protein was then eluted with elution buffer (DPBS, 500 mM Imidazole) from resin. Elution fractions were pooled and concentrated using Amicon Ultracentrifugal filters (10,000 Da MWCO, Millipore Sigma). This concentrated protein was further purified by size exclusion chromatography (SEC) using a HiLoad 16 / 600 Superdex 200 pg column), with DPBS as its running buffer. The SEC profile of WHT2483 produced a single peak at ~75 mL identified as the protein of interest by SDS-PAGE and confirmed the expected molecular weight of ~37 kDa. The total yield of protein was 12.6 mg at a concentration of 0.92 mg / mL. The total yield of protein was 12.6 mg at a concentration of 0.92 mg / mL.

[0267] To assess the potential of the synthetic antigens of the disclosure to be used as vaccines the following experiments could be undertaken.

[0268] The protein mixture is mixed with Freund's adjuvant (FA) before injection into a rabbit. A typical three dose schedule could be carried out, followed with one immunization in complete FA followed at 28-day intervals with two immunization in incomplete FA. To assess whether protective antibodies can be raised against any the proteins of this disclosure, serum is obtained from blood of the rabbits taken two weeks after the third immunization and the antibody is purified by protein A sepharose chromatography. To assess if the antibodies inhibit the growth of P. vivax, the parasites can be incubated with serially diluted antibody in culture wells. The parasitaemia can be observed after 96 hours (2 invasion cycles). The growth inhibition single cycle assay can be performed according to methods described in Malkin et al. (2005). Alternative experiments to assess the protective effects of any of the proteins / antigens can be found in WO2022169790.

[0269] Essentially mice are infected with Plasmodium sporozoites and infection are allowed to grow. Mice are then administered certain doses of antigen. Some mice are then challenged three months later with bites from 10 mosquitoes infected with PbmCherryLuci sporozoites. A reduction in infection upon challenge, demonstrating partial immunity, can then be observed since all mice will ultimately develop malaria disease. The mice can be infected again 6 weeks later with 20,000 sporozoites and immunised with the antigen that cures them of this second infection. This second infection and treatment is considered a secondary ‘boost’ immunization, and when these mice are challenged 3 months later with the bites from infected mosquitos they could show signs of completely protection from infection.

[0270] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0271] All publications discussed and / or referenced herein are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0272] REFERENCES

[0273] Adams et al. (2010) Acta Crystallogr D Biol Crystallogr 66, 213-221.

[0274] Brune et al. (2016) Sci. Rep. 19: 19234.

[0275] Chan et al. (2021) Nat Commun 12: 1538.

[0276] Cross et al. (2016) J Infect Dis 214: S210-S217.

[0277] Cutts et al. (2014) BMC Med 12: 150.

[0278] Dauparas et al (2022) Science 378:49-56.

[0279] Galinski et al. (1992) Cell 69: 1213-1226.

[0280] Geall et al. (2012) Proc Natl Acad Sci USA 109: 14604-9.

[0281] Goodman and Draper (2010) Ann Trop Med Parasitol 104: 189.

[0282] Grant et al. (2016) Vaccine 34: 5656-5663.

[0283] Gruszczyk et al. (2018), Nature, 559,135-139

[0284] Gruszczyk et al. (2018) Science, 359,6371, 48-55

[0285] Hajitou et al. (2006) Cell 125:385-398.

[0286] Kabsch. (2010) Acta Crystallogr D Biol Crystallogr 66, 125-132.

[0287] Jumper et al. (2021) Nature 596, 583-589Longley et al. (2020) Nat Med 26:741-9.

[0288] Malkin et al . (2005) Vaccine 23:3131-3138.

[0289] Mazhari et al. (2020) PLoS One 15, e0238010 (2020).

[0290] McCoy et al. (2007) J Appl Crystallogr 40, 658-674

[0291] Needleman and Wunsch (1970) J. Mol Biol. 45:443-453.

[0292] Robison et al. (2015) PLoS Med 27:el001891.

[0293] The Lancet (2024) Lancet 403 : 10426.

Claims

CLAIMS1. A synthetic) antigen which comprises at least one amino acid substitution when compared to a wild type P. vivax reticulocyte binding protein 2b (PvRBP2b) having a sequence of amino acids provided as SEQ ID NO: 1, wherein the at least one amino acid substitution is at a position corresponding to the following amino acid number of SEQ ID NO: 1 : 243, 247, 248, 264, 269, 270, 276, 278, 280, 283, 291, 305, 309, 313, 317, 319, 322, 324, 337, 339, 347, 352, 354, 356, 357, 361, 367, 369, 371, 372, 377, 378, 382, 393, 398, 402, 404, 405, 406, 407, 409, 413, 417, 418, 428, 431, 436, 439, 444, 459, 463 or 467.

2. The synthetic antigen of claim 1 which has a substitution at one or more or all of position(s): 243, 247, 248, 270, 309, 322, 324, 337, 347, 354, 357, 361, 367, 369, 371, 372, 377, 378, 398, 402, 405, 406, 418, 431, 436, or 439.

3. The synthetic antigen of claim 1 or claim 2 which has a substitution at one or more or all of position(s): 243, 247, 322, 337, 347, 354, 357, 367, 377, 378, 398, 406, 418, 431 or 436.

4. The synthetic antigen according to any one of claims 1 to 3 which has a substitution at all of position(s): 243, 247, 270, 322, 337, 347, 354, 357, 367, 369, 371,377, 378, 398, 406, 418, 431, 436 and 439.

5. The synthetic antigen according to any one of claims 1 to 4 which is at least 80% identical to the amino acid sequence provided as SEQ ID NO:3.

6. The synthetic antigen according to any one of claims 1 to 5 which comprises or consists of the amino acid sequence provided as SEQ ID NO:3.

7. The synthetic antigen according to any one of claims 1 to 3 which has a substitution at all of position(s): 243, 247, 270, 322, 337, 347, 354, 357, 361, 367, 377,378, 398, 406, 418, 431 and 436.

8. The synthetic antigen according to any one of claims 1 to 3 or 7 which is at least 80% identical to the amino acid sequence provided as SEQ ID NO:4.

9. The synthetic antigen according to any one of claims 1 to 3, 7 or 8 which comprises or consists of the amino acid sequence provided as SEQ ID NO:4.

10. The synthetic antigen according to any one of claims 1 to 3 which has a substitution at all of position(s): 243, 247, 248, 270, 309, 322, 324, 337, 347, 354, 357, 361, 367, 369, 371, 372, 377, 378, 398, 402, 405, 406, 418, 431, 436 and 439.

11. The synthetic antigen according to any one of claims 1 to 3 or 10 which is at least 80% identical to the amino acid sequence provided as SEQ ID NO: 5.

12. The synthetic antigen according to any one of claims 1 to 3, 10 or 11 which comprises or consists of the amino acid sequence provided as SEQ ID NO:5.

13. The synthetic antigen according to any one of claims 1 to 12, wherein one or more of the following apply: i) the amino acid at position 243 is leucine, ii) the amino acid at position 247 is leucine, iii) the amino acid at position 248 is leucine, iv) the amino acid at position 264 is asparagine, v) the amino acid at position 269 is glutamic acid, vi) the amino acid at position 270 is leucine, vii) the amino acid at position 276 is threonine, viii) the amino acid at position 278 is leucine, ix) the amino acid at position 280 is glutamic acid, x) the amino acid at position 283 is asparagine, xi) the amino acid at position 291 is glutamic acid, xii) the amino acid at position 305 is lysine, xiii) the amino acid at position 309 is arginine, xiv) the amino acid at position 313 is threonine, xv) the amino acid at position 317 is serine, xvi) the amino acid at position 319 is lysine, xvii) the amino acid at position 322 is tryptophan, xviii) the amino acid at position 324 is phenylalanine, xix) the amino acid at position 337 is leucine, xx) the amino acid at position 339 is aspartic acid, xxi) the amino acid at position 347 is phenylalanine,xxii) the amino acid at position 352 is asparagine, xxiii) the amino acid at position 354 is tyrosine or phenylalanine, xxiv) the amino acid at position 356 is asparagine, xxv) the amino acid at position 357 is leucine, xxvi) the amino acid at position 361 is tyrosine, xxvii) the amino acid at position 367 is aspartic acid, xxviii) the amino acid at position 369 is isoleucine or aspartic acid, xxix) the amino acid at position 371 is leucine, xxx) the amino acid at position 372 is glutamine, xxxi) the amino acid at position 377 is valine, xxxii) the amino acid at position 378 is leucine, xxxiii) the amino acid at position 382 is lysine, xxxiv) the amino acid at position 393 is glutamic acid or arginine, xxxv) the amino acid at position 398 is alanine, xxxvi) the amino acid at position 402 is isoleucine, xxxvii) the amino acid at position 404 is aspartic acid, xxxviii) the amino acid at position 405 is isoleucine, xxxix) the amino acid at position 406 is alanine, xl) the amino acid at position 407 is asparagine, xli) the amino acid at position 409 is glutamic acid, xlii) the amino acid at position 413 is tyrosine, xliii) the amino acid at position 417 is aspartic acid, xliv) the amino acid at position 418 is glutamic acid or threonine, xlv) the amino acid at position 428 is tyrosine, xlvi) the amino acid at position 431 is alanine, xlvii) the amino acid at position 436 is alanine or serine, xlviii) the amino acid at position 439 is glutamine or asparagine, xlix) the amino acid at position 444 is glutamic acid or arginine, 1) the amino acid at position 459 is serine, li) the amino acid at position 463 is serine, or lii) the amino acid at position 467 is lysine, wherein the amino acid position is a position corresponding to the amino acid sequence provided as SEQ ID NO: 1.

14. The synthetic antigen of claim 13, wherein the amino acid at position 354 is tyrosine.

15. The synthetic antigen of claim 13 or claim 14, wherein the amino acid at position 369 is isoleucine.

16. The synthetic antigen according to any one of claims 13 to 15, wherein the amino acid at position 418 is glutamic acid.

17. The synthetic antigen according to any one of claims 13 to 16, wherein the amino acid at position 436 is alanine.

18. The synthetic antigen according to any one of claims 13 to 17, wherein the amino acid at position 439 is glutamine.

19. The synthetic antigen according to any one of claims 1 to 18 which has one or more of a higher expression level in cell culture, higher thermostability or higher affinity to an mh-l’lasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody when compared to an antigen having the amino acid sequence of SEQ ID NO:2.

20. The synthetic antigen according to any one of claims 1 to 18 which has a higher thermostability when compared to an antigen having the amino acid sequence of SEQ ID NO:2.

21. The synthetic antigen according to any one of claims 1 to 20 which is at least 302 amino acids in length.

22. A polynucleotide encoding the synthetic antigen according to any one of claims 1 to 2.

23. A vector comprising the polynucleotide of claim 22.

24. A host cell comprising the polynucleotide of claim 22 and / or the vector of claim 23.

25. A method of producing the synthetic antigen according to any one of claims 1 to 22, the method comprising expressing in a cell or cell free expression system the polynucleotide of claim 22.

26. A method of producing the cell of claim 24, the method comprising the step of introducing the polynucleotide of claim 22 and / or the vector of claim 23, into a cell.

27. A composition comprising one or more of the synthetic antigens according to any one of claims 1 to 22, the polynucleotide of claim 22, the vector of claim 23, or the cell of claim 24, and a suitable carrier.

28. The composition of claim 27 which is a vaccine composition.

29. The composition according to any one of claims 27 to 28 which comprises the antigen according to any one of claims 1 to 22.

30. A solid support comprising the synthetic antigen according to any one of claims 1 to 22.

31. The solid support of claim 30 which is a dipstick, test strip, or micro-well plate.

32. The solid support of claim 31, wherein the test strip is a lateral flow test strip.

33. A diagnostic device comprising the solid support according to any one of claims 30 to 32.

34. A kit comprising one or more of the synthetic antigens according to any one of claims 1 to 22, the polynucleotide of claim 22, the vector of claim 23, the cell of claim 43, the composition according to any one of claims 27 to 29, the solid support according to any one of claims 30 to 32 or the diagnostic device of claim 33.

35. A method of determining whether a subject has, or has had, a Plasmodium sp. infection, the method comprising determining the presence or absence of an antibody which binds the synthetic antigen according to any one of claims 1 to 21 in a sample obtained from the subject, wherein the presence of the antibody is indicative of the subject having, or previously having, a. Plasmodium sp. infection.

36. A method of treating a Plasmodium sp. infection in a subject, the method comprising administering an anti- Plasmodium sp. agent to the subject if they have been determined to have a Plasmodium sp. infection using the method of claim 35.

37. A method of raising an immune response in a subject, the method comprising administering to the subject one or more of the synthetic antigens according to any one of claims 1 to 22, the polynucleotide of claim 22, the vector of claim 23, the cell of claim 24, or the composition according to any one of claims 27 to 29.

38. A method of treating or preventing a Plasmodium sp. infection in a subject, the method comprising administering to the subject one or more of the synthetic antigens according to any one of claims 1 to 22, the polynucleotide of claim 22, the vector of claim 23, the cell of claim 24, or the composition according to any one of claims 27 to 29.

39. The method according to any one of claims 35 to 38, wherein the Plasmodium sp. is Plasmodium vixax.

40. Use of the synthetic antigen according to any one of claims 1 to 22, the polynucleotide of claim 22, the vector of claim 23, the cell of claim 24, or the composition according to any one of claims 27 to 29 in the manufacture of a medicament for raising an immune response.

41. Use of the synthetic antigen according to any one of claims 1 to 22, the polynucleotide of claim 22, the vector of claim 23, the cell of claim 24, or the composition according to any one of claims 27 to 29 in the manufacture of a medicament for treating or preventing Plasmodium sp. infection in a subject.

42. A method for selecting a synthetic antigen which binds an &nA-Plasmodium sp. reticulocyte binding protein 2b (RBP2b) antibody, the method comprising i) producing a synthetic antigen according to any one of claims 1 to 22, ii) determining if the synthetic antigen binds the anti-RBP2b antibody, and iii) selecting the synthetic antigen if it binds the anti-RBP2b antibody.

43. The method of claim 42 which further comprises expressing the synthetic antigen in a cell and determining if more antigen is produced when compared to a corresponding wild type antigen produced under the same conditions.

44. The method of claim 42 or claim 43 which further comprises detecting the thermostability of the synthetic antigen, and step iii) comprises selecting the synthetic antigen if it has a higher thermostability when compared to a corresponding wild type antigen.

45. The method according to any one of claims 42 to 44 which further comprises detecting the affinity of the synthetic antigen-antibody binding, and step iii) comprises selecting the synthetic antigen if it has a higher affinity when compared to a corresponding wild type antigen.

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