WO2025257561A1

WO2025257561A1 - Modified pvdbp-rii subdomain 3 p. vivax polypeptide

Info

Publication number: WO2025257561A1
Application number: PCT/GB2025/051297
Authority: WO
Inventors: Matthew Kenneth HIGGINS; Natalie Michaela Ena BARBER; Tossapol PHOLCHAREE
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2024-06-14
Filing date: 2025-06-12
Publication date: 2025-12-18
Anticipated expiration: 2026-12-14
Also published as: GB202408571D0

Abstract

The invention relates to a modified subdomain 3 P. vivax polypeptide, antigens, pharmaceutical formulations and vaccines. The present invention also provides methods of treating or preventing Plasmodium infection and/or disease by administering the modified subdomain 3 P. vivax polypeptide antigens, pharmaceutical formulations and vaccines.

Description

Modified PvDBP-RII Subdomain 3 P. vivax Polypeptide

The present invention provides modified subdomain 3 P. vivax polypeptide, antigens, pharmaceutical formulations and vaccines. The present invention also provides methods of treating or preventing Plasmodium infection and/or disease by administering the modified subdomain 3 P. vivax polypeptide antigens, pharmaceutical formulations and vaccines.

Plasmodium vivax is the predominant cause of human malaria outside Africa, leading to around 14.5 million annual cases [1], While it does not receive the same attention as its more deadly relative, Plasmodium falciparum, it causes significant human suffering and an effective vaccine is urgently required [2, 3], The blood stage of the Plasmodium vivax life cycle is a promising point of intervention. The symptoms of malaria occur as the parasite invades and replicates within human reticulocytes [4], In addition, differentiation of blood-stage parasites into gametocytes allows their uptake and development in mosquitos. A vaccine which prevents reticulocyte invasion would therefore prevent the symptoms and transmission of malaria [5], Reticulocyte invasion involves binding of the Plasmodium vivax Duffy binding protein, PvDBP, to the human Duffy antigen receptor for chemokines, DARC, found on the reticulocyte surface [6], The importance of this interaction in vivax malaria is emphasised by the effect of the Duffynegative phenotype [7], This polymorphism in DARC is common through much of Africa, and there is a close geographical correlation between Duffy-negativity and the reduced prevalence of Plasmodium vivax across most of the continent [2], Indeed, knockout of the orthologue PkDBPa prevents invasion of Duffy-positive erythrocytes by transgenic Plasmodium knowlesi [8-10], PvDBP is the most developed blood stage vaccine candidate in the quest to prevent vivax malaria.

PvDBP has a large modular ectodomain. Within this lies a -350 amino acid residue Duffy- binding-like domain known as PvDBP-RII, which interacts with the 60 residue DARC ectodomain [11 , 12], Immunisation of mice, rabbits and non-human primates with PvDBP-RII induces inhibitory antibodies that block binding of PvDBP to DARC [13, 14], In humans, high- titres of naturally-acquired PvDBP-RII-targeting antibodies reduce DARC binding in vitro and are associated with decreased risk of Plasmodium vivax infection [15], lower parasite densities and reduced risk of clinical malaria [16, 17], Immunisation of human volunteers with recombinant viral vectors expressing PvDBP-RII induces strain-transcending antibodies which prevent PvDBP-RII from binding to DARC, while human antibodies, from either vaccination or natural infection, inhibit invasion [9, 18], More recently, human volunteers have been vaccinated with PvDBP-RII, either delivered through a viral vector, as a protein with the Matrix M adjuvant. On challenge with Plasmodium vivax parasites, the volunteers vaccinated with the protein vaccine showed a reduction in the mean parasite multiplication rate of 51 % when compared with the unvaccinated controls [19],

While human vaccine trials with PvDBP have shown efficacy, they also indicate that the vaccine induced responses from PvDBP-RII are not sufficient to allow sterile protection. These findings encourage a rational, structure-guided approach to the design of improved PvDBP- based immunogens. Structural studies have shown that PvDBP-RII consists of three subdomains, with subdomains 1 and 2 forming a single structural unit and subdomain 3 a separate unit [20], The ectodomain of DARC binds to PvDBP-RII, with the interface centred around a sulphated tyrosine residue [21] (Figure 1A). In in vitro studies, DARC binding has been shown to induce PvDBP-RII dimer formation, with the DARC peptide located at the dimerization interface [22, 23], While there is currently no data to show that dimerization is functionally relevant in vivo during invasion, the binding site for DARC and the dimerization surface are both proposed as potential sites to target with vaccine-induced antibodies. Indeed, screening a linear peptide array with non-inhibitory and inhibitory human serum identified peptides which recognise antibodies found specifically in inhibitory serum [24] and are located in regions of subdomain 2 involved in PvDBP-RII dimerization and DARC19-30 binding [23], In contrast, monoclonal antibodies derived from PvDBPII-immunised mice which prevent PvDBP-RII from binding to DARC in vitro, bind to subdomain 3 [25] (Figure 1A).

Human neutralising monoclonal antibodies can also target epitopes on different subdomains of PvDBP-RII (Figure 1A). One study isolated monoclonal antibodies from a human volunteer from a malaria endemic region, finding that these bind predominantly to subdomain 2 and overlap with the binding site for DARC19-30 and the proposed dimerization site [18], In contrast, a second study isolated a panel of ten monoclonal antibodies from human volunteers vaccinated with PvDBP-RII, and showed that one of these, DB9, was most effective at neutralising blood stage growth of a sequence diverse set of Plasmodium vivax parasites. DB9 binds to the outer surface of subdomain 3, distant from the characterised DARC binding site [9], Given these findings and the morbidity and mortality associated with P. vivax, as well as the formation of hypnozites with the potential for relapse, supports the urgent unmet need for an improved P. vivax vaccine and/or treatment.

Brief summary of the disclosure

The invention is based on the surprising finding that a modified PvDBP-RII subdomain 3 Plasmodium vivax polypeptide can be used as therapies and/or preventatives for P. vivax. The modified PvDBP-RII subdomain 3 Plasmodium vivax polypeptide of the present invention can be produced readily in a correctly folded form, thereby allowing effective vaccination.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

In one aspect the present invention provides a modified PvDBP-RII subdomain 3 Plasmodium vivax polypeptide comprising modification at any one of or more of residues 388, 391 , 392, 397, 448, 449, 452 or 453 with reference to SEQ ID NO:1 or positions corresponding thereto.

In another aspect the invention provides an isolated nucleic acid sequence encoding the modified PvDBP-RII subdomain 3 protein described herein.

In another aspect the invention provides a pharmaceutical composition comprising the modified PvDBP-RII subdomain 3 protein described herein.

In another aspect the invention provides a vector comprising the nucleic acid sequence described herein.

In another aspect the invention provides a recombinant DNA molecule comprising: i) said isolated nucleic acid sequence of the present invention; and ii) a vector. In another aspect the invention provides a host cell comprising the recombinant DNA molecule according to the present invention, which expresses said protein encoded in said recombinant DNA molecule.

In another aspect the invention provides a vaccine against malaria comprising the modified PvDBP-RII subdomain 3 of the present invention or nucleic acid sequence of the present invention, in an amount sufficient to induce immunization against said disease, and a pharmaceutically acceptable carrier.

In another aspect the invention provides an immunogenic composition comprising the modified PvDBP-RII subdomain 3 of the present invention or nucleic acid sequence of the present invention.

In another aspect the invention provides a vaccine composition comprising an immunogenically effective amount of the modified PvDBP-RII subdomain 3 of the present invention or nucleic acid sequence of the present invention and a pharmacologically acceptable carrier.

In another aspect the invention provides a method of prevention or amelioration of a disease in a subject, the method comprising administering a therapeutically effective dose of a vaccine composition or immunogenic composition according to the present invention to the subject.

In another aspect the invention provides a method of prophylaxis of a disease, the method comprising administering a therapeutically effective dose of a vaccine composition or immunogenic composition of the present invention to a subject in need of prophylaxis.

In another aspect the invention provides the use of a vaccine composition or immunogenic composition of the present invention for the treatment or prophylaxis of disease.

In another aspect the invention provides the vaccine composition or immunogenic composition of the present invention for use in a method of inducing an antigen-specific immune response in a subject.

In another aspect the invention provides a recombinant method for making a modified PvDBP- RII subdomain 3, comprising: expressing the vector of the present invention in a host cell; and isolating the modified PvDBP-RII subdomain 3 from said host cell. Brief description of the Figures

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 Design of stable variants of PvDBP subdomain 3 a) A composite model of PvDBP-RII with subdomain 3 in medium grey and highlighted in an oval with dotted lines and subdomains 1 and 2 also in medium grey. A peptide from DARC is shown in black. The variable domains of inhibitory antibody DB9 are shown in light grey. This is derived from a composite of PDB codes 6R2S and 8A44, aligned on PvDBP-RII. b). A close-up of subdomain 3 of PvDBP-RII, showing the five residues which contact subdomain 2 and are mutated in the interface protein, c) A Western blot showing subdomain 3 (SD3) and interface (INT) expression in E. coli. In each case, Total is whole lysed cells while Soluble is whole lysed cells centrifuged with the supernatant loaded onto the blot, d) Analysis by SECMALLS of subdomain 3 and interface. In each case the black line shows the absorbance for the sample analysed on a superdex 75 column while the grey line shows the mass determined by light scattering, e). Analysis by circular dichroism of subdomain 3 and interface proteins showing a predominantly a-helical fold.

Figure 2 Subdomain 3 and interface bind antibody DB9 a) Analysis by surface plasmon resonance of the binding of PvDBP-RII, subdomain 3 and interface to immobilised monoclonal antibody DB9. Each set of curves shows a 2-fold dilution series from a maximum concentration of 1 M. b) The structure of subdomain 3 (dark grey) in complex with the Fab fragments of antibody DB9 (light grey).

Figure 3 Subdomain 3-based immunogens are more effective than PvDBP-RII a) Growth inhibitory activity for different concentrations of IgG induced in rabbits through immunisation with subdomain 3 (light grey, triangles), interface (dark grey, rhombus) and PvDBP-RII (mid-grey, circles). The left hand panels are tested against a Plasmodium knowlesi strain in which the PkDBPs have been deleted and replaced with the PvDBP-RII from the Sall strain of Plasmodium vivax, while the right hand panels are for an equivalent strain which uses PvDBP-RII from the W1 strain of Plasmodium vivax. The upper panels show growth inhibitory activity measured against total IgG concentration while the lower panels show the same data collected for the specific quantity of PvDBP-RII specific IgG. b) Total IgG (dark grey, circles), purified from rabbits immunised with PvDBP-RII, was separated using a column displaying subdomain 3 into subdomain 3-specific IgG (mid-grey, hexagons) and IgG depleted of antibodies binding subdomain 3 (light grey, squares). Their growth inhibitory activity was measured against Plasmodium knowlesi expressing PvDBP from the Sall strain. The left- panel shows the inhibitory activity as a factor of total IgG for each sample, which the righthand panel is corrected for IgG specific for PvDBP-RII. c) An equivalent depletion experiment conducted for a human serum sample from a volunteer vaccinated with PvDBP-RII.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

Various aspects of the invention are described in further detail below.

Detailed Description

The positions of modifications and residues corresponding thereto are provided with reference to the full PvDBP protein as shown in SEQ ID NO: 1. Therefore, for example, position 388 herein refers to position 388 of SEQ ID NO: 1. PvDBP-RII Subdomain 3 (S3) is composed of residues Pro387-Ser508 of PvDBP (i.e. 387 to 508 of SEQ ID NO: 1), as such in aspects of the invention that provide a PvDBP-RII subdomain 3 protein, position 387 of SEQ ID NO: 1 is equivalent to position 1 of the PvDBP-RII subdomain 3 protein (i.e. as shown in SEQ ID NO: 2) and so on. This is a standard means of referring to subdomain residue positions in the field (Batchelor et al., Nat Struct Mol Biol. 2011 Aug; 18(8): 908-914). Therefore position 388 corresponds to position 2 of SEQ ID NO: 2, position 391 corresponds to position 5 of SEQ ID NO: 2, position 392 corresponds to position 6 of SEQ ID NO: 2, position 397 corresponds to position 11 of SEQ ID NO: 2, position 448 corresponds to position 62 of SEQ ID NO: 2, position 449 corresponds to position 63 of SEQ ID NO: 2, position 452 corresponds to position 66 of SEQ ID NO: 2, position 453 corresponds to position 67 of SEQ ID NO: 2.

In one embodiment the modification is configured to solubilise and refold the protein. In another embodiment the modification is generated by insertion, deletion, substitution or any combination thereof, preferably the modification is a substitution of the amino acid. In a preferred embodiment the modification is a substitution to a charged or polar residue. In one embodiment, the one or more modification is selected from the group comprising Q388D with reference to SEQ ID NO:1 or to another charged or polar residue R391 E with reference to SEQ ID NO:1 or to another charged or polar residue W392K with reference to SEQ ID NO:1 or to another charged or polar residue G397 with reference to SEQ ID NO:1 changed to another charged or polar residue N448 with reference to SEQ ID NO:1 changed to another charged or polar residue Q449E with reference to SEQ ID NO:1 or to another charged or polar residue V452E with reference to SEQ ID NO:1 or to another charged or polar residue L453 with reference to SEQ ID NO:1 changed to another charged or polar residue.

A charged residue is an amino acid with a charged residue, a charged residue may be selected from the list comprising: R, K, H, D and E. A polar residue is an amino acid with a charged hydrophilic residue, a polar residue may be selected from the list comprising: S, T, N, and Q.

In some embodiments, the modification comprises a substitution of position 388 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, R, K, H, D, E, S, T or N. In some embodiments, the modification comprises a substitution of position 388 of SEQ ID NO: 1 with Aspartic acid (D). In some embodiments, the modification comprises a Q388D of SEQ ID NO: 1.

In some embodiments, the modification comprises a substitution of position 391 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, K, H, D, E, S, T, N or Q. In some embodiments, the modification comprises a substitution of position 391 of SEQ ID NO: 1 with Glutamic acid (E). In some embodiments, the modification comprises a R391 E of SEQ ID NO: 1.

In some embodiments, the modification comprises a substitution of position 392 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, K, H, D, E, S, T, N or Q. In some embodiments, the modification comprises a substitution of position 392 of SEQ ID NO: 1 with Lysine (K). In some embodiments, the modification comprises a W392K of SEQ ID NO: 1.

In some embodiments, the modification comprises a substitution of position 397 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, R, K, H, D, E, S, T or N. In some embodiments, the modification comprises a substitution of position 448 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, R, K, H, D, E, S, T or N.

In some embodiments, the modification comprises a substitution of position 449 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, K, H, D, E, S, T, N or Q. In some embodiments, the modification comprises a substitution of position 449 of SEQ ID NO: 1 with Glutamic acid (E). In some embodiments, the modification comprises a Q449E of SEQ ID NO: 1.

In some embodiments, the modification comprises a substitution of position 452 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, K, H, D, E, S, T, N or Q. In some embodiments, the modification comprises a substitution of position 452 of SEQ ID NO: 1 with Glutamic acid (E). In some embodiments, the modification comprises a V452E of SEQ ID NO: 1.

In some embodiments, the modification comprises a substitution of position 453 of SEQ ID NO: 1 with a polar or charged amino acid residue, for example, R, K, H, D, E, S, T or N.

In one embodiment, the modification can be any one or more of the modifications as described herein to one or more of positions 388, 391 , 392, 397, 448, 449, 452 and 453 or any combination thereof.

In another embodiment the modified PvDBP-RII subdomain 3 comprises amino acid sequence SEQ ID NO: 2. In another embodiment the modified PvDBP-RII subdomain 3 consists of amino acid sequence SEQ ID NO: 2.

In another aspect the invention provides an isolated nucleic acid sequence encoding the modified PvDBP-RII subdomain 3 protein described herein. In one embodiment the nucleic acid sequence comprises SEQ ID NO: 3. In one embodiment the nucleic acid sequence consists of SEQ ID NO: 3.

In another aspect the invention provides a pharmaceutical composition comprising the modified PvDBP-RII subdomain 3 protein described herein. In one embodiment the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In one embodiment the pharmaceutical composition further comprises an adjuvant. In another embodiment the modified PvDBP-RII subdomain 3 comprises amino acid sequence SEQ ID NO: 2. In another embodiment the modified PvDBP-RII subdomain 3 consists of the amino acid sequence SEQ ID NO: 2.

In another aspect the invention provides a vector comprising the nucleic acid sequence described herein. In one embodiment the vector is an mRNA vaccine.

In another aspect the invention provides a recombinant DNA molecule comprising: i) said isolated nucleic acid sequence of the present invention; and ii) a vector.

In another aspect the invention provides a host cell comprising the recombinant DNA molecule according to the present invention, which expresses said protein encoded in said recombinant DNA molecule. In another embodiment the host cell is a prokaryote cell. In another embodiment the host cell is a eukaryotic cell. In one embodiment the host cell produces an amino acid sequence comprising sequence of SEQ ID NO:2. In one embodiment the host cell produces an amino acid sequence consisting of sequence of SEQ ID NO:2.

In another aspect the invention provides a vaccine against malaria comprising the modified PvDBP-RII subdomain 3 of the present invention or nucleic acid sequence of the present invention, in an amount sufficient to induce immunization against said disease, and a pharmaceutically acceptable carrier. In another embodiment the vaccine further comprises an adjuvant.

In another embodiment the vaccine composition or immunogenic composition of the present invention wherein the vaccine compositions or immunogenic compositions are lyophilized or freeze-dried. In another embodiment the vaccine composition or immunogenic composition further comprises at least one adjuvant. In another embodiment the pharmaceutical composition, vaccine or immunogenic composition of the present invention, wherein the adjuvant is selected from the group comprising Freund's complete adjuvant, Freund's incomplete adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, saponins, mineral oil, vegetable oil, carbopol aluminium hydroxide, aluminium phosphate, aluminium oxide, oil-emulsions saponins, vitamin- E solubilisate or any combination thereof.

In another embodiment the vaccine composition or immunogenic composition is administered intranasally, opthalmically, intradermally, intraperitoneally, intravenously, subcutaneously, orally, cloacally, by aerosol (spray vaccination) or intramuscularly.

In one embodiment the subject in need thereof may be infected with or at risk of being infected with a species of Plasmodium. In another embodiment the disease is malaria.

In another embodiment the amount of the modified PvDBP-RII subdomain 3 administered is an amount effective to induce an immune response in a subject.

In another embodiment the amount of the modified PvDBP-RII subdomain 3 administered is an amount effective to induce antibody production in a subject.

In another embodiment the amount of the modified PvDBP-RII subdomain 3 administered is an amount sufficient to induce a protective immune response to Plasmodium vivax merozoites in a mammal.

In one embodiment the use of a vaccine composition or immunogenic composition of the present invention, wherein the disease is malaria.

In one embodiment the antigen-specific immune response comprises a T cell response or a B cell response. In another aspect the invention provides a recombinant method for making a modified PvDBP- RII subdomain 3, comprising: expressing the vector of the present invention in a host cell; and isolating the modified PvDBP-RII subdomain 3 from said host cell.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole.

Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, botany and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

Definitions

As used herein, “therapeutically effective amount” can refer to the amount of a composition and/or therapy provided herein sufficient to result in the prevention, reduction, mitigation, and/or elimination of one or more symptoms of malaria. The term “therapeutically effective amount” can also refer to the amount of a composition and/or therapy provided herein sufficient to prevent and/or treat an infection, a disease, and/or a symptom thereof caused by an organism of the genus Plasmodium, including but not limited to P. vivax.

As used herein, “cell,” “cell line,” and “cell culture” can include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.

As used herein, “control” can refer to an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.

As used herein, “effective amount” can refer to an amount sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term also includes within its scope amounts effective to enhance normal physiological function.

The terms “sufficient” and “effective”, as used interchangeably herein, can refer to an amount (e.g. mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s). For example, a therapeutically effective amount refers to an amount needed to achieve one or more therapeutic effects.

As used herein, “expression” can refer to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.

As used herein, “encode,” “encoding,” and the like refers to biological relationship between nucleic acids that form codons and the proteins that they translate into.

As used herein, “codon” can refer to a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis. It will be appreciated that one codon translates into only one amino acid. However, one amino acid can be translated from more than one codon. This phenomena is also known in the art as Codon degeneracy. It will also be appreciated that due to codon degeneracy, where a polypeptide sequence is given, unless specified otherwise, all possible nucleic acid sequences that can encode the polypeptide are contemplated and within the scope of this disclosure.

As used herein, “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.

As used herein, “separated” refers to the state of being physically divided from the original source or population such that the separated compound, agent, particle, or molecule can no longer be considered part of the original source or population.

As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism.

As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, side effect, and/or symptom thereof, and/or to decreasing in the rate of advancement of a disease, disorder, condition, side effect, and/or symptom thereof. The term also can include enhancing normal physiological function, palliative treatment, and partial remediation of a disease, disorder, condition, side effect, and/or symptom thereof. The disease, disorder, condition, can be infection with a species of the genus Plasmodium, including but not limited to P. vivax or a symptom thereof. The disease, disorder, or condition can be malaria or a symptom thereof. The terms “treating” and “treatment” as used herein refer generally to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as disease or disorders resulting from infection with a species of the genus Plasmodium, including but not limited to P. vivax and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. For example the disease or disorder can be malaria. The term “treatment” as used herein can cover any treatment of Malaria and/or infection with a species of the genus Plasmodium, including but not limited to P. vivax in a mammal, particularly a human, and can include: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term “treatment” as used herein can refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.

As used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, nontoxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.

As used herein, “preventative” and “prevent” refers to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.

As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. As used herein, “chemotherapeutic agent” or “chemotherapeutic” refer to a therapeutic agent utilized to prevent or treat cancer.

As used herein, “aptamer” refers to single-stranded DNA or RNA molecules that can bind to pre-selected targets including proteins with high affinity and specificity. Their specificity and characteristics are not directly determined by their primary sequence, but instead by their tertiary structure.

As used herein, “immunomodulator,” refers to an agent, such as a therapeutic agent, which is capable of modulating or regulating one or more immune function or response.

As used herein, “protein” can refer to a molecule composed of one or more chains of amino acids in a specific order. The term protein is used interchangeable with “polypeptide.” The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has a unique function.

As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and doublestranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, doublestranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein.

As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.

As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.

As used herein, “DNA molecule” includes nucleic acids/polynucleotides that are made of DNA. As used herein, “gene” can refer to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. “Genes” do not necessarily have to be translated into proteins can also produce only RNA products.

As used herein, the term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc.). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

As used herein, “variant” refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. “Variant” can include functional and structural variants.

As used herein, “identity,” is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 1970, 48: 443-453,) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.

As used herein, the term “vector” or is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both.

As used herein, “wild-type” can refer to the typical form of an organism, variety, strain, gene, protein, or characteristic as it occurs in nature, as distinguished from mutant forms that may result from selective breeding or transformation with a transgene. As used herein, “purified” or “purify” can be used in reference to a nucleic acid sequence, peptide, or polypeptide that has increased purity relative to the natural environment.

As used herein, “specifically binds” or “specific binding” refers to binding that occurs between such paired species such as enzyme/substrate, receptor/agonist or antagonist, antibody/antigen, lectin/carbohydrate, oligo DNA primers/DNA, enzyme or protein/DNA, and/or RNA molecule to other nucleic acid (DNA or RNA) or amino acid, which may be mediated by covalent or non-covalent interactions or a combination of covalent and non- covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding that occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen, enzyme/substrate, DNA/DNA, DNA/RNA, DNA/protein, RNA/protein, RNA/amino acid, receptor/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.

As used herein “induces,” “inducing,” or “induced” refers to activating or stimulating a process or pathway within a cell, such as endocytosis, secretion, and exocytosis.

The term “immune response” can refer to the reaction of the molecules, components, pathways, organs, fluids and/or cells of the body to the presence of a substance that is foreign or recognized by the body as foreign to the body.

The phrase “modulate or modulation of the immune response” can refer to change in the immune response that results from the introduction of a composition, vaccine, or other compound or formulation described herein in a recipient subject as compared to a suitable control.

As used herein, the term “vaccine” can refer to a compound, molecule, compositions, and formulations that are capable of inducing an immune response in a subject. The term “vaccine” can also be used to refer to a compound, molecule, compositions, and formulations that are capable of providing protective immunity against an organism. The vaccine may provide protection against a same (i.e. homologous) or different (i.e. heterologous) strain of an organism. The vaccine can be capable of providing protection against homologous and heterologous species, variants or strains.

As used herein, the term “antigen” refers to a molecule with one or more epitopes that stimulate a host's immune system to make a secretory, humoral and/or cellular antigenspecific response, or to a DNA molecule that is capable of producing such an antigen in a vertebrate. The term is also used interchangeably with “immunogen.” For example, a specific antigen can be complete protein, portions of a protein, peptides, fusion proteins, glycosylated proteins and combinations thereof.

As used herein, the term “immunization” can refer to the process of inducing a continuing protective level of antibody and/or cellular immune response which is directed against a strain of a species of the genus Plasmodium, including a P. vivax, or antigen thereof, either before or after exposure of the subject to P. vivax.

As used herein, “administering” can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intraarteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g. by diffusion) a composition the perivascular space and adventitia. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

As used herein, “adjuvant” can refer to an additional compound, composition, or ingredient that can facilitate stimulation an immune response in addition to the main antigen of a composition, formulation, or vaccine. Generally, an adjuvant can increase the immune response of an antigen as compared to the antigen alone. This can improve and/or facilitate any protective immunity developed in the recipient subject in response to the antigen. “Adjuvant” as used herein can refer to a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune response(s).

As used herein, “promoter” includes all sequences capable of driving transcription of a gene. In particular, the term “promoter” as used herein can refer to a DNA sequence generally described as the 5' regulator region of a gene, located proximal to the start codon. The transcription of an adjacent gene sequence is initiated at the promoter region. The term “promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene. The term “promoter” can encompass constitutive promoters and inducible promoters.

The term “operatively linked” as used herein can refer to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other.

For example, a promoter is operatively linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operatively linked to regulatory sequences in a sense or antisense orientation. In one example, the complementary RNA regions can be operatively linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA. The term “operatively linked” as used herein can also refer to the direct or indirect linkage of any two nucleic acid sequences on a singly nucleic acid fragment such that they are indirectly or directly physically connected on the same nucleic acid fragment. The term “operatively linked” as used herein can also refer to the insertion of a nucleic acid within the 5' and 3' end of another nucleic or the direct coupling of a nucleic acid to the 5' or 3' end of another nucleic acid.

Provided herein are synthetic P. vivax antigens that can stimulate antibody production within a subject. The antibodies produced by the subject in response to the synthetic P. vivax antigen can specifically bind and/or otherwise interfere with binding of DBP to DARC and thus can be neutralizing to P. vivax invasion and/or infection. In embodiments, the synthetic antigens can overcome the issues related to strain specific immunity and can provide broad protection against various strains of P. vivax.

Synthetic P. vivax Antigens

Provided herein are P. vivax synthetic antigens and nucleic acids encoding the synthetic peptide antigens. In embodiments, the P. vivax synthetic antigen can include or be composed entirely of a modified subdomain 3 polypeptide (where the base polypeptide is SEQ ID NO: 1) or fragment of at least 10 amino acids thereof. The modified subdomain 3 polypeptide can include one or more of the following mutations, where the mutations are referenced as to SEQ ID NO.: 1 :

Q388D with reference to SEQ ID NO:1 or to another charged or polar residue R391 E with reference to SEQ ID NO:1 or to another charged or polar residue W392K with reference to SEQ ID NO:1 or to another charged or polar residue

G397 with reference to SEQ ID NO:1 changed to another charged or polar residue N448 with reference to SEQ ID NO:1 changed to another charged or polar residue Q449E with reference to SEQ ID NO:1 or to another charged or polar residue V452E with reference to SEQ ID NO:1 or to another charged or polar residue L453 with reference to SEQ ID NO:1 changed to another charged or polar residue.

In some embodiments, the P. vivax synthetic antigens can be a polypeptide that can include or be composed entirely of a polypeptide having a sequence that is about 90% to 100% identical to SEQ ID NO: 2 or a fragment of at least 10 amino acids thereof.

The synthetic P. vivax antigen polypeptides provided herein can be encoded by a polynucleotide. The polynucleotide can be expressed and translated in a suitable in vitro or in vivo expression system. Such systems are generally known in the art. In addition to the nucleotides that encode the synthetic P. vivax antigen polypeptide, the polynucleotide can include additional nucleotides that can be regulatory and/or encode additional transcribed proteins, such as selectable markers and/or reporter proteins. Example selectable markers and reporter molecules include, but are not limited to, Examples of selectable markers include, but are not limited to, DNA and/or RNA segments that contain restriction enzyme sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and/or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and/or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as p-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. FLAG- and His-tags), and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art. Additional nucleotides can be operatively linked to the synthetic P. vivax antigen encoding nucleotides at the 5' and/or 3' end of the synthetic P. vivax antigen encoding nucleotides. In embodiments, the synthetic P. vivax antigen polynucleotide can include a polyadenylation region at the 3'end of the coding region of the synthetic P. vivax antigen polynucleotide. In addition to the synthetic P. vivax antigen polynucleotide, nucleotides for linkers and/or polynucleotides that improves or otherwise regulates synthesis, purification, expression, and/or identification of the translated protein can be operatively linked to the synthetic P. vivax antigen polynucleotide. In some embodiments, the polynucleotide can include or be composed entirely of a polynucleotide that is 90-100% identical to SEQ ID NO: 3 or a fragment of at least 10 nucleotides thereof.

The polynucleotides provided herein can be incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector can contain one or more regulatory sequences or one or more other sequences used to facilitate the expression of the polynucleotide. The expression vector can contain one or more regulatory sequences or one or more other sequences used to facilitate the replication of the expression vector. The expression vector can be suitable for expressing the polynucleotide in a bacterial cell. In other embodiments, the expression vector can be suitable for expressing the polynucleotide in a yeast cell. In further embodiments, the expression vector can be suitable for expressing the polynucleotide in a plant cell. In other embodiments, the expression vector can be suitable for expressing the polynucleotide in a mammalian cell. In another embodiment, the vector can be suitable for expressing the polynucleotide in a fungal cell. Suitable expression vectors are generally known in the art. All or part of the vectors can be capable of being transcribed in vitro without a host cell or in a host cell. The vectors can be capable of being replicated by a host cell. All or part of the vector or a RNA molecule produced from the vector template can be capable of being integrated directly or indirectly into a host cell genome. The vectors can be viral vectors, i.e. vectors that are virus based or incorporate viral proteins or nucleic acids corresponding to a viral protein. Suitable viral vectors can include adenoviral, lentiviral, retroviral, and alpha viral vectors.

In some aspects the invention is an mRNA vaccine comprising a polynucleotide encoding the synthetic P. vivax antigen polypeptides of the present invention. While not wishing to be bound by theory, it is believed that the RNA (e.g ., mRNA) vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon translation as the RNA (e.g ., mRNA) vaccines co-opt natural cellular machinery. Unlike traditional vaccines, which are manufactured ex vivo and may trigger unwanted cellular responses, RNA (e.g ., mRNA) vaccines are presented to the cellular system in a more native fashion. In some aspects the invention is a P. vivax antigen polypeptide vaccine, comprising at least one RNA polynucleotide having an open reading frame encoding at least one P. vivax antigenic polypeptide of the present invention, formulated in a cationic lipid nanoparticle. Some embodiments provide RNA (e.g ., mRNA) vaccines that include at least one RNA (e.g ., mRNA) polynucleotide having an open reading frame encoding at least one P. vivax antigenic polypeptide of the present invention or an immunogenic fragment thereof (e.g ., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide) and at least one RNA (e.g ., mRNA polynucleotide) having an open reading frame encoding a flagellin adjuvant. Herein, the use of the tern "antigenic polypeptide" encompasses immunogenic fragments of the antigenic polypeptide (an immunogenic fragment that induces (or is capable of inducing) an immune response to P. vivax. In some embodiments, at least one P. vivax antigenic polypeptide comprises an amino acid sequence identified by any one of SEQ ID NO: 1-2. In some embodiments, the amino acid sequence of the P. vivax antigenic polypeptide is, or is a fragment of, or is a homolog or variant having at least 80% (e.g ., 85%, 90%, 95%, 98%, 99%) identity to, the amino acid sequence identified by any one of SEQ ID NO: 1-2. In some embodiments, a RNA (e.g ., mRNA) vaccine further comprising an adjuvant. Also provided herein is a RNA (e.g ., mRNA) vaccine of any one of the foregoing paragraphs formulated in a nanoparticie (e.g ., a lipid nanoparticle).

Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject any of the RNA (e.g ., mRNA) vaccine as provided herein in an amount effective to produce an antigen-specific immune response. In some embodiments, an antigen-specific immune response comprises a T cell response or a B cell response. In some embodiments, a RNA (e.g ., mRNA) vaccine is administered to a subject by intradermal or intramuscular injection. In some embodiments, a method of producing an antigen-specific immune response comprises administering to a subject a single dose (no booster dose) of a RNA (e.g ., mRNA) vaccine of the present disclosure. In some embodiments, a method further comprises administering to the subject a second (booster) dose of a RNA (e.g ., mRNA) vaccine. Additional doses of a RNA (e.g ., mRNA) vaccine may be administered. In some embodiments, the subjects exhibit a seroconversion rate of at least 80% (e.g ., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine. Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood. After seroconversion has occurred, a virus can be detected in blood tests for the antibody. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.

Some embodiments, of the present disclosure provide methods of inducing an antigen specific immune response in a subject, including administering to a subject a RNA (e.g ., mRNA) vaccine in an effective amount to produce an antigen specific immune response in a subject. Antigen-specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titer following administration to the subject of any of the RNA (e.g ., mRNA) vaccines of the present disclosure. In some embodiments, the anti- antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control. In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has not been administered a RNA (e.g ., mRNA) vaccine of the present disclosure.

The synthetic P. vivax antigens described herein can be provided in a pharmaceutical formulation. As such, also provided herein are pharmaceutical formulations and vaccines that can contain an amount of one or more synthetic P. vivax antigens described herein. The amount of synthetic P. vivax antigen(s) can be an amount effective to stimulate an immune response in a subject when administered to a subject and/or prevent and/or treat P. vivax invasion of reticulocytes, P. vivax infection, and/or malaria.

Pharmaceutical formulations can be formulated for delivery via a variety of routes and can contain a pharmaceutically acceptable carrier. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20th Ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.

Suitable pharmaceutically acceptable carriers include, but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxyl methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.

The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition.

The pharmaceutical formulations can be administered to a subject in need thereof. The subject in need thereof can have a disease, disorder, or a symptom thereof. Example disease or disorder is malaria.

A pharmaceutical formulation can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The construct, biologic molecules and pharmaceutical formulations thereof described herein can be disposed on or otherwise integrated with or coupled to a medical device such as, but not limited to, a catheter or stent, such that the construct, biological molecule can be released to the surrounding local area or systemically over a period of time after insertion or implantation into a subject in need thereof. These can also be referred to as drug eluting medical devices.

Pharmaceutical formulations suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers can include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Injectable pharmaceutical formulations can be sterile and can be fluid to the extent that easy syringability exists. Injectable pharmaceutical formulations can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by incorporating an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating any of the synthetic P. vivax antigen(s) as described herein in an amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the nucleic acid vectors into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fluidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the synthetic P. vivax antigen(s) described herein can be formulated into ointments, salves, gels, or creams as generally known in the art. In some embodiments, the synthetic P. vivax antigen(s) described can be applied via transdermal delivery systems, which can slowly release the synthetic P. vivax antigens described herein for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media.

Administration of the P. vivax antigen(s) described herein is not restricted to a single route, but may encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan.

The pharmaceutical formulations can be administered to a subject by any suitable method that allows the agent to exert its effect on the subject in vivo. For example, the formulations or other compositions described herein can be administered to the subject by known procedures including, but not limited to, by oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation, via nasal delivery, vaginally, rectally, and intramuscularly. The formulations or other compositions described herein can be administered parenterally, by epifascial, intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymatous, and/or sublingual delivery. Delivery can be by injection, infusion, catheter delivery, or some other means, such as by tablet or spray.

For oral administration, a formulation as described herein can be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulation can contain conventional additives, such as lactose, mannitol, cornstarch or potato starch, binders, crystalline cellulose, cellulose derivatives, acacia, cornstarch, gelatins, disintegrators, potato starch, sodium carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch glycolate, lubricants, and/or or magnesium stearate.

For parenteral administration (i.e., administration by through a route other than the alimentary canal), the formulations described herein can be combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation can be prepared by dissolving the active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering the solution sterile. The formulation can be presented in unit or multi-dose containers, such as sealed ampoules or vials. The formulation can be delivered by injection, infusion, or other means known in the art.

For transdermal administration, the formulation described herein can be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increase the permeability of the skin to the nucleic acid vectors of the invention and permit the nucleic acid vectors to penetrate through the skin and into the bloodstream. The formulations and/or compositions described herein can be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which can be dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch.

The pharmaceutical formulations or other compositions described herein can be administered to a subject either as a single agent, or in combination with one or more other agents. Additional agents include but are not limited to DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, antiinflammatories, anti-histamines, anti-infectives, and chemotherapeutics.

The synthetic P. vivax antigens can be formulated as a vaccine. The vaccine can contain an effective amount or an effective concentration of one or more of the P. vivax antigens provided herein. The amount can be effective to stimulate an immune response, stimulate antibody production, provide protective immunity, immunize, treat, and/or prevent P. vivax invasion, infection, and/or malaria in the subject and/or offspring thereof. The effective amount can range from about 0.001 pg to about 1 ,000 g or more of the composition described herein. In some embodiments, the effective amount of the composition described herein can range from about 0.001 mg/kg body weight to about 1 ,000 mg/kg body weight. In yet other embodiments, the effective amount of the composition can range from about 1% w/w to about 99% or more w/w, w/v, or v/v of the total vaccine formulation. The vaccines, described herein can be effective to stimulate an immune response, stimulate antibody production, provide protective immunity against one or more P. vivax strains, immunize a subject against one or more P. vivax strains, treat and/or prevent invasion and/or infection by one or more P. vivax strains in the subject and/or offspring thereof. In other words, in embodiments, the vaccines described herein can be capable of providing protection against multiple strains of P. vivax as opposed to being strain specific.

The vaccines described herein can include one or more additional agents. The vaccines provided herein can include one or more suitable adjuvants. Suitable adjuvants are generally known in the art and can include, but are not limited to aluminum salts (e.g, aluminum phosphate and aluminum hydroxide), organic adjuvants (e.g. squalene), and oil-based (e.g., MF59). In embodiments, the compositions can contain a suitable pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxyl methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition. Other suitable pharmaceutically acceptable carriers are identified elsewhere herein and will be appreciated by those of ordinary skill in the art.

The vaccines can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition. The vaccines can also include an amount, including an effective amount, of one or more of auxiliary active agents, including but not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, and chemotherapeutics. Suitable compounds for the auxiliary active agents have been previously described herein in relation to the pharmaceutical formulations.

In some embodiments, the vaccines provided herein can be included in a combination vaccine or other combination formulation. In some embodiments, the vaccines provided herein can be included in a combination vaccine or other combination formulation with a P. falciparum vaccine or antigen or other treatment.

The synthetic P. vivax antigens, formulations and vaccines thereof provided herein can be administered to a subject by any suitable route. The subject can be a subject in need thereof. The subject in need thereof can be exposed, will be exposed, and/or is at risk of being exposed to a species of the genus Plasmodium, including but not limited to, P. vivax. Administration of the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein can induce or otherwise stimulate an immune response in the recipient subject and/or an offspring of the recipient subject. Administration of the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein can stimulate antibody production in the recipient subject. In other embodiments, administration of the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein can provide protective immunity against one or more strains of P. vivax in a recipient subject and/or an offspring of the recipient subject.

Accordingly, provided herein are methods of inducing or otherwise stimulating an immune response in a subject and/or an offspring of the subject that include the step of administering the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein to a subject one or more times. Also provided herein are methods of stimulating antibody production in a subject and/or offspring of the subject that includes the step of administering the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein to a subject one or more times. Also provided herein are methods of stimulating protective immunity a subject and/or offspring thereof by administering the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein to a subject one or more times. Also provided herein are methods of treating and/or preventing P. vivax infection and/or disease by administering the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein to a subject one or more times. In embodiments, the amount of the compound, compositions, formulation and/or vaccine can be an amount effective to stimulate an immune response, stimulate antibody production, provide protective immunity, immunize, treat, and/or prevent infection and/or disease by one or more strains of P. vivax in the subject and/or offspring thereof.

The synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein can be administered to the subject by any suitable routes. In some embodiments, about 0.01 cc to 10 cc or more of the synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein can be administered to a subject. In some embodiments, an amount effective to induce an immune response in the recipient subject and/or offspring thereof. The synthetic P. vivax antigen(s), formulation(s) and/or vaccine(s) thereof provided herein can be administered to the subject one or more times. Where administration occurs more than once the time period between each does can each independently range from days (e.g. 1-7 days), weeks (e.g. 1-52 weeks, or years (e.g. 1-5 years) apart. Administration can occur during any life stage of the subject. Administration can be simultaneously or in series with other vaccines. Suitably the modified subdomain 3 P. vivax polypeptide comprises an amino acid sequence having at least 30% identity to amino acid sequence SEQ ID NO: 2 or a functional fragment thereof, and comprises two or more of the modifications described herein.

Suitably the modified subdomain P. vivax polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence SEQ ID NO: 2 or a functional fragment thereof, and comprises two or more of the modifications described herein.

Suitably the modified subdomain P. vivax polypeptide may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid SEQ ID NO: 2 or a functional fragment thereof, and comprises two or more of the modifications described herein.

Suitably the two or more modifications are mutations. Suitably any of the modifications herein may be referred to interchangeably as ‘mutations’. Suitably the mutations are selected from deletions, insertions, substitutions etc. In one embodiment the mutations are amino acid substitutions.

As used herein, "heterologous" in reference to a polypeptide or polynucleotide sequence is a sequence that originates, for example, from a cell or an organism from a foreign species. Alternatively, if the sequence originates from the same species, it is derived from a cell organism having a different genetic background; or if from the same genetic background, it is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. As such, heterologous sequences are in a configuration not found in nature.

Mutations may include deletions or substitutions or combinations thereof. For example, the mutations may be conservative or non-conservative amino acid substitutions.

“Conservative amino acid substitutions” refer to the interchangeability of residues having similar side chains, and thus typically involves substitution of an amino acid in a polypeptide with amino acids within the same or similar defined class of amino acids. By way of example, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acids having aromatic side chains may be substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid may be substituted with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided below:

RESIDUE POSSIBLE CONSERVATIVE SUBSTITUTIONS

A, L, V, I Other aliphatic (A? L7V, I ) i Other non-polar (A, L, V, I, G, M)

G, M i Other non-polar (A, L, V, I, G, M)

D, E i Other acidic (D, E)

K, R, H j Other basic (K, R, H)

N, Q, S, T i Other polar

H, Y, W, F | Other aromatic (H, Y, W, F)

C i None

P i None

“Non-conservative substitution” refers to substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and may affect (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

“Deletion” refers to modification of a polypeptide by removal of one or more amino acids in comparison to a wild-type or control polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, or 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids of the polypeptide while retaining enzymatic activity. Deletions can comprise a continuous segment or can be discontinuous.

“Identity” or “percent identity” refers to the degree of sequence variation between two given nucleic acid or amino acid sequences. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by visual inspection. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the world wide web at ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al., J. Mol. Biol.215: 403-410 (1990)). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1 , In one embodiment less than about 0.01, and In one embodiment less than about 0.001.

Unless otherwise stated, “percent identity” as used herein refers to the value obtained using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) implemented in the EMBOSS Needle alignment tool using default matrix files EBLOSUM62 for protein with default parameters (Gap Open = 10, Gap Extend =0.5, End Gap Penalty = False, End Gap Open = 10, End Gap Extend = 0.5) or DNAfull for nucleic acids with default parameters (Gap Open = 10, Gap Extend =0.5, End Gap Penalty = False, End Gap Open = 10, End Gap Extend = 0.5); or any equivalent program thereof. EMBOSS Needle is available, e.g., from EMBL-EBI such as at the following website: ebi.ac.uk/Tools/psa/emboss_needle/ and as described in the following publication: “The EMBL-EBI search and sequence analysis tools APIs in 2019.” Madeira et al. Nucleic Acids Research, June 2019, 47(W1):W636-W641. The term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by EMBOSS Needle

A modified subdomain 3 P. vivax polypeptide encoded by a nucleic acid or a modified subdomain 3 P. vivax polypeptide of the invention may be a functional fragment of a modified subdomain 3 P. vivax polypeptide as described herein. A "functional fragment" refers to a protein fragment that retains protein function.

The term “equivalent amino acids” or “corresponding amino acids” refers to amino acids in a sequence of interest, which correspond to those amino acids of an identified reference sequence, typically herein the reference sequence is SEQ ID NO:1 for P. vivax PvDBP polypeptide. A region of equivalent or corresponding amino acids may be determined by aligning the amino acid sequences of the proteins from the different species, using an alignment program such as BLAST® or ClustalW. Note that the corresponding positions in a sequence of interest should be determined by comparison with a like for like reference sequence.

A “corresponding” amino acid position to a given SEQ ID NO can be determined using Geneious as a global alignment with free end gaps having the following parameters: cost matrix Blossum 62, gap open penalty 12, gap extension penalty 3, refinement iterations 2; or an equivalent program thereof or an equivalent program thereof.

Unless otherwise stated, a “corresponding” amino acid position to a given SEQ ID NO is determined using EMBOSS Needle default parameters: BLOSUM62; Gap Open 10, GAP EXTEND 0.5; END GAP OPEN 10 and END GAP EXTEND 0.5, or an equivalent program thereof. See, Madeira et al. (2022) Nucleic Acids Research, 01 Jul 2022, 50(W1):W276-W279; PMID: 35412617 PMCID: PMC9252731. The term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical corresponding nucleotide or amino acid residue matches when co“Sequencing DNA" refers to determining the nucleic acid sequence of a piece of DNA, e.g. of a gene. Standard methods and commercial services are known in the art. Basic methods for DNA sequencing include the Maxam-Gilbert method and the chain termination method. High-throughput techniques have also been developed and may be used in the method of the present invention. These high-throughput techniques include, but are not limited to, Massively parallel signature sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, Combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing and Nanopore DNA sequencing.

Sequencing may be carried out using primers that are capable of binding to an isolated polynucleotide of the invention. For example, primers that complimentary to at least a portion of an isolated polynucleotide of the invention.

As used herein, the term “primer" refers to an oligonucleotide which is capable of annealing to a polynucleotide target and serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH). A primer (in some examples an extension primer and in some examples an amplification primer) may be single stranded for maximum efficiency in extension and/or amplification. The primer may be an oligodeoxyribonucleotide. A primer is typically sufficiently long to prime the synthesis of extension and/or amplification products in the presence of the agent for polymerization. The minimum length of the primer can depend on many factors, including, but not limited to temperature and composition (A/T vs. G/C content) of the primer. In the context of amplification primers, these are typically provided as a pair of bi-directional primers consisting of one forward and one reverse primer or provided as a pair of forward primers as commonly used in the art of DNA amplification such as in PCR amplification.

As such, it will be understood that the term "primer," as used herein, can refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified. Hence, a "primer" can include a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical base pairing. Primers can be prepared by any suitable method known in the art. Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, the phospho di- or tri-ester method, the diethylphosphoramidate method and the solid support method disclosed in U.S. Patent No. 4,458,066.

Primers can be labelled, if desired, by incorporating detectable moieties by for instance spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical moieties. The PCR method is well described in handbooks and known to the skilled person. After amplification by PCR, target polynucleotides can be detected by hybridization with a probe polynucleotide, which forms a stable hybrid with the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes are essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions can be used.

The invention further provides isolated polynucleotides that encode a modified subdomain 3 P. vivax polypeptide or fragment thereof as defined in any aspect or embodiment herein. As such, also provided or positions corresponding thereto are modified subdomain 3 P. vivax polypeptides or functional fragments thereof that may be expressed from such isolated polynucleotides. An “isolated” polynucleotide is substantially separated away from other polynucleotide sequences with which the polynucleotide is normally associated, such as, from the chromosomal or extrachromosomal DNA of a cell in which the polynucleotide naturally occurs. A polynucleotide may be an isolated polynucleotide when it comprises a transgene or part of a transgene present in the genome of another organism. The term also embraces polynucleotides that are biochemically purified so as to substantially remove contaminating polynucleotides and other cellular components. Isolated polynucleotides are substantially free of sequences (such as protein encoding sequences) that naturally flank the nucleic acid (i.e. , sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. The isolated polynucleotide may be flanked by its native genomic sequences that control its expression in the cell, for example, the native promoter, or native 3 ' untranslated region.

An “isolated polypeptide” as used herein refers to a protein which is free of at least some proteins with which it would normally be found, is essentially free of other proteins from the same source, e.g., from the same cell or species, has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell, is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the “isolated polypeptide” is linked in nature,. Preferably, the isolated protein is substantially free from other contaminating proteins or polypeptides or other contaminants that are found in its natural environment.

The invention describe herein also relates to a kit comprising a container and instructions for use, the container comprising a modified subdomain 3 P. vivax polypeptide of the present invention or nucleic acid encoding the polypeptide of the present invention, and the instructions for use.

Aspects of the invention are demonstrated by the following non-limiting examples.

EXAMPLES

Surface remodelling allows generation of a soluble version of subdomain 3 Subdomain 3 of PvDBP-RII forms an autonomous structural unit, consisting of two long antiparallel a-helices, along which runs a region of loops and short helices (Figure 1 A). Subdomain 3 packs against subdomain 2 through a small hydrophobic core and hydrogen bonds and we reasoned that expressing subdomain 3 alone would expose this small hydrophobic patch and might impact its solubility. We therefore designed a version of subdomain 3 in which we resurfaced the hydrophobic patch by replacing hydrophobic residues with hydrophilic alternatives (W392K and V452E) (Figure 1 B). In addition, we altered three residues within this interface region to increase their charge (Q388D, R391 E and Q449E), with the surprising effect of increasing the solubility of the isolated domain. We name this variant interface and compared it with unaltered subdomain 3.

We expressed both interface and subdomain 3 in E. coli. Small scale expression trials demonstrated that subdomain 3 expressed in an insoluble form in the E. coli pellet, while interface was expressed in a soluble form (Figure 1C). We next purified both proteins. In the case of interface, we purified the soluble component, while subdomain 3 was refolded from inclusion bodies. This yielded 3 mg/l for subdomain 3 and 20 mg/l for interface. Both purified proteins were monomeric and monodispersed as demonstrated by SEC-MALLS (Figure 1 D) and both showed circular dichroism spectra characteristic of a-helical proteins (Figure 1 E), with similar thermal stability, with a denaturing transition at >70°C. Therefore, while both subdomain 3 and interface can be produced from E. coli in a folded form, subdomain 3 requires refolding from inclusion bodies. In contrast, surface remodelling allows interface to be produced in a readily scalable form by ensuring that it is expressed as a soluble protein. While subdomain 3 is only compatible with delivery as a protein-based vaccine after refolding, interface is compatible with delivery by all currently used platforms, including as an RNA vaccine or as a viral vector.

Both interface and subdomain 3 bind to antibody DB9

We next used surface plasmon resonance analysis to assess the binding of interface and subdomain 3 to monoclonal antibody DB9, allowing us to assess whether the epitope is correctly folded. We immobilised DB9 onto the surface of a protein A/G-coated chip and flowed increasing concentrations of PvDBP-RII, subdomain 3 and interface over this chip. All three bound with similar dissociation constants in the nanomolar range (2.63 nM for PvDBP-RII, 1.11 nM for subdomain 3 and 2.67 nM for interface) (Figure 2A and Table 1). Table 1: kinetic parameters measured by surface plasmon resonance analysis for binding to monoclonal antibody DB9

To check the conformation of subdomain 3 we determined its structures using x-ray crystallography (Figure 2B and Table 2).

Table 2: crystallographic statistics We prepared Fab fragments from DB9 and mixed with subdomain 3. Crystals formed and the structures were determined by molecular replacement. Alignment of this structure with that of PvDBP-RII bound to DB9 showed them to align with a root mean square deviation of 0.43A. A similar approach did not yield crystals of interface, perhaps because some of the mutations which generate the interface were involved in forming crystal contacts within the subdomain 3:DB9 crystals.

Both interface and subdomain 3 can therefore be generated in a correctly folded form which retain the ability to bind to neutralising antibody DB9.

Interface and subdomain 3 generate a more potent neutralising antibody response than PvDBP-RII

We next compared the antibody responses induced in rabbits on immunisation with three 20 .g doses of either interface, subdomain 3 or PvDBP-RII. In each case, the immunogens were mixed with the Freund’s adjuvant and dosing was conducted on days 14, 28 and 42, with sera harvested on day 56. We immunised two rabbits with each immunogen.

To test the efficacy of these sera at preventing erythrocyte invasion, we used a Plasmodium knowlesi model. While Plasmodium vivax cannot be cultured, transgenic Plasmodium knowlesi in which the three PkDBP proteins have been replaced with PvDBP can be studied using in vitro growth inhibitory assays [10] and, when used to analyse a panel of monoclonal antibodies, gave similar outcomes to an ex vivo Plasmodium vivax invasion assay [9], Our vaccine immunogens were based on the Sall PvDBP sequence and we therefore used two Plasmodium knowlesi strains, designed to express PvDBP from strains Sall and W1 , providing us with a homologous and a heterologous test strain. The PvDBP-RII from these strains differ in 10 positions, and they were chosen to represent the maximum sequence diversity found in different geographical locations [19],

We started by purifying total IgG from the rabbit sera and assessing their efficacy in a growth inhibition assay, assessing two-fold dilutions of sera from a maximum concentration of 10mg/ml. In both cases, IgG from rabbits immunised with PvDBP-RII produced only 20-30% growth inhibition at 10mg/ml. In contrast, IgG from rabbits immunised with subdomain 3 and interface were substantially more effective, giving 100% growth inhibition at 10mg/ml and with IC50 values of 3.5mg/ml for P knowlesi expressing W1 and ~5mg/ml for the strain expressing Sall. Therefore subdomain 3 and interface were equivalently effective as vaccine immunogens and both outperformed PvDBP-RII. We next asked whether the better performance of subdomain 3 and interface was due to the induction of a larger titre of PvDBP-targeting antibody (i.e. antibody quantity) or due to a greater proportion of growth-inhibitory antibody (i.e. antibody quality). To answer this, we used calibration free concentration analysis to assess the quantity of PvDBP-RII specific IgG in each sample. This was done using surface plasmon resonance, with PvDBP-RII conjugated to the chip surface at high density to ensure conditions in which mass transport effects were evident, followed by injection of two different concentrations of IgG. Replotting the growthinhibition data as a factor of PvDBP-RII-specific IgG showed that the curves for rabbits immunised with all three immunogens were equivalent, albeit with PvDBP-RII not inducing sufficient antibodies to reach 50% growth-inhibition. Therefore the quality of antibodies induced by PvDBP-RII, interface and subdomain 3 are all equivalent and both interface and the improved performance of subdomain 3 is due to high quantities of growth-inhibitory antigen specific IgG.

The finding that subdomain 3-based immunogens induce IgG of a similar quality to PvDBP- RII led to us as whether the growth-inhibitory antibodies induced by PvDBP-RII immunisation all target subdomain 3 or whether there are also growth-inhibitory antibodies targeting other regions of PvDBP-RII. To answer this question, we conducted depletion experiments. Total IgG from rabbits immunised with PvDBP-RII, or human volunteers immunised with PvDBP-RII as part of a clinical trial, were passed over a column which had been coupled to subdomain 3 protein. The IgG which did not stick to the column (subdomain 3-depleted IgG) and those eluted from the column (subdomain 3-specific IgG) were assessed for the presence of antibodies that bind to subdomain 3 by ELISA, confirming efficient depletion. These antibodies were next tested for growth-inhibitory activity, using the Plasmodium knowlesi transgenic using the Sall variant. Both rabbit (Figure 3b) and human (Figure 3c) subdomain 3-specific IgG showed effective growth inhibition with EC50 values of ~1 mg/ml. In contract, in neither case was growth inhibition observed for subdomain 3 depleted IgG at the maximum concentration that could be achieved. Therefore all detectable growth-inhibition obtained from either human or rabbit IgG from PvDBP-RII-immunised individuals was due to antibodies binding to subdomain 3.

Conclusions

Controlled human malaria infection of volunteers vaccinated with a protein vaccine consisting of the PvDBP-RII immunogen formulated with Matrix M adjuvant have provided the first evidence that PvDBP-based vaccines can affect the multiplication rate of Plasmodium vivax in vaccinated humans [19], Nevertheless, this study also highlights that current vaccines fall short of inducing the levels of immunity required for sterile protection and emphasise the need for improved PvDBP-based vaccine immunogens [19], In this study, we attempt to produce such an immunogen using a rational, structure-guided approach.

Structure-guided vaccine design often starts with structural studies to reveal how the most effective neutralising, or in this case growth-inhibitory, monoclonal antibodies function. In the case of PvDBP-RII, studies have been conducted of both mouse and human antibodies, resulting in structures of the epitopes of five antibodies [9, 18, 21 , 25], These studies are much smaller in scope those conducted for other malaria antigens, such as PfRH5 and PfCSP, where hundreds of monoclonal antibodies have been analysed, and the outcomes are less clear. Antibodies that bind to various regions of PvDBP can be growth-inhibitory, including those that target subdomain 2 [18], where the DARC binding site [21] and proposed dimerization interface lie [22], or those that target subdomain 3 [9], It is also not clear how each of these growth-inhibitory antibodies functions, with steric hinderance of membrane approach proposed as a possible mechanism [9], Despite this, we decided to follow up our finding that the broadly-reactive, growth-inhibitory antibody DB9 binds to subdomain 3 of PvDBP-RII [9] and to design and test subdomain 3 as a vaccine immunogen.

Subdomain 3 adopts a discrete three helical architecture which interacts with other parts of PvDBP-RII through a small hydrophobic patch. Subdomain 3 alone expressed in an insoluble form and required refolding to produce a functional protein. In contrast, resurfacing of the exposed hydrophobic patch, through five amino acid changes, resulted in a soluble, stable, highly expressed subdomain 3 immunogen, which we call interface. In our hands PvDBP-RII is challenging to express and is often not stable on storage, which may be limiting its effectiveness as an immunogen after formulation with adjuvant or immunisation of human volunteers. Surprisingly, interface is extremely stable and scalable production is likely to be effective.

Side-by-side comparison in a well-established model of growth-inhibition, revealed the surprising outcome that interface induces a more effective growth-inhibitory response than PvDBP-RII. Indeed, at the maximum concentration of IgG tested, PvDBP-RII was 20-30% effective and interface was 100% effective, with an equivalent EC30 around five-fold lower for interface. When we separated antibodies induced using PvDBP-RII into those that did and didn’t bind to subdomain 3, we found that growth-inhibitory antibodies predominantly bound to subdomain 3. Indeed, when we assessed the growth-inhibitory effect of antibodies induced using PvDBP-RII and interface, as a function of subdomain 3-reactive antibodies, we found that PvDBP-RII and interface formed equivalently. Therefore, to our surprise, the better efficacy of antibodies induced by subdomain 3 was not due to improvement in the quality of the antibody response, through focusing onto a more effective epitope region. Instead, both PvDBP-ll and interface induced predominantly interface targeting growth-inhibitory antibodies and interface induced these in greater quantities.

In summary, this study uses rational, structure-based immunogen design to produce a novel form of PvDBP which is stable, readily produced and induces a more growth-inhibitory antibody response than previous PvDBP-based immunogens. No previous study had conducted a side-by-side comparison of vaccine immunogens consisting of PvDBP-RII and a subdomain 3 immunogen and other PvDBP-based vaccine immunogen design programs have focused on full-length PvDBP-RII, or on fragments containing the DARC binding site in subdomain 2. It is therefore very surprising that subdomain 3 outperforms PvDBP-RII to the degree that we show here. In addition, our rational structure-guided design of a soluble form of subdomain 3 which can be produced without refolding, opens the way to use this immunogen directly in a variety of vaccine platforms, including those such as mRNA or viral vector delivery, which require the production of an antigen in a soluble form within the vaccinated individual. As a result of these innovations, this immunogen is now available for clinical testing as a versatile component of vaccines to prevent Plasmodium vivax.

Materials and methods

Expression and purification of PvDBP-RII, subdomain 3 and interface

PvDBP-RII and antibody DB9 were produced as [9], Gene sequences for PvDBP-RII, subdomain 3 and interface were obtained from GeneArt and were cloned into a modified version of the pEt15b vector to provide an N-terminal his-tag followed by a TEV cleavage site. These were expressed in E. coli BL21-DE3 cells, induced with 1mM IPTG at OD 1.0 and grown overnight at 25°C.

Subdomain 3 was found in the insoluble fraction and was purified by refolding. Cells were resuspended in 20mM Tris pH 8.0, 300mM NaCI, 20mM imidazole and broken by sonication. After centrifugation at 50,000g for 30 minutes, the pellet was resuspended in 6M GdnHCI, 20mM Tris pH 8, 20mM imidazole, 10mM p-mercaptoethanol by incubation at room temperature for 2 hours before centrifugation at 50,000g for 30 minutes at 4°C. The soluble fraction was incubated with Ni-NTA beads, and the bound material was washed in the 6M GdnHCI, 20mM Tris pH 8, 20mM imidazole, 300pM oxidised glutathione, 3mM reduced glutathione. It was then refolded while attached to the Ni-NTA column with a slow decreasing concentration of GdnHCI, while maintaining other buffer components unchanged, before eluting in 20mM Tris pH 8, 300 mM NaCI, 200mM imidazole. This yielded ~1 mg of protein per litre of cells.

Interface was expressed in a soluble form. Cells were lysed as for subdomain 3 and the soluble fraction was applied to a Ni-NTA column. This was washed using 20mM Tris pH 8, 300mM NaCI, 20mM imidazole and the protein was eluted using 20mM Tris pH 8, 300 mM NaCI, 200mM imidazole, yielding around 2.2mg per litre of cells.

In both cases, the proteins were next dialysed into PBS, supplemented with 300pM oxidised glutathione, 3mM reduced glutathione and cleaved with TEV protease overnight at room temperature. They were then passed through a Ni-NTA column and the flow through was collected. This was concentrated and applied to a superdex 75 (Cytiva) in 20mM Tris pH 8.0, 50mM NaCI.

Circular dichroism analysis

Circular dichroism analysis was conducted using a J-815 spectropolarimeter (JASCO, Japan) with an attached Pelitier water bath. Proteins were buffer exchanged using PD10 columns into 10mM sodium phosphate pH 7.5, 150mM NaF and were diluted to a final concentration of 0.2mg/ml. Spectra were collected from 190nm to 260nm wavelengths at 25°C and four independent measurements were averaged together to obtain the final curve. To use circular dichroism analysis to study thermal stability, spectra were collected from 200nm and 250nm wavelengths at 2°C intervals at temperatures from 20°C to 90°C. The ellipticity at 220nm wavelength was plotted against temperate to determine the melting temperature.

Surface plasmon resonance

Surface plasmon resonance analysis was conducted using a Biacore T200 instrument (GE healthcare) using 20mM HEPES pH 7.4, 150mM NaCI, 0.005% Tween-20. A Biacore chip was prepared by using amine coupling to coat a CM5 chip in protein A/G. Monoclonal antibody DB9 was then coupled onto flow path 2, with flow path 1 left as a negative control. To analyse binding of PvDBP-RII, subdomain 3 and interface to DB9, these were then flowed across the chip surface using a two-fold dilution series from a maximum concentration of 1 pM. Dataware analysed using the BIAevaluation software.

Crystallisation and structure determination For crystallisation, subdomain 3 and the Fab fragment of DB9 were mixed in a ratio of 1.1 :1 and were incubated at room temperature for 30 minutes. The mixture was loaded onto a superdex 200 10/30 column, run in 20mM Tris pH 8.0, 50mM NaCI (Cytiva). The protein was concentrated to 11mg/ml and subjected to crystallistion trials. Crystals grew with reservoir solution of 0.2M ammonium acetate, 0.1M sodium acetate pH 4.0, 15% PEG 4000. These were transferred into a cryo-protection solution containing 0.2M ammonium acetate, 0.1M sodium acetate pH 4.0, 15% PEG 4000, 25% glycerol. A dataset was collected to a final resolution of 1.55A on beamline I03 at Diamond Light Source. Molecular replacement was conducted in Phaser using the previous structure of the Fab fragment of DB9 bound to PvDBP- RII (PDB:6R2S) as a search model. DB9 Fab:SD3 with loops removed was used as a search model. Modelling building was conducted in coot and refinement in buster.

Growth inhibitory assays

• Endotoxin removal using the Pierce High-capacity endotoxin removal resin (thermo)

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Sequences SEQ ID No: 1 - The amino acid sequence of PvDBP

MKGKNRSLFVLLVLLLLHKVNNVLLERTIETLLECKNEYVKGENGYKLAKGHHCVEEDNLERWLQGTN ERRSEENIKYKYGVTELKIKYAQMNGKRSSRILKESIYGAHNFGGNSYMEGKDGGDKTGEEKDGEHKT DSKTDNGKGANNLVMLDYETSSNGQPAGTLDNVLEFVTGHEGNSRKNSSNGGNPYDIDHKKTISSAII NHAFSGNTVMKNCNYKRKRRERDWDCNTKKDVCIPDRRYQLCMKELTNLVNNTDTNFHRDITFRKLYL KRKLIYDAAVEGDLLLKLNNYRYNKDFCKDIRWSLGDFGDIIMGTDMEGIGYSKVVENNLRSIFGTDE KAQQRRKQWWNESKAQIWTAMMYSVKKRLKGNFIWICKLNVAVNIEPQIYRWIREWGRDYVSELPTEV QKLKEKCDGKIAYTDKKVCKVPPCQNACKSYDQWITRKKNQWDVLSNKFISVKNAEKVQTAGIVTPYD ILKQELDEFNEVAFENEINKRDGAYIELCVCSVEEAKKNTQEVVTNVDNAAKSQATNSNPISQPVDSS KAEKVPGDSTHGNVNSGQDSSTTGKAVTGDGQNGNQTPAESDVQRSDIAESVSAKNVDPQKSVSKRSD DTASVTGIAEAGKENLGASNSRPSESTVEANSPGDDTVNSASIPVVSGENPLVTPYNGLRHSKDNSDS DGPAESMANPDSNSKGETGKGQDNDMAKATKDSSNSSDGTSSATGDTTDAVDREINKGVPEDRDKTVG SKDGGGEDNSANKDAATVVGEDRIRENSAGGSTNDRSKNDTEKNGASTPDSKQSEDATALSKTESLES TESGDRTTNDTTNSLENKNGGKEKDLQKHDFKSNDTPNEEPNSDQTTDAEGHDRDSIKNDKAERRKHM NKDTFTKNTNSHHLNSNNNLSNGKLDIKEYKYRDVKATREDI ILMSSVRKCNNNISLEYCNSVEDKIS SNTCSREKSKNLCCSISDFCLNYFDVHSYEYLSCMKKEFEDPSYKCFTIGGFKDKTYFAAAGALLILL LLIASRKMIKNDSEEATFNEFEEYCDNIHRIPLMPNNIEHMQPSTPLDYS

SEQ ID No: 2 - the amino acid sequence of interface (modified subdomain 3): 387-PDIYEKIREWGRDYVSELPTEVQKLKEKCDGKIAYTDKKVCKVPPCQNACKSY DQWITRKKNEWDELSNKFISVKNAEKVQTAGIVTPYDILKQELDEFNEVAFENEINKRDGAYI ELCVC-507

SEQ ID No: 3 - the nucleic acid sequence of interface (modified subdomain 3)

CCTGATATCTATGAAAAAATTCGTGAATGGGGTCGCGATTATGTTAGCGAACTGCCG AC C G AAG T T C AG AAAC T G AAAG AAAAAT G T GAT G G C AAAAT C G C C TAG AC C GAT AAA AAAGTTTGTAAAGTTCCGCCTTGTCAGAATGCCTGTAAAAGCTATGATCAGTGGATT AC C C G C AAAAAAAAC GAATGGGAT G AAC T G AG C AAC AAG TTCATTAGCGT T AAAAAC G C C G AAAAAG T T CAGAC C G C AG GTATTGTTACCCCGTAT GAT AT T C T G AAAC AAGAG C T G G AT GAAT T T AAC G AAG T G G C C T T T G AAAAC G AG AT CAAT AAAC G T GAT G G T G C C TATATTGAACTGTGCGTTTGTTCTTGA

References

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2. Howes, R.E., et al., Plasmodium vivax Transmission in Africa. PLoS Negl Trop Dis, 2015. 9(11): p. e0004222.

3. Price, R.N., N.M. Douglas, and N.M. Anstey, New developments in Plasmodium vivax malaria: severe disease and the rise of chloroquine resistance. Curr Opin Infect Dis, 2009. 22(5): p. 430-5. 4. Flannery, E.L., M.B. Markus, and A.M. Vaughan, Plasmodium vivax. Trends Parasitol, 2019. 35(7): p. 583-584.

5. De, S.L., et al., Progress towards the development of a P. vivax vaccine. Expert Rev Vaccines, 2021. 20(2): p. 97-112.

6. Horuk, R., et al., A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor. Science, 1993. 261(5125): p. 1182-4.

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8. Singh, A.P., et al., Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion. Mol Microbiol, 2005. 55(6): p. 1925-34.

9. Rawlinson, T.A., et al., Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody. Nat Microbiol, 2019. 4(9): p. 1497- 1507.

10. Mohring, F., et al., Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research. Elife, 2019. 8.

11. Chitnis, C.E. and A. Sharma, Targeting the Plasmodium vivax Duffy-binding protein. Trends Parasitol, 2008. 24(1): p. 29-34.

12. Chitnis, C.E. and L.H. Miller, Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion. J Exp Med, 1994. 180(2): p. 497-506.

13. de Cassan, S.C., et al., Preclinical Assessment of Viral Vectored and Protein Vaccines Targeting the Duffy-Binding Protein Region II of Plasmodium Vivax. Front Immunol, 2015. 6: p. 348.

14. Moreno, A., et al., Preclinical assessment of the receptor-binding domain of Plasmodium vivax Duffy-binding protein as a vaccine candidate in rhesus macaques. Vaccine, 2008. 26(34): p. 4338-44.

15. Cole-Tobian, J.L., et al., Strain-specific duffy binding protein antibodies correlate with protection against infection with homologous compared to heterologous plasmodium vivax strains in Papua New Guinean children. Infect Immun, 2009. 77(9): p. 4009-17.

16. King, C.L., et al., Naturally acquired Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage Plasmodium vivax infection. Proc Natl Acad Sci U S A, 2008. 105(24): p. 8363-8.

17. Nicolete, V.C., et al., Naturally Acquired Binding-Inhibitory Antibodies to Plasmodium vivax Duffy Binding Protein and Clinical Immunity to Malaria in Rural Amazonians. J Infect Dis, 2016. 214(10): p. 1539-1546. 18. Urusova, D., et al., Structural basis for neutralization of Plasmodium vivax by naturally acquired human antibodies that target DBP. Nat Microbiol, 2019. 4(9): p. 1486- 1496.

19. Hou, M.M., et al., Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection. Sci Transl Med, 2023. 15(704): p. eadf1782.

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21. Moskovitz, R., et al., Structural basis for DARC binding in reticulocyte invasion by Plasmodium vivax. Nat Commun, 2023. 14(1): p. 3637.

22. Batchelor, J.D., J. A. Zahm, and N.H. Tolia, Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC. Nat Struct Mol Biol, 2011. 18(8): p. 908-14.

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Claims

1. A modified PvDBP-RII subdomain 3 Plasmodium vivax polypeptide comprising modification at any one of or more of residues 388, 391, 392, 397, 448, 449, 452 or 453 with reference to SEQ ID NO:1 or positions corresponding thereto.

2. The modified PvDBP-RII subdomain 3 according to claim 1, wherein the modification is configured to solubilise and refold the protein.

3. The modified PvDBP-RII subdomain 3 according to claim 1 or 2, wherein the modification is generated by insertion, deletion, substitution or any combination thereof, preferably the modification is a substitution of the amino acid.

4. The modified PvDBP-RII subdomain 3 according to any preceding claim, wherein the modification is a substitution to a charged or polar residue.

5. The modified PvDBP-RII subdomain 3 according to any preceding claim, wherein one or more modification is selected from the group comprising

Q388D with reference to SEQ ID NO:1 or to another charged or polar residue

R391E with reference to SEQ ID NO:1 or to another charged or polar residue

W392K with reference to SEQ ID NO:1 or to another charged or polar residue

G397 with reference to SEQ ID NO:1 changed to another charged or polar residue

N448 with reference to SEQ ID NO:1 changed to another charged or polar residue

Q449E with reference to SEQ ID NO:1 or to another charged or polar residue

V452E with reference to SEQ ID NO:1 or to another charged or polar residue

L453 with reference to SEQ ID NO:1 changed to another charged or polar residue

6. The modified PvDBP-RII subdomain 3 according to any preceding claim, comprising amino acid sequence SEQ ID NO: 2.

7. An isolated nucleic acid sequence encoding the protein according to any preceding claim.

8. The nucleic acid sequence of claim 7 comprising SEQ ID NO: 3.

9. A pharmaceutical composition comprising the protein according to anyone of claims 1 to 6.

10. The pharmaceutical composition of claim 9, further comprising a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 9, wherein the modified PvDBP-RII subdomain

3 consists of the amino acid sequence SEQ ID NO: 2.

12. A vector comprising the nucleic acid sequence according to claims 7 or 8.

13. A recombinant DNA molecule comprising: i) said isolated nucleic acid sequence of claims 7 or 8; and ii) a vector.

14. A host cell comprising the recombinant DNA molecule according to claim 13, which expresses said protein encoded in said recombinant DNA molecule.

15. A vaccine against malaria comprising the modified PvDBP-RII subdomain 3 of claims 1 to 6 or nucleic acid sequence of claims 7 or 8, in an amount sufficient to induce immunization against said disease, and a pharmaceutically acceptable carrier.

16. An immunogenic composition comprising the modified PvDBP-RII subdomain 3 of claims 1 to 6 or nucleic acid sequence of claims 7 or 8.

17. A vaccine composition comprising an immunogenically effective amount of the modified PvDBP-RII subdomain 3 of claims 1 to 6 or nucleic acid sequence of claims 7 or 8 and a pharmacologically acceptable carrier.

18. A method of prevention or amelioration of a disease in a subject, the method comprising administering a therapeutically effective dose of a vaccine composition or immunogenic composition according to claims 16 or 17 to the subject.

19. A method of prophylaxis of a disease, the method comprising administering a therapeutically effective dose of a vaccine composition or immunogenic composition according to claims 16 or 17 to a subject in need of prophylaxis.

20. The method of claim 18 or 19, wherein the subject in need thereof is infected with or at risk of being infected with a species of Plasmodium.

21. The method according to claim 18 or 19, wherein the disease is malaria.

22. The vaccine composition or immunogenic composition of any one of claims 16 or 17 for use in a method of inducing an antigen-specific immune response in a subject.

23. The vaccine composition or immunogenic composition for use according to claim 22, wherein the antigen-specific immune response comprises a T cell response or a B cell response.

24. A recombinant method for making a modified PvDBP-RII subdomain 3, comprising: expressing the vector of claim 14 in a host cell; and isolating the modified PvDBP-RII subdomain 3 from said host cell.