WO2025231359A2

WO2025231359A2 - A novel method to target pfgbp130 as a vaccine for p. falciparum malaria

Info

Publication number: WO2025231359A2
Application number: PCT/US2025/027490
Authority: WO
Inventors: Jonathan Kurtis; Dipak K. RAJ
Original assignee: Brown University
Current assignee: Brown University
Priority date: 2024-05-03
Filing date: 2025-05-02
Publication date: 2025-11-06
Anticipated expiration: 2026-11-03
Also published as: WO2025231359A3

Abstract

The present invention relates to a malaria vaccine composition designed to elicit an immune response against Plasmodium falciparum by providing proteins or amino acid sequences, including PfGBP130 or PfGBP130-A surface antigen sequences. Upon administration to a human subject, the vaccine composition induces the production of specific anti-malaria antibodies. These antibodies are configured to inhibit the invasion of red blood cells by Plasmodium falciparum parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells, thereby offering a preventive and/or therapeutic effect against malaria. The composition aims to enhance the immune defense mechanism in humans, reducing the incidence and severity of malaria infections by targeting critical stages of the parasite's lifecycle.

Description

A NOVEL METHOD TO TARGET PFGBP130 AS A VACCINE FOR P. FALCIPARUM MALARIA

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent Application No.: 63/642,438, filed 03- May-2024, the entire disclosure of which is incorporated by reference as if fully set forth herein in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates to a malaria vaccine composition and more particularly, to a composition configured to provide Plasmodium falciparum proteins, fragments thereof, or amino acid sequences to produce specific anti-malaria antibodies in a human subject.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant number R01AI144014- 01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0004] This submission will, in the future, be accompanied by a “Sequence Listing XML” file containing SEQ ID NOs: 1-Y.

BACKGROUND OF THE INVENTION

[0005] Malaria vaccines have been a significant focus of research due to the global burden of malaria, particularly caused by the Plasmodium falciparum parasite. Traditional approaches to malaria vaccination have primarily focused on targeting the sporozoite stage of the parasite's life cycle, aiming to prevent the initial infection of liver cells. These vaccines often utilize attenuated sporozoites or derivatives thereof to elicit an immune response. However, the complexity of the Plasmodium life cycle and its ability to evade the immune system have posed challenges in achieving high efficacy with these vaccines.

[0006] Another approach has involved targeting the blood-stage of the parasite, which is responsible for the clinical symptoms of malaria. Blood-stage vaccines aim to induce an immune response that can prevent the parasite from invading red blood cells. Various antigens have been explored for this purpose, including merozoites and some blood-stage antigens. Despite promising preclinical results, many of these candidates have struggled to demonstrate significant efficacy in clinical trials, often due to the high genetic variability of the parasite and the need for a robust and sustained immune response.

[0007] Recent advancements have explored the use of multi-antigen vaccines, which combine several different antigens to enhance the breadth and strength of the immune response. These vaccines aim to target multiple stages of the parasite's life cycle or multiple antigens within a single stage. While this strategy has shown potential in increasing the efficacy of malaria vaccines, the complexity of formulation and the need for precise antigen selection remain significant hurdles. What is urgently needed are new malaria vaccines to save human lives, and none of these previous approaches have provided a comprehensive solution that combines the features described in this disclosure.

BRIEF SUMMARY OF THE INVENTION

[0008] The following brief summary presents a simplified summary of the innovation in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify all key or critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

[0009] Malaria remains a critical global health issue, claiming hundreds of thousands of lives annually, with the World Health Organization estimating over 400,000 deaths each year, predominantly affecting children under five in sub-Saharan Africa. This life-threatening disease is caused by Plasmodium parasites, transmitted through the bites of infected female Anopheles mosquitoes. The most lethal of these parasites, Plasmodium falciparum, is responsible for the majority of malaria-related fatalities. Despite ongoing efforts to combat malaria, the emergence of drug-resistant parasite strains and insecticide-resistant mosquitoes complicates control measures.

[0010] The need for an effective malaria vaccine is critical, as it would provide a powerful tool in the fight against this disease. Current malaria control strategies rely heavily on the use of insecticide-treated bed nets, indoor residual spraying, and antimalarial drugs. However, these methods have limitations, including the potential for resistance development and the need for continuous implementation and monitoring. A vaccine that can provide long-lasting protection against malaria would be a game-changer, reducing the incidence of the disease and saving countless lives. The development of such a vaccine requires a deep understanding of the Plasmodium parasite's biology and the human immune response to infection.

[0011] The history of malaria vaccines is a testament to the complexity and challenges associated with developing a vaccine for a parasitic disease. Efforts to create a malaria vaccine date back several decades, with researchers exploring various approaches to stimulate an immune response capable of preventing infection. Early attempts focused on whole parasite vaccines, which involved using attenuated or inactivated forms of the parasite to elicit immunity. However, these approaches faced significant hurdles, including safety concerns and difficulties in producing the vaccine at scale.

[0012] In the 1980s, advances in molecular biology and immunology led to a shift towards subunit vaccines, which use specific proteins or antigens from the parasite to trigger an immune response. One of the most notable efforts in this area was the development of the RTS.S/AS01 vaccine, which targets the circumsporozoite protein of Plasmodium falciparum. This vaccine underwent extensive clinical trials and, in 2021 , received a positive recommendation from the World Health Organization for broader use in children in sub-Saharan Africa.

[0013] Despite this limited progress, the quest for a highly effective malaria vaccine continues. Herein we have explored novel approaches, such as using viral vectors to deliver malaria antigens, developing multi-stage vaccines that target different life cycle stages of the parasite, and employing cutting-edge technologies like mRNA platforms. The ongoing research and development efforts are driven by the urgent need to overcome the limitations of existing control measures and provide a sustainable solution to the global malaria burden.

[0014] The journey disclosed herein towards a malaria vaccine has been marked by scientific breakthroughs, setbacks, and a relentless pursuit of innovation. It underscores the importance of continued investment in research and collaboration among scientists, governments, and international organizations to achieve the ultimate goal of malaria eradication.

[0015] Working in the actual areas most affected, the urgency for an effective malaria vaccine is underscored by the limitations of current strategies, such as insecticide-treated bed nets and antimalarial drugs, which face challenges like resistance and require sustained implementation. A vaccine offering durable protection could significantly reduce malaria incidence and mortality. Research disclosed herein indicates that understanding the Plasmodium parasite's biology and the human immune response is crucial for vaccine development. These studies highlight the potential impact of a successful vaccine in altering the course of malaria and saving countless lives.

[0016] The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant proteinbased immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies.

[0017] The malaria vaccine composition may be designed to provide Plasmodium falciparum proteins or amino acid sequences, including PfGBP130 or PfGBP130-A surface antigen sequences. When administered to a human subject, the vaccine can stimulate the production of specific anti-malaria antibodies. These antibodies may inhibit the invasion of red blood cells by Plasmodium falciparum malaria parasites and/or kill the parasite within the red blood cells, thereby preventing and/or treating malaria in the human subject. Some examples of the malaria vaccine composition may include Plasmodium falciparum proteins or amino acid sequences derived from PfGBP130 or PfGBP130-A. The vaccine can also include sequences from PfGBP130 or PfGBP130-A formulated as a recombinant protein.

[0018] The malaria vaccine composition may also include Plasmodium falciparum proteins or amino acid sequences derived from a nucleic acid coding sequence. This can include at least one of all possible coding sequences for these amino acid sequences, and the vaccine may include mRNA encoding for PfGBP130-A or a PfGBP130-ecto. The Plasmodium falciparum proteins or amino acid sequences in the malaria vaccine composition may comprise PfGBP130 surface antigen sequences. The malaria vaccine composition may also comprise PfGBP130-A surface antigen sequences. The antibodies generated within the human subject by the malaria vaccine composition may be specific to the Plasmodium falciparum proteins or amino acid sequences.

[0019] The antibodies generated within the human subject may also be specific to the PfGBP130 or PfGBP130-A surface antigen sequences. The vaccine can include a DNA plasmid encoding PfGBP130-A. The antibodies produced by the malaria vaccine composition may inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites. The antibodies may also inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites.

[0020] The malaria vaccine composition may include amino acids 111-374 of PfGBP130-A and/or amino acids 89-824 of PfGBP130. The vaccine may include a nucleic acid coding sequence with at least one of all possible coding sequences for these amino acid sequences. It can also include mRNA and/or DNA that encodes for amino acids 89-824 of PfGBP130-ecto and/or amino acids 111-374 of PfGBP130-A. The malaria vaccine composition may treat malaria in the human subject. The vaccine can be formulated in a lipid nanoparticle encapsulation, a viruslike particle, a nanoparticle, a conjugate, a plasmid vector, or a combination thereof.

[0021] A method of preventing and/or treating malaria in a human subject may involve administering a malaria vaccine composition designed to provide Plasmodium falciparum proteins or amino acid sequences, including PfGBP130 or PfGBP130-A surface antigen sequences. When administered, the vaccine can stimulate the production of specific anti-malaria antibodies that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites. Some examples of the method may include using Plasmodium falciparum proteins or amino acid sequences derived from PfGBP130 or PfGBP130-A formulated as a recombinant protein.

[0022] The method may also involve using Plasmodium falciparum proteins or amino acid sequences derived from a nucleic acid coding sequence, which can include at least one of all possible coding sequences for these amino acid sequences. The vaccine may include mRNA encoding for PfGBP130-A or a PfGBP130-ecto.

[0023] The Plasmodium falciparum proteins or amino acid sequences used in the method may comprise PfGBP130 surface antigen sequences. The method may also use Plasmodium falciparum proteins or amino acid sequences comprising PfGBP130-A surface antigen sequences. The antibodies generated within the human subject by the method may be specific to the Plasmodium falciparum proteins or amino acid sequences.

[0024] The antibodies generated may also be specific to the PfGBP130 or PfGBP130-A surface antigen sequences. The vaccine can include a DNA plasmid encoding PfGBP130-A. The antibodies produced by the method may inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites. The antibodies may also inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites. The malaria vaccine composition used in the method may include amino acids 111-374 of PfGBP130-A and/or amino acids 89-824 of PfGBP130. The method may involve using a vaccine that includes mRNA and/or DNA that encodes for amino acids 89-824 of PfGBP130-ecto and/or amino acids 111-374 of PfGBP130-A.

[0025] The malaria vaccine composition used in the method may treat malaria in the human subject. The vaccine can be formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector, or a combination thereof.

[0026] As such, keeping in mind possible combination therapies and the above discussion, as an additional brief summary or to provide discussion points for a brief summary, some example features of the technology disclosed herein can be briefly summarized by the following list of features, any of which can be inter-combined or discussed optionally with any other feature, Figure, Drawing, detail, embodiment, aspect, or example disclosed herein:

[0027] Feature 1 : A malaria vaccine composition configured to provide Plasmodium falciparum proteins or amino acid sequences including PfGBP130 or PfGBP130-A surface antigen sequences, wherein when the malaria vaccine composition is administered to a human subject specific anti-malaria antibodies are produced; the malaria vaccine composition being configured to provide a production of antibodies within the human subject that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells, thereby preventing and/or treating malaria in the human subject.

[0028] Feature 2: The malaria vaccine composition of feature 1 , wherein the Plasmodium falciparum proteins or amino acid sequences are derived from PfGBP130 or PfGBP130-A and/or wherein the vaccine includes sequences from PfGBP130 or PfGBP130-A formulated as a recombinant protein.

[0029] Feature 3: The malaria vaccine composition of feature 1 , wherein the Plasmodium falciparum proteins or amino acid sequences are derived from a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences and/or wherein the vaccine includes mRNA encoding for PfGBP130-A (SEQ ID NO: 1) or a PfGBP130- ecto (SEQ ID NO: 2).

[0030] Feature 4: The malaria vaccine composition of feature 1 , wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130 surface antigen sequences (SEQ ID NO: 3).

[0031] Feature 5: The malaria vaccine composition of feature 1 , wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130-A surface antigen sequences (SEQ ID NO: 4).

[0032] Feature 6: The malaria vaccine composition of feature 1, wherein the antibodies generated within the human subject are specific to the Plasmodium falciparum proteins or amino acid sequences.

[0033] Feature 7: The malaria vaccine composition of feature 1 , wherein the antibodies generated within the human subject are specific to the PfGBP130 or PfGBP130-A surface antigen sequences and/or wherein the vaccine includes a DNA plasmid (SEQ ID NO: 5) encoding PfGBP130-A.

[0034] Feature 8: The malaria vaccine composition of feature 1 , wherein the antibodies inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells.

[0035] Feature 9: The malaria vaccine composition of feature 1 , wherein the antibodies inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells.

[0036] Feature 10: The malaria vaccine composition of feature 1 , wherein the malaria vaccine composition includes amino acids (aa) 111-374 of PfGBP130-A (SEQ ID NO: 6) and/or includes aa 89-824 of PfGBP130 (SEQ ID NO: 7).

[0037] Feature 11: The malaria vaccine of feature 1, wherein the vaccine includes a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences or includes mRNA (SEQ ID NO: 8) and/or DNA (SEQ ID NO: 9) that encodes for aa 89-824 of PfGBP130-ecto (SEQ ID NO: 10) and/or encodes for aa 111-374 of PfGBP130-A (SEQ ID NO: 11).

[0038] Feature 12: The malaria vaccine composition of feature 1, wherein the malaria vaccine composition treats malaria in the human subject and/or wherein the malaria vaccine is formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector including SEQ ID NO: 12, or a combination thereof.

[0039] Feature 13: A method of preventing and/or treating malaria in a human subject, the method comprising administering to the human subject a malaria vaccine composition configured to provide Plasmodium falciparum proteins or amino acid sequences including PfGBP130 or PfGBP130-A surface antigen sequences, wherein when the malaria vaccine composition is administered to the human subject specific anti-malaria antibodies are produced; the malaria vaccine composition being configured to provide a production of antibodies within the human subject that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells.

[0040] Feature 14: The method of feature 13, wherein the Plasmodium falciparum proteins or amino acid sequences are derived from PfGBP130 or PfGBP130-A formulated as a recombinant protein.

[0041] Feature 15: The method of feature 13, wherein the Plasmodium falciparum proteins or amino acid sequences are derived from a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences or wherein the vaccine includes mRNA encoding for PfGBP130-A (SEQ ID NO: 1) or a PfGBP130-ecto (SEQ ID NO: 2).

[0042] Feature 16: The method of feature 13, wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130 surface antigen sequences (SEQ ID NO: 3).

[0043] Feature 17: The method of feature 13, wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130-A surface antigen sequences (SEQ ID NO: 4).

[0044] Feature 18: The method of feature 13, wherein the antibodies generated within the human subject are specific to the Plasmodium falciparum proteins or amino acid sequences.

[0045] Feature 19: The method of feature 13, wherein the antibodies generated within the human subject are specific to the PfGBP130 or PfGBP130-A surface antigen sequences and/or wherein the vaccine includes a DNA plasmid (SEQ ID NO: 5) encoding PfGBP130-A.

[0046] Feature 20: The method of feature 13, wherein the antibodies inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells.

[0047] Feature 21: The method of feature 13, wherein the antibodies inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites and/or the antibodies binding to the antigens can cause the death of the parasites within red blood cells.

[0048] Feature 22: The method of feature 13, wherein the malaria vaccine composition includes amino acids (aa) 111-374 of PfGBP130-A (SEQ ID NO: 6) and/or includes aa 89-824 of PfGBP130 (SEQ ID NO: 7).

[0049] Feature 23: The method of feature 13, wherein the vaccine includes mRNA (SEQ ID NO: 8) and/or DNA (SEQ ID NO: 9) that encodes for aa 89-824 of PfGBP130-ecto (SEQ ID NO: 10) and/or encodes for aa 111-374 of PfGBP130-A (SEQ ID NO: 11).

[0050] Feature 24: The method of feature 13, wherein the malaria vaccine composition treats malaria in the human subject and/or wherein the malaria vaccine is formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector including SEQ ID NO: 12, or a combination thereof.

[0051] Any of the vaccines or methods can include sterilization even just before an administration. Any of the features, methods and/or details herein can be provided in a kit. While the summary examples disclosed above provide some introduction to embodiments of the invention, other implementations are also contemplated, described, and recited herein. These and other features and advantages will be apparent from a reading of the following detailed description, the example claims, and a review of the associated drawings. It is to be understood that both the foregoing general description and the following detailed description are explanatory only and are not restrictive of aspects as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] For the purpose of illustration, certain discernable embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, data, dimensions, and illustrations shown. In the examples of the drawings:

[0053] FIG. 1A shows a plot illustrating polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 79-89% in multiple parasite strains in vitro. FIG. 1B shows a plot of parasite growth vs. % serum. FIG. 1C shows how polyclonal anti- PfGBP130-A antibodies generated by recombinant protein immunization in mice resulted in no or modest (22-29%) growth inhibition in multiple parasite strains in vitro.

[0054] FIG. 2 shows an example domain structure of PfGBP130.

[0055] FIG. 3A shows mice immunized with a DNA plasmid encoding PfGBPI 30-A generated low titer (1:8,000) antibodies against PfGBPI 30-A and PfGBP130-ecto coated beads with no reactivity against negative control protein (PfGARP-ecto) coated beads. FIG. 3B shows mice immunized with recombinant protein encoding PfGBPI 30-A generated high titer (1 :512,000) antibodies against PfGBP130-A and PfGBPI 30-ecto coated beads with negligible binding to a negative control protein (PfGARP-ecto) coated beads.

[0056] FIG. 4 shows a plot illustrating how antibodies to PfGBPI 30-A inhibit merozoite invasion.

[0057] FIG. 5A shows lipid encapsulated mRNA encoding PfGBP130-A fluorescence results. FIG. 5B, shows PfGBP130-ecto generates high titer (both 1 :512,000, fluorescence units vs. titer). FIG. 5C shows polyclonal murine anti-PfGBP130 antibodies generated by immunization with LNPs containing mRNA encoding PfGBP130-A or PfGBP130-ecto inhibit parasite growth by 80% in vitro.

[0058] FIG. 6 shows a table illustrating epidemiologic characteristics of resistant and susceptible individuals used in differential screening assays. FIG. 7 shows a table illustrating Parasite genes identified following three rounds of differential bio-panning of P.

[0059] FIG. 8A shows expression of PfGBP130-A. FIG. 8B shows expression of PfGBP130- ecto in HEK293 cells. FIG. 8C shows expression of PfGARP-ecto in HEK293 cells.

[0060] FIG. 9A shows polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 81-95% while no significant growth inhibition was observed for anti PfGBP130-A antibodies generated by recombinant protein immunization or with antisera raised against a negative control DNA vaccine construct, PfPHISTc. FIG. 9B shows polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 94% compared to antisera from mice immunized with the empty plasmid vector (anti pVR2001).

[0061] FIG. 10 shows characterization of anti-PfGBP130-A antisera by western blot.

[0062] FIG. 11A shows SEQ ID. NO: 1. FIG. 11B shows SEQ ID NO: 2. FIG. 11C shows SEQ ID. NO: 3. FIG. 11 D shows SEQ ID NO: 4. FIG. 11 E shows SEQ ID. NO: 5. FIG. 11 F shows SEQ ID NO: 6. FIG. 11G shows SEQ ID. NO: 7. FIG. 11H shows SEQ ID NO: 8. FIG. 111 shows SEQ ID. NO: 9. FIG. 11 J shows SEQ ID NO: 10. FIG. 11K shows SEQ ID. NO: 11. FIG. 11L shows SEQ ID NO: 12.

[0063] It should be understood that while illustrations can sometimes be used in the example figures above to describe different embodiments and different aspects of the technology, any aspect from any figure can be optionally inter-combined with an aspect from any other figure or text. Any example disclosed herein can be inter-combined with any other. All trademarks, images, likenesses, words, and depictions that could be construed in the drawings and the disclosure are plainly in fair use and are provided solely for the purposes of illustration of the invention in view of an urgent need to prevent injuries and to treat subjects as further discussed in more detail below. DETAILED DESCRIPTION OF THE INVENTION

[0064] The subject innovation is now described, in some examples with reference to the drawings, wherein examples can be used to refer to the aspects of the breadth of concepts of the invention. In the following description, for purposes of explanation, specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.

DEFINITIONS

[0065] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention can be determined by the claims. Unless otherwise defined, 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 belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0066] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0067] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0068] As used herein, the term “or” means “and/or.” The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0069] As used herein, a “range” may be provided. A statement may include “in the range from about A to about B”. All points from A to B are subsumed by the range, and all those points can define preferred ranges. Within said range, any range subsumed therein means any range that is within the stated range. Endpoints within the range can define a new range. For example, the following are all subsumed within the range of about 10 to about 50. 10 to 20; 15 to 35; 23 to 40; or 50 to 31; or any other range or set of ranges within the stated range. As such, within the range any set of endpoints subsumed therein can be used as an exemplary range.

[0070] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Any method described herein can be claimed and/or described as a composition and vice versa.

[0071] The term "consisting of" as it is known in the practice refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0072] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. In specific examples, “consisting essentially of” can be explained herein for each example or can be defined broadly, for example, by stating that an administration to a subject (in a method herein) does not include any other active pharmaceutical ingredient or therapeutic agent in addition to the one specified. In another example, the term “consisting essentially of” can be utilized to indicate a nanocarrier and a therapeutic agent with no other ingredients that are listed in a claim and yet including any other ingredients that are not specifically listed.

[0073] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference. The term “feature” and the term “detail” can be interchanged with a “claim”. Any list of features, details, examples, embodiments, and/or aspects herein can be placed into a “claim”.

[0074] As used herein, the term "subject" refers to a mammal, bird, or the like, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), racing mammals, laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex. In another example, the term “subject” can refer to a connective tissue culture, and the methods disclosed herein, while claimed towards subjects, contemplate use in the laboratory in synthetic tissue(s). As used herein, a female cell can refer to a cell with 2X chromosomes; a male cell can refer to a cell with 1X and 1Y chromosome.

[0075] As used herein, the terms "effective amount" and “therapeutically effective amount” include an amount sufficient to modulate a treatment or prevent or ameliorate a manifestation of disease or medical condition, such as a connective tissue condition or a risk of a connective tissue injury. Such a condition (or risk) may not be readily discernable and may take years, statistical analysis, and/or machine learning to determine a prevention, treatment, or amelioration. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the condition and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0076] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, manage, modulate, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect (undesirable characteristic) or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (/.e., not worsening) state of a condition or decay, delay or slowing of a progression and/or risk of injury, and an increased lifespan/enjoyment as compared to that expected in the absence of treatment. [0077] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. The therapeutic agent or drug may refer to a formulation, composition, or agent. The formulation can be changed to a fresh formulation during administration. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long- acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1 , 2, 3, 5, 7 or 10 years, or more.

[0078] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by any technique known in the art that is subsequently applied to a subject, topical application, absorption, injection, ingestion, transfusion, implantation or transplantation. In an example embodiment, compositions are applied as a tablet or drug in capsule. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subdermal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. It is known in the art that therapeutic agents can be rapidly deployed through the skin and directly into joint/ligaments by use of DMSO (dimethyl sulfoxide) as a carrier solvent applied (with the therapeutic agent) to the skin near to or surrounding a joint. While DMSO is rarely used anymore for these purposes because of its nature as a universal solvent and its tendency to carry any residual chemicals present on the skin into the bloodstream (along with the intended agent), the technology contemplates such uses. In one contemplated embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tissue, lymph node, or site of treatment. In another example, administration is provided in the form of a natural product, vitamin, supplement, food, aerosol, inhalation, vapor, or drink. Formulations disclosed herein can be ready made or require mixing just before administration.

[0079] Any of the methods disclosed herein can be carried out in part or completely by including a dietary change, a food, natural product, precursor, or prodrug of a therapeutic agent. As used herein, a precursor or a prodrug is intended to encompass compounds or therapeutic agents which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., a compound for any of the present claims or features). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host subject. For example, esters or carbonates (e.g., esters or carbonates of alcohols or of carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the small-molecule chemical structures selected from this disclosure can be replaced with the corresponding suitable prodrug, for example, wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. A common method of making a precursor/prodrug that can be used herein is to use a carrier/nanocarrier (e.g., mesoporous silica particles). The precursor/prodrug can be released from a carrier to form the active therapeutic agent. A precursor or prodrug can be metabolized to the active parent compound (therapeutic agent) in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid). No argument can be made that the term “prodrug” is not enabled herein based on an assertion that actual prodrugs were not made and tested.

[0080] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0081] The terms: “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0082] As used herein, the term: “small molecule” refers to a molecule that has a molecular weight < 1000. As used herein, the term: “large molecule” refers to a molecule that has repeating units or that has a molecular weight > 1000, and the term includes biologies such as the examples of oligonucleotides, peptides, antibodies, linkers, oligosaccharides, polymers, DNA chains, and RNA chains. The term: “therapeutic agent” may refer to small molecule, element, large molecule, biologic, formulation, composition, agent, or a combination thereof.

PHARMACEUTICAL COMPOSITIONS

[0083] The compositions and methods of the present invention may be utilized to prevent a need for other treatment, to provide benefit when other treatment(s) fail, or to treat an individual in need thereof. In some embodiments, the individual is suspected of needing treatment. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. A compound can represent a combination therapy herein. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (/.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. Compositions can be in gas forms. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0084] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0085] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0086] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible compositions employed in pharmaceutical formulations.

[0087] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally, for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue; absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermal administration (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. Inhalation can include inhalation of a liquid (droplets or aerosol). Inhalation can include a micronized powder adhered to carrier particles or can be without carrier particles. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541 ,231 , 5,427,798, 5,358,970 and 4,172,896, each and all of which are incorporated herein by reference in their entireties, as well as in patents cited therein.

[0088] In the embodiments discussed and in any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0089] Other terms are defined herein within the description of the various aspects of the invention or are used as would be understood by an ordinary person.

A NOVEL METHOD TO TARGE PFGBP130 AS A VACCINE FOR P. FALCIPARUM MALARIA

[0090] Malaria remains a devastating global health challenge, with Plasmodium falciparum being the most virulent species responsible for the majority of malaria-related morbidity and mortality. Traditional approaches to malaria vaccination have focused on targeting various stages of the parasite's lifecycle, including the pre-erythrocytic, erythrocytic, and sexual stages. Early vaccine candidates primarily targeted the circumsporozoite protein (CSP) of the sporozoite stage, aiming to prevent the parasite from establishing infection in the liver. While some of these candidates, such as RTS,S/AS01 , have shown partial efficacy, they have not provided comprehensive protection against malaria.

[0091] Another approach has involved the development of vaccines targeting the blood-stage of the parasite, which is responsible for the clinical symptoms of malaria. These vaccines have aimed to induce immune responses against merozoite surface proteins, such as MSP1 and AMA1, which are involved in the invasion of red blood cells. Despite extensive research, these candidates have faced challenges in achieving high levels of efficacy, partly due to the genetic diversity of the parasite and the complexity of the immune responses required to effectively neutralize the parasite.

[0092] Recent efforts have also explored the use of whole-parasite vaccines, which involve the administration of attenuated or inactivated parasites to elicit a broad immune response. While these approaches have shown promise in preclinical and early clinical trials, they present logistical challenges in terms of production, storage, and delivery, which have hindered their widespread implementation. However, none of these approaches have provided a comprehensive solution that combines the novel and life-saving features described in this disclosure.

[0093] In some embodiments, the present invention relates to a malaria vaccine composition comprising Plasmodium falciparum PfGBP130 or PfGBP130-A surface antigen sequences. Upon administration to a human subject, the vaccine composition stimulates the production of antibodies that inhibit the invasion of red blood cells by Plasmodium falciparum parasites. Additionally, the antibodies bind to and kill parasites residing within red blood cells. This dual action of inhibiting invasion and targeting intracellular parasites effectively prevents and/or treats malaria infection in the vaccinated subject. The vaccine composition offers a promising approach to malaria prevention and treatment by targeting critical stages of the parasite's lifecycle within the human host.

[0094] In some embodiments, the techniques described herein relate to a malaria vaccine composition including: Plasmodium falciparum PfGBP130 or PfGBP130-A surface antigen sequences, wherein administration of the vaccine composition to a human subject stimulates production of antibodies that inhibit red blood cell invasion by Plasmodium falciparum parasites and/or bind to and kill parasites within red blood cells, thereby preventing and/or treating malaria infection in the vaccinated subject.

[0095] According to some example aspects, the techniques described herein relate to a malaria vaccine composition, wherein the antibodies block parasite invasion of red blood cells by binding to specific epitopes on the PfGBP130 or PfGBP130-A surface antigens, thereby preventing the parasites from entering and infecting the red blood cells, and subsequently inhibiting the growth and replication of the parasites within the host's bloodstream.

[0096] In some embodiments, the techniques described herein relate to a malaria vaccine composition, wherein the antibodies kill parasites inside red blood cells by recognizing and binding to the PfGBP130 or PfGBP130-A surface antigens expressed on the surface of infected red blood cells, thereby triggering antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mechanisms that lead to the destruction of the parasitized red blood cells and the elimination of the parasites from the host's body.

[0097] According to some example aspects, the techniques described herein relate to a malaria vaccine composition, wherein the vaccine composition prevents malaria infection in the vaccinated subject by inducing a robust and long-lasting humoral immune response characterized by high titers of PfGBP130 or PfGBP130-A-specific antibodies that circulate in the bloodstream and provide protection against future Plasmodium falciparum infections.

[0098] In some embodiments, the techniques described herein relate to a malaria vaccine composition, wherein the vaccine composition treats malaria infection in the vaccinated subject by stimulating the production of PfGBP130 or PfGBP130-A-specific antibodies that target and eliminate Plasmodium falciparum-infected red blood cells, thereby reducing the parasite burden, alleviating the clinical symptoms of malaria, and promoting the resolution of the infection.

[0099] According to some example aspects, the techniques described herein relate to a malaria vaccine composition, wherein the Plasmodium falciparum antigens include PfGBP130 surface antigen sequences, which are highly conserved among different Plasmodium falciparum strains and are critical for the parasite's ability to invade and infect red blood cells, making them ideal targets for vaccine- induced antibodies.

[0100] In some embodiments, the techniques described herein relate to a malaria vaccine composition, wherein the Plasmodium falciparum antigens include PfGBP130-A surface antigen sequences, which are specific to the asexual blood stages of the Plasmodium falciparum life cycle and play a crucial role in the parasite's survival and propagation within the human host, thus representing promising vaccine candidates for eliciting protective anti-malaria immunity.

[0101] According to some example aspects, the techniques described herein relate to a malaria vaccine composition, wherein the antibodies produced are anti-malaria antibodies that specifically recognize and bind to the PfGBP130 or PfGBP130-A surface antigens, exhibiting high affinity and avidity for their target epitopes and mediating various effector functions that contribute to the control and clearance of Plasmodium falciparum infections.

[0102] In some embodiments, the techniques described herein relate to a method of preventing and/or treating malaria infection in a human subject, the method including: administering to the human subject a malaria vaccine composition including Plasmodium falciparum PfGBP130 or PfGBP130-A surface antigen sequences, wherein the administration stimulates production of antibodies that inhibit red blood cell invasion by Plasmodium falciparum parasites and/or bind to and kill parasites within red blood cells, thereby preventing and/or treating the malaria infection in the subject.

[0103] According to some example aspects, the techniques described herein relate to a method, wherein the antibodies block parasite invasion of red blood cells by recognizing and binding to specific epitopes on the PfGBP130 or PfGBP130-A surface antigens that are involved in the initial attachment and entry of the parasites into the red blood cells, thereby interfering with the parasite's ability to establish a successful infection.

[0104] In some embodiments, the techniques described herein relate to a method, wherein the antibodies kill parasites inside red blood cells by opsonizing the infected red blood cells and facilitating their phagocytosis by macrophages and other immune cells, as well as by activating complement-mediated lysis of the parasitized red blood cells, leading to the release and exposure of the parasites to the host's immune system for elimination.

[0105] According to some example aspects, the techniques described herein relate to a method, wherein the vaccine composition prevents malaria infection in the vaccinated subject by eliciting a strong and durable antibody response that can neutralize the invading Plasmodium falciparum sporozoites and merozoites, preventing them from infecting the liver cells and red blood cells, respectively, and thus breaking the parasite's life cycle and transmission.

[0106] In some embodiments, the techniques described herein relate to a method, wherein the vaccine composition treats malaria infection in the vaccinated subject by boosting the production of PfGBP130 or PfGBP130-A-specific antibodies that can recognize and target the infected red blood cells, leading to their destruction and the release of the parasites, which can then be eliminated by the host's immune system, ultimately resulting in the resolution of the infection and the alleviation of the clinical symptoms.

[0107] According to some example aspects, the techniques described herein relate to a method, wherein the Plasmodium falciparum antigens include PfGBP130 surface antigen sequences that are expressed on the surface of the parasite at different stages of its life cycle, including the sporozoite, merozoite, and gametocyte stages, making them attractive targets for vaccine-induced antibodies that can interfere with the parasite's development and transmission.

[0108] In some embodiments, the techniques described herein relate to a method, wherein the Plasmodium falciparum antigens include PfGBP130-A surface antigen sequences that are exclusively expressed on the surface of the infected red blood cells during the asexual blood stages of the parasite's life cycle, serving as specific markers for the identification and targeting of the parasitized cells by the vaccine-induced antibodies.

[0109] According to some example aspects, the techniques described herein relate to a method, wherein the antibodies produced are anti-malaria antibodies that belong to different immunoglobulin classes and subclasses, including lgG1 , lgG2, lgG3, and lgG4, each with distinct effector functions and half-lives, providing a comprehensive and long-lasting protective immunity against Plasmodium falciparum infections.

[0110] In some embodiments, the techniques described herein relate to a method, wherein administering the vaccine composition delivers the Plasmodium falciparum antigens to the human subject in a safe, stable, and immunogenic form, such as recombinant proteins, synthetic peptides, or virus-like particles, along with appropriate adjuvants and delivery systems that enhance the immunogenicity and efficacy of the vaccine.

[0111] According to some example aspects, the techniques described herein relate to a method, wherein administering the vaccine composition provides immunity against malaria to the human subject by stimulating the production of PfGBP130 or PfGBP130-A-specific memory B cells and long-lived plasma cells that can maintain high levels of circulating antibodies and mount rapid and robust anamnestic responses upon re-exposure to the parasite antigens.

[0112] In some embodiments, the techniques described herein relate to a method, wherein the vaccine composition stimulates antibody production in the human subject through the activation of antigen-specific B cells that undergo clonal expansion, affinity maturation, and differentiation into antibody-secreting plasma cells, as well as the generation of memory B cells that can provide long-term protection against future Plasmodium falciparum infections.

[0113] According to some example aspects, the techniques described herein relate to a method, wherein the antibodies produced in the human subject are malaria-specific antibodies that can cross-react with multiple Plasmodium falciparum strains and isolates from different geographical regions, providing broad-spectrum protection against diverse parasite populations and reducing the risk of vaccine escape and resistance.

[0114] In some embodiments, the techniques described herein relate to a malaria vaccine composition, wherein the vaccine composition is formulated as a sterile, aqueous suspension containing the PfGBP130 or PfGBP130-A surface antigen sequences at a concentration of 50- 200 pg/mL, along with suitable excipients, stabilizers, and preservatives that maintain the stability, integrity, and immunogenicity of the antigens during storage and administration.

[0115] According to some example aspects, the techniques described herein relate to a malaria vaccine composition, wherein the vaccine composition is administered to the human subject via intramuscular, subcutaneous, or intradermal routes, using a prime-boost regimen that includes at least two doses of the vaccine given at least 4 weeks apart, to ensure the induction of a robust and long-lasting antibody response against the PfGBP130 or PfGBP130-A surface antigens.

[0116] In some embodiments, the techniques described herein relate to a method, wherein administering the vaccine composition to the human subject is performed in endemic areas with high transmission rates of Plasmodium falciparum malaria, as part of a comprehensive malaria control and elimination program that includes vector control measures, case management, and surveillance, to maximize the impact and effectiveness of the vaccine in reducing the burden and transmission of the disease.

[0117] According to some example aspects, the techniques described herein relate to a method, wherein the vaccine composition is co-administered with other malaria vaccine candidates targeting different stages of the Plasmodium falciparum life cycle, such as pre- erythrocytic vaccines that prevent liver cell infection and asexual blood-stage vaccines that inhibit red blood cell invasion and parasite growth, to provide a multi-stage, multi-antigen vaccine approach that enhances the overall protective efficacy and durability of the immune response.

[0118] In another example, this disclosure provides a malaria vaccine composition that may be configured to deliver Plasmodium falciparum proteins or amino acid sequences, including PfGBP130 or PfGBP130-A surface antigen sequences, a portion of or coding sequences for either. This composition may be administered to a human subject to produce anti-malaria antibodies. The antibodies generated can inhibit the invasion of red blood cells by Plasmodium falciparum malaria parasites or can cause the death of the parasites within red blood cells, potentially preventing and/or treating malaria in the human subject. The vaccine composition may include sequences derived from PfGBP130 or PfGBP130-A, formulated as recombinant proteins, mRNA, or DNA plasmids. The antibodies produced may specifically target the Plasmodium falciparum proteins or amino acid sequences, thereby neutralizing the malaria parasites. The vaccine composition may be formulated in various delivery systems, such as lipid nanoparticle encapsulation, virus-like particles, nanoparticles, conjugates, or plasmid vectors, to enhance its efficacy in preventing malaria.

[0119] According to an embodiment, the production of anti-malaria antibodies may be initiated when the malaria vaccine composition is administered to a human subject. This process may involve the antibodies being produced in response to the presence of Plasmodium falciparum proteins or amino acid sequences, including PfGBP130 or PfGBP130-A surface antigen sequences. The antibodies may then function to inhibit the invasion of red blood cells by Plasmodium falciparum malaria parasites or can cause the death of the parasites within red blood cells. This inhibition may occur through the binding of the antibodies to the parasites, potentially neutralizing their ability to invade red blood cells. The vaccine composition may be configured to include sequences from PfGBP130 or PfGBP130-A, which may be formulated as a recombinant protein or derived from a nucleic acid coding sequence. The antibodies generated may be specific to the Plasmodium falciparum proteins or amino acid sequences, thereby providing a targeted response to the malaria parasites. The vaccine may also include mRNA or DNA encoding for specific sequences, which may enhance the production of antibodies within the human subject. The overall process may aim to prevent and/or treat malaria by effectively inhibiting the red blood cell invasion by the malaria parasites.

[0120] In a discussion, study or a reading of the details, features, embodiments, aspects, FIGs., and/or examples of the technology disclosed herein, any of the features, embodiments, aspects, and/or examples herein can be optionally inter-combined (or inter-discussed) with the example details listed below, and any portion (or aspect) of any detail below can be intercombined with any portion of any feature or example disclosed herein:

[0121] Detail 1 : A malaria vaccine composition configured to provide Plasmodium falciparum proteins or amino acid sequences including PfGBP130 or PfGBP130-A surface antigen sequences, wherein when the malaria vaccine composition is administered to a human subject, specific anti-malaria antibodies are produced; the malaria vaccine composition being configured to provide a production of antibodies within the human subject that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites and/or can cause the death of the parasites within red blood cells, thereby preventing and/or treating malaria in the human subject, wherein the vaccine composition optionally comprises one or more adjuvants, for example, selected from the group consisting of aluminum hydroxide, aluminum phosphate, calcium phosphate, oil emulsions, toll-like receptor (TLR) agonists, and/or saponins.

[0122] Detail 2: The malaria vaccine composition of detail 1 , wherein the Plasmodium falciparum proteins or amino acid sequences are derived from PfGBP130 or PfGBP130-A and/or wherein the vaccine includes sequences from PfGBP130 or PfGBP130-A formulated as a recombinant protein expressed in a suitable expression system selected from the group consisting of bacteria, yeast, insect cells, and/or mammalian cells.

[0123] Detail 3: The malaria vaccine composition of detail 1 , wherein the Plasmodium falciparum proteins or amino acid sequences are derived from a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences and/or wherein the vaccine includes mRNA encoding for PfGBP130-A (SEQ ID NO: 1) or a PfGBP130- ecto (SEQ ID NO: 2), wherein the mRNA is modified to enhance stability and/or translation efficiency.

[0124] Detail 4: The malaria vaccine composition of detail 1 , wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130 surface antigen sequences (SEQ ID NO: 3), wherein the PfGBP130 surface antigen sequences are glycosylated and/or conjugated to a carrier protein to enhance immunogenicity.

[0125] Detail 5: The malaria vaccine composition of detail 1 , wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130-A surface antigen sequences (SEQ ID NO: 4), wherein the PfGBP130-A surface antigen sequences are formulated as viruslike particles (VLPs) or nanoparticles to enhance uptake by antigen-presenting cells.

[0126] Detail 6: The malaria vaccine composition of detail 1 , wherein the antibodies generated within the human subject are specific to the Plasmodium falciparum proteins or amino acid sequences and have a high affinity and/or avidity for their target antigens, with dissociation constants in the nanomolar or picomolar range.

[0127] Detail 7: The malaria vaccine composition of detail 1 , wherein the antibodies generated within the human subject are specific to the PfGBP130 or PfGBP130-A surface antigen sequences and/or wherein the vaccine includes a DNA plasmid (SEQ ID NO: 5) encoding PfGBP130-A, wherein the DNA plasmid is optimized for expression in human cells and includes a strong promoter and enhancer elements.

[0128] Detail 8: The malaria vaccine composition of detail 1 , wherein the antibodies inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites and/or can cause the death of the parasites within red blood cells, wherein the antibodies recognize conformational epitopes on the parasite surface that are critical for invasion.

[0129] Detail 9: The malaria vaccine composition of detail 1 , wherein the antibodies inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites and/or can cause the death of the parasites within red blood cells, wherein the antibodies block key interactions between parasite ligands and red blood cell receptors that are essential for invasion.

[0130] Detail 10: The malaria vaccine composition of detail 1 , wherein the malaria vaccine composition includes amino acids (aa) 111-374 of PfGBP130-A (SEQ ID NO: 6) and/or includes aa 89-824 of PfGBP130 (SEQ ID NO: 7), wherein these amino acid regions contain critical epitopes recognized by protective antibodies.

[0131] Detail 11 : The malaria vaccine of detail 1 , wherein the vaccine includes a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences or includes mRNA (SEQ ID NO: 8) and/or DNA (SEQ ID NO: 9) that encodes for aa 89-824 of PfGBP130-ecto (SEQ ID NO: 10) and/or encodes for aa 111-374 of PfGBP130-A (SEQ ID NO: 11), wherein the nucleic acid sequences are codon-optimized for expression in human cells.

[0132] Detail 12: The malaria vaccine composition of detail 1 , wherein the malaria vaccine composition treats malaria in the human subject and/or wherein the malaria vaccine is formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector including SEQ ID NO: 12, or a combination thereof, wherein the formulation enhances delivery and/or stability of the vaccine components.

[0133] Detail 13: A method of preventing and/or treating malaria in a human subject, the method comprising administering to the human subject a malaria vaccine composition configured to provide Plasmodium falciparum proteins or amino acid sequences including PfGBP130 or PfGBP130-A surface antigen sequences, wherein when the malaria vaccine composition is administered to the human subject specific anti-malaria antibodies are produced; the malaria vaccine composition being configured to provide a production of antibodies within the human subject that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites and/or can cause the death of the parasites within red blood cells, wherein the vaccine composition is administered in a prime-boost regimen to enhance the magnitude and durability of the immune response.

[0134] Detail 14: The method of detail 13, wherein the Plasmodium falciparum proteins or amino acid sequences are derived from PfGBP130 or PfGBP130-A formulated as a recombinant protein purified using affinity chromatography and/or size-exclusion chromatography to enhance purity and minimize contaminants.

[0135] Detail 15: The method of detail 13, wherein the Plasmodium falciparum proteins or amino acid sequences are derived from a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences or wherein the vaccine includes mRNA encoding for PfGBP130-A (SEQ ID NO: 1) or a PfGBP130-ecto (SEQ ID NO: 2), wherein the mRNA is formulated in lipid nanoparticles to enhance delivery and stability. [0136] Detail 16: The method of detail 13, wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130 surface antigen sequences (SEQ ID NO: 3), wherein the PfGBP130 surface antigen sequences are conjugated to a toll-like receptor (TLR) agonist to enhance immune activation.

[0137] Detail 17: The method of detail 13, wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130-A surface antigen sequences (SEQ ID NO: 4), wherein the PfGBP130-A surface antigen sequences are formulated with a saponin-based adjuvant to enhance T cell responses.

[0138] Detail 18: The method of detail 13, wherein the antibodies generated within the human subject are specific to the Plasmodium falciparum proteins or amino acid sequences and are predominantly of the lgG1 and lgG3 subclasses, which are known to have potent effector functions.

[0139] Detail 19: The method of detail 13, wherein the antibodies generated within the human subject are specific to the PfGBP130 or PfGBP130-A surface antigen sequences and/or wherein the vaccine includes a DNA plasmid (SEQ ID NO: 5) encoding PfGBP130-A, wherein the DNA plasmid is delivered using electroporation to enhance cellular uptake and expression.

[0140] Detail 20: The method of detail 13, wherein the antibodies inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites and/or can cause the death of the parasites within red blood cells, wherein the antibodies induce complement-mediated lysis of the parasites.

[0141] Detail 21: The method of detail 13, wherein the antibodies inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites and/or can cause the death of the parasites within red blood cells, wherein the antibodies induce antibody-dependent cellular cytotoxicity (ADCC) against the parasites.

[0142] Detail 22: The method of detail 13, wherein the malaria vaccine composition includes amino acids (aa) 111-374 of PfGBP130-A (SEQ ID NO: 6) and/or includes aa 89-824 of PfGBP130 (SEQ ID NO: 7), wherein these amino acid regions are highly conserved among Plasmodium falciparum strains to provide broad protection.

[0143] Detail 23: The method of detail 13, wherein the vaccine includes mRNA (SEQ ID NO: 8) and/or DNA (SEQ ID NO: 9) that encodes for aa 89-824 of PfGBP130-ecto (SEQ ID NO: 10) and/or encodes for aa 111-374 of PfGBP130-A (SEQ ID NO: 11), wherein the mRNA and/or DNA is formulated with a cationic lipid to enhance delivery and expression. [0144] Detail 24: The method of detail 13, wherein the malaria vaccine composition treats malaria in the human subject and/or wherein the malaria vaccine is formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector including SEQ ID NO: 12, or a combination thereof, wherein the formulation targets the vaccine components to key immune cells such as dendritic cells.

[0145] Detail 25: The method of detail 13, wherein the malaria vaccine composition is administered intramuscularly, intradermally, subcutaneously, or using a needle-free delivery device to enhance patient compliance and/or reduce injection site reactions.

[0146] Detail 26: The method of detail 13, wherein the malaria vaccine composition is administered in a single dose or in multiple doses spaced several weeks or months apart to optimize the immune response and/or align with existing vaccination schedules.

[0147] Detail 27: The method of detail 13, wherein the malaria vaccine composition is coadministered with other vaccines targeting different life cycle stages of the malaria parasite and/or other endemic diseases to provide comprehensive protection.

[0148] Detail 28: The method of detail 13, wherein the efficacy of the malaria vaccine composition is assessed by measuring the incidence of clinical malaria, the density of parasitemia, and/or the level of anti-malaria antibodies in the human subject following vaccination.

[0149] Detail 29: A malaria vaccine composition comprising: Plasmodium falciparum surface antigen sequences, wherein the Plasmodium falciparum surface antigen sequences are selected from the group consisting of PfGBP130, PfGBP130-A, and combinations thereof; a pharmaceutically acceptable carrier; and optionally, one or more adjuvants; wherein when the malaria vaccine composition is administered to a human subject, the malaria vaccine composition induces production of antibodies specific to Plasmodium falciparum in the human subject, and wherein the antibodies inhibit invasion of red blood cells by Plasmodium falciparum parasites and/or can cause the death of the parasites within red blood cells, thereby preventing or treating malaria in the human subject.

[0150] Detail 30: The malaria vaccine composition of detail 29, wherein the PfGBP130-A surface antigen sequences comprise amino acids 111-374 of SEQ ID NO: 6.

[0151] Detail 31: The malaria vaccine composition of detail 29, wherein the PfGBP130 surface antigen sequences comprise amino acids 89-824 of SEQ ID NO: 7.

[0152] Detail 32: The malaria vaccine composition of detail 29, wherein the Plasmodium falciparum surface antigen sequences are recombinantly expressed in a host cell selected from the group consisting of bacterial cells, yeast cells, insect cells, and mammalian cells.

[0153] Detail 33: The malaria vaccine composition of detail 29, wherein the Plasmodium falciparum surface antigen sequences are conjugated to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid, diphtheria toxoid, and CRM 197.

[0154] Detail 34: The malaria vaccine composition of detail 29, wherein the one or more adjuvants are selected from the group consisting of aluminum salts, oil-in-water emulsions, water- in-oil emulsions, saponins, liposomes, microparticles, nanoparticles, Toll-like receptor (TLR) agonists, and combinations thereof.

[0155] Detail 35: The malaria vaccine composition of detail 29, wherein the malaria vaccine composition is formulated for administration via a route selected from the group consisting of intramuscular, subcutaneous, intradermal, oral, intranasal, sublingual, and transdermal.

[0156] Detail 36: The malaria vaccine composition of detail 29, wherein the malaria vaccine composition is lyophilized.

[0157] Detail 37: The malaria vaccine composition of detail 29, wherein the malaria vaccine composition is a monovalent vaccine or a multivalent vaccine.

[0158] Detail 38: The malaria vaccine composition of detail 29, wherein the malaria vaccine composition is a nucleic acid vaccine, a protein vaccine, a peptide vaccine, or a virus-like particle vaccine.

[0159] Detail 39: A method of preventing or treating malaria in a human subject, the method comprising: administering to the human subject a malaria vaccine composition comprising Plasmodium falciparum surface antigen sequences, wherein the Plasmodium falciparum surface antigen sequences are selected from the group consisting of PfGBP130, PfGBP130-A, and combinations thereof; wherein the malaria vaccine composition induces production of antibodies specific to Plasmodium falciparum in the human subject, and wherein the antibodies inhibit invasion of red blood cells by Plasmodium falciparum parasites and/or can cause the death of the parasites within red blood cells, thereby preventing or treating malaria in the human subject.

[0160] Detail 40: The method of detail 39, wherein the PfGBP130-A surface antigen sequences comprise amino acids 111-374 of SEQ ID NO: 6.

[0161] Detail 41 : The method of detail 39, wherein the PfGBP130 surface antigen sequences comprise amino acids 89-824 of SEQ ID NO: 7.

[0162] Detail 42: The method of detail 39, wherein the Plasmodium falciparum surface antigen sequences are recombinantly expressed in a host cell selected from the group consisting of bacterial cells, yeast cells, insect cells, and mammalian cells.

[0163] Detail 43: The method of detail 39, wherein the Plasmodium falciparum surface antigen sequences are conjugated to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid, diphtheria toxoid, and CRM197.

[0164] Detail 44: The method of detail 39, wherein the malaria vaccine composition further comprises one or more adjuvants selected from the group consisting of aluminum salts, oil-in- water emulsions, water-in-oil emulsions, saponins, liposomes, microparticles, nanoparticles, Tolllike receptor (TLR) agonists, and combinations thereof.

[0165] Detail 45: The method of detail 39, wherein administering the malaria vaccine composition to the human subject comprises one or more of intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, sublingual administration, or transdermal administration.

[0166] Detail 46: The method of detail 39, wherein the malaria vaccine composition is administered in a single dose or in multiple doses.

[0167] Detail 47: The method of detail 46, wherein the multiple doses are administered in a prime-boost vaccination regimen.

[0168] Detail 48: The method of detail 39, wherein the malaria vaccine composition is a monovalent vaccine or a multivalent vaccine.

[0169] Detail 49: The method of detail 39, wherein the malaria vaccine composition is a nucleic acid vaccine, a protein vaccine, a peptide vaccine, or a virus-like particle vaccine.

[0170] Detail 50: The method of detail 39, wherein the human subject is a child under the age of 5 years, a pregnant woman, or an immunocompromised individual.

[0171] Detail 51 : The method of detail 39, wherein the human subject resides in or travels to a malaria-endemic region.

[0172] Detail 52: The method of detail 39, wherein the malaria vaccine composition is administered in combination with one or more additional antimalarial interventions selected from the group consisting of insecticide-treated bed nets, indoor residual spraying, and antimalarial drugs.

[0173] Detail 53: The method of detail 39, further comprising: determining the efficacy of the malaria vaccine composition by measuring one or more of the following: (i) the level of antibodies specific to Plasmodium falciparum in the human subject; (ii) the inhibition of red blood cell invasion by Plasmodium falciparum parasites in the human subject and/or the antibodies binding can cause the death of the parasites within red blood cells; (iii) the reduction in the incidence of clinical malaria episodes in the human subject; and (iv) the reduction in the severity of malaria symptoms in the human subject.

[0174] Detail 54: The method of detail 39, further comprising: monitoring the safety of the malaria vaccine composition by assessing the occurrence of adverse events in the human subject following administration of the malaria vaccine composition.

[0175] Detail 55: A kit comprising: the malaria vaccine composition of detail 29; and instructions for administering the malaria vaccine composition to a human subject to prevent or treat malaria.

[0176] Detail 56: A malaria vaccine composition comprising: Plasmodium falciparum surface antigen sequences, wherein the Plasmodium falciparum surface antigen sequences are selected from the group consisting of PfGBP130, PfGBP130-A, and combinations thereof, and/or wherein the Plasmodium falciparum surface antigen sequences are recombinantly expressed in a host cell selected from the group consisting of bacterial cells, yeast cells, insect cells, and mammalian cells, and/or wherein the Plasmodium falciparum surface antigen sequences are purified to a purity of at least 95% as determined by SDS-PAGE analysis, and/or wherein the Plasmodium falciparum surface antigen sequences are formulated at a concentration of 1 pg/mL to 1000 pg/mL; a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from the group consisting of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof; and one or more adjuvants selected from the group consisting of aluminum salts, oil-in-water emulsions, water-in-oil emulsions, saponins, liposomes, microparticles, nanoparticles, Toll-like receptor (TLR) agonists, and combinations thereof, wherein the one or more adjuvants are present at a concentration of 1 pg/mL to 1000 pg/mL; wherein when the malaria vaccine composition is administered to a human subject via a route selected from the group consisting of intramuscular, subcutaneous, intradermal, oral, intranasal, sublingual, and transdermal, the malaria vaccine composition induces production of IgG antibodies specific to Plasmodium falciparum in the human subject at a titer of at least 1:10,000 as determined by ELISA, and/or wherein the IgG antibodies inhibit invasion of red blood cells by Plasmodium falciparum parasites by at least 90% as determined by a growth inhibition assay, thereby preventing or treating malaria in the human subject, and/or wherein the malaria vaccine composition provides protection against malaria for at least 6 months after administration.

[0177] Detail 57: The malaria vaccine composition of detail 56, wherein the PfGBP130-A surface antigen sequences comprise amino acids 111-374 of SEQ ID NO: 6, and/or wherein the PfGBP130-A surface antigen sequences are glycosylated.

[0178] Detail 58: The malaria vaccine composition of detail 56, wherein the PfGBP130 surface antigen sequences comprise amino acids 89-824 of SEQ ID NO: 7, and/or wherein the PfGBP130 surface antigen sequences are lipidated.

[0179] Detail 59: The malaria vaccine composition of detail 56, wherein the Plasmodium falciparum surface antigen sequences are conjugated to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid, diphtheria toxoid, and CRM197, and/or wherein the conjugation is performed using a bifunctional crosslinking agent selected from the group consisting of succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMOG), succinimidyl 6- ((iodoacetyl)amino)hexanoate (SIAX), and succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB).

[0180] Detail 60: The malaria vaccine composition of detail 56, wherein the aluminum salts are selected from the group consisting of aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate, and/or wherein the aluminum salts are present at a concentration of 100 pg/mL to 1000 pg/mL.

[0181] Detail 61 : The malaria vaccine composition of detail 56, wherein the oil-in-water emulsions comprise squalene, polysorbate 80, and sorbitan trioleate, and/or wherein the squalene is present at a concentration of 2% to 10% (v/v), the polysorbate 80 is present at a concentration of 0.1 % to 3% (v/v), and the sorbitan trioleate is present at a concentration of 0.1 % to 3% (v/v).

[0182] Detail 62: The malaria vaccine composition of detail 56, wherein the water-in-oil emulsions comprise mineral oil, mannide monooleate, and Arlacel A, and/or wherein the mineral oil is present at a concentration of 30% to 90% (v/v), the mannide monooleate is present at a concentration of 5% to 30% (v/v), and the Arlacel A is present at a concentration of 1% to 10% (v/v).

[0183] Detail 63: The malaria vaccine composition of detail 56, wherein the saponins are selected from the group consisting of QS-21 , QS-7, QS-17, QS-18, and QS-L1, and/or wherein the saponins are present at a concentration of 1 pg/mL to 500 pg/mL.

[0184] Detail 64: The malaria vaccine composition of detail 56, wherein the liposomes comprise phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and/or wherein the liposomes have a mean diameter of 50 nm to 2000 nm.

[0185] Detail 65: The malaria vaccine composition of detail 56, wherein the microparticles are selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA) microparticles, chitosan microparticles, and alginate microparticles, and/or wherein the microparticles have a mean diameter of 0.5 pm to 10 pm.

[0186] Detail 66: The malaria vaccine composition of detail 56, wherein the nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, silica nanoparticles, and carbon nanoparticles, and/or wherein the nanoparticles have a mean diameter of 1 nm to 200 nm.

[0187] Detail 67: The malaria vaccine composition of detail 56, wherein the TLR agonists are selected from the group consisting of imiquimod, resiquimod, CpG oligodeoxynucleotides, poly(l:C), and monophosphoryl lipid A, and/or wherein the TLR agonists are present at a concentration of 1 pg/mL to 1000 pg/mL.

[0188] Detail 68: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is lyophilized, and/or wherein the lyophilized malaria vaccine composition is reconstituted with a diluent selected from the group consisting of water, saline, and phosphate- buffered saline prior to administration.

[0189] Detail 69: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is a monovalent vaccine comprising only one type of Plasmodium falciparum surface antigen sequence.

[0190] Detail 70: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is a multivalent vaccine comprising two or more types of Plasmodium falciparum surface antigen sequences.

[0191] Detail 71: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is a nucleic acid vaccine comprising a nucleic acid sequence encoding the Plasmodium falciparum surface antigen sequences, and/or wherein the nucleic acid sequence is operably linked to a promoter sequence. [0192] Detail 72: The malaria vaccine composition of detail 71 , wherein the nucleic acid sequence is a DNA sequence or an RNA sequence, and/or wherein the nucleic acid sequence is codon-optimized for expression in human cells.

[0193] Detail 73: The malaria vaccine composition of detail 71 , wherein the nucleic acid sequence is formulated in a lipid nanoparticle comprising ionizable cationic lipids, neutral lipids, cholesterol, and polyethylene glycol (PEG)-lipids.

[0194] Detail 74: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is a protein vaccine comprising the Plasmodium falciparum surface antigen sequences in the form of recombinant proteins.

[0195] Detail 75: The malaria vaccine composition of detail 74, wherein the recombinant proteins are expressed in a prokaryotic expression system or a eukaryotic expression system, and/or wherein the recombinant proteins are purified using affinity chromatography, ion-exchange chromatography, or size-exclusion chromatography.

[0196] Detail 76: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is a peptide vaccine comprising synthetic peptides corresponding to epitopes of the Plasmodium falciparum surface antigen sequences, and/or wherein the synthetic peptides are between 8 and 50 amino acids in length.

[0197] Detail 77: The malaria vaccine composition of detail 76, wherein the synthetic peptides are chemically synthesized using solid-phase peptide synthesis, and/or wherein the synthetic peptides are purified using high-performance liquid chromatography (HPLC).

[0198] Detail 78: The malaria vaccine composition of detail 56, wherein the malaria vaccine composition is a virus-like particle (VLP) vaccine comprising the Plasmodium falciparum surface antigen sequences displayed on the surface of a VLP selected from the group consisting of hepatitis B virus core antigen VLPs, human papillomavirus L1 VLPs, and bacteriophage Q|3 VLPs.

[0199] Detail 79: The malaria vaccine composition of detail 78, wherein the VLPs are produced by expressing the Plasmodium falciparum surface antigen sequences as fusion proteins with the VLP monomers in a suitable expression system, and/or wherein the VLPs are purified using ultracentrifugation or chromatography.

[0200] Detail 80: A method of preventing or treating malaria in a human subject, the method comprising: administering to the human subject a malaria vaccine composition comprising Plasmodium falciparum surface antigen sequences, wherein the Plasmodium falciparum surface antigen sequences are selected from the group consisting of PfGBP130, PfGBP130-A, and combinations thereof, and/or wherein the Plasmodium falciparum surface antigen sequences are recombinantly expressed in a host cell selected from the group consisting of bacterial cells, yeast cells, insect cells, and mammalian cells, and/or wherein the Plasmodium falciparum surface antigen sequences are purified to a purity of at least 95% as determined by SDS-PAGE analysis, and/or wherein the Plasmodium falciparum surface antigen sequences are formulated at a concentration of 1 pg/mL to 1000 pg/mL; wherein the malaria vaccine composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof, and one or more adjuvants selected from the group consisting of aluminum salts, oil-in-water emulsions, water-in-oil emulsions, saponins, liposomes, microparticles, nanoparticles, Toll-like receptor (TLR) agonists, and combinations thereof, wherein the one or more adjuvants are present at a concentration of 1 pg/mL to 1000 pg/mL; wherein the malaria vaccine composition is administered to the human subject via a route selected from the group consisting of intramuscular, subcutaneous, intradermal, oral, intranasal, sublingual, and transdermal, and/or wherein the malaria vaccine composition is administered in a single dose or in multiple doses, and/or wherein the multiple doses are administered in a prime-boost vaccination regimen; wherein the malaria vaccine composition induces production of IgG antibodies specific to Plasmodium falciparum in the human subject at a titer of at least 1 :10,000 as determined by ELISA within 4 weeks after administration, and/or wherein the IgG antibodies inhibit invasion of red blood cells by Plasmodium falciparum parasites by at least 90% as determined by a growth inhibition assay and/or can cause the death of the parasites within red blood cells, thereby preventing or treating malaria in the human subject, and/or wherein the malaria vaccine composition provides protection against malaria for at least 6 months after administration.

[0201] Detail 81 : The method of detail 80, wherein the PfGBP130-A surface antigen sequences comprise amino acids 111-374 of SEQ ID NO: 6, and/or wherein the PfGBP130-A surface antigen sequences are glycosylated.

[0202] Detail 82: The method of detail 80, wherein the PfGBP130 surface antigen sequences comprise amino acids 89-824 of SEQ ID NO: 7, and/or wherein the PfGBP130 surface antigen sequences are lipidated.

[0203] Detail 83: The method of detail 80, wherein the Plasmodium falciparum surface antigen sequences are conjugated to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid, diphtheria toxoid, and CRM 197, and/or wherein the conjugation is performed using a bifunctional crosslinking agent selected from the group consisting of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate (SMCC), succinimidyl 6-((iodoacetyl)amino)hexanoate (SIAX), and succinimidyl 4-(p- maleimidophenyl)butyrate (SMPB).

[0204] Detail 84: The method of detail 80, wherein the aluminum salts are selected from the group consisting of aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate, and/or wherein the aluminum salts are present at a concentration of 100 pg/mL to 1000 pg/mL.

[0205] Detail 85: The method of detail 80, wherein the oil-in-water emulsions comprise squalene, polysorbate 80, and sorbitan trioleate, and/or wherein the squalene is present at a concentration of 2% to 10% (v/v), the polysorbate 80 is present at a concentration of 0.1% to 3% (v/v), and the sorbitan trioleate is present at a concentration of 0.1 % to 3% (v/v).

[0206] Detail 86: The method of detail 80, wherein the water-in-oil emulsions comprise mineral oil, mannide monooleate, and Arlacel A, and/or wherein the mineral oil is present at a concentration of 30% to 90% (v/v), the mannide monooleate is present at a concentration of 5% to 30% (v/v), and the Arlacel A is present at a concentration of 1% to 10% (v/v).

[0207] Detail 87: The method of detail 80, wherein the saponins are selected from the group consisting of QS-21 , QS-7, QS-17, QS-18, and QS-L1, and/or wherein the saponins are present at a concentration of 1 pg/mL to 500 pg/mL.

[0208] Detail 88: The method of detail 80, wherein the liposomes comprise phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and/or wherein the liposomes have a mean diameter of 50 nm to 2000 nm.

[0209] Detail 89: The method of detail 80, wherein the microparticles are selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA) microparticles, chitosan microparticles, and alginate microparticles, and/or wherein the microparticles have a mean diameter of 0.5 pm to 10 pm.

[0210] Detail 90: The method of detail 80, wherein the nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, silica nanoparticles, and carbon nanoparticles, and/or wherein the nanoparticles have a mean diameter of 1 nm to 200 nm.

[0211] Detail 91: The method of detail 80, wherein the TLR agonists are selected from the group consisting of imiquimod, resiquimod, CpG oligodeoxynucleotides, poly(l:C), and monophosphoryl lipid A, and/or wherein the TLR agonists are present at a concentration of 1 pg/mL to 1000 pg/mL

[0212] Detail 92: The method of detail 80, wherein the human subject is a child under the age of 5 years, a pregnant woman, or an immunocompromised individual.

[0213] Detail 93: The method of detail 80, wherein the human subject resides in or travels to a malaria-endemic region selected from the group consisting of sub-Saharan Africa, Southeast Asia, the Pacific Islands, and South America.

[0214] Detail 94: The method of detail 80, wherein the malaria vaccine composition is administered in combination with one or more additional antimalarial interventions selected from the group consisting of insecticide-treated bed nets, indoor residual spraying, and antimalarial drugs, and/or wherein the antimalarial drugs are selected from the group consisting of artemisininbased combination therapies, chloroquine, amodiaquine, mefloquine, primaquine, and tafenoquine.

[0215] Detail 95: The method of detail 80, further comprising: determining the efficacy of the malaria vaccine composition by measuring one or more of the following: (i) the level of IgG antibodies specific to Plasmodium falciparum in the human subject at one or more time points after administration of the malaria vaccine composition using ELISA; (ii) the inhibition of red blood cell invasion by Plasmodium falciparum parasites in the human subject at one or more time points after administration of the malaria vaccine composition using a growth inhibition assay and/or the antibodies can cause the death of the parasites within red blood cells; (iii) the reduction in the incidence of clinical malaria episodes in the human subject over a period of 6 months to 2 years after administration of the malaria vaccine composition compared to a control group that did not receive the malaria vaccine composition; and (iv) the reduction in the severity of malaria symptoms in the human subject over a period of 6 months to 2 years after administration of the malaria vaccine composition compared to a control group that did not receive the malaria vaccine composition, wherein the severity of malaria symptoms is assessed using a standardized clinical scoring system.

[0216] Detail 96: The method of detail 80, further comprising: monitoring the safety of the malaria vaccine composition by assessing the occurrence of adverse events in the human subject following administration of the malaria vaccine composition, wherein the adverse events are selected from the group consisting of injection site reactions, fever, headache, myalgia, arthralgia, nausea, vomiting, diarrhea, and allergic reactions, and/or wherein the adverse events are graded according to the Common Terminology Criteria for Adverse Events (CTCAE) scale.

[0217] Detail 97: A kit comprising: the malaria vaccine composition of detail 56; and instructions for administering the malaria vaccine composition to a human subject to prevent or treat malaria, wherein the instructions comprise information on the dosage, route of administration, and schedule of administration of the malaria vaccine composition, and/or wherein the instructions further comprise information on the potential adverse events associated with the malaria vaccine composition and guidance on how to manage them.

[0218] Detail 98: The kit of detail 97, further comprising one or more additional components selected from the group consisting of: (i) a syringe or needle for administering the malaria vaccine composition; (ii) a sterile diluent for reconstituting the malaria vaccine composition; (iii) an antimalarial drug for use in combination with the malaria vaccine composition; and (iv) an insecticide-treated bed net or indoor residual spraying kit for use in combination with the malaria vaccine composition.

[0219] Detail 99: The kit of detail 97, wherein the instructions are provided in a written format, an electronic format, or a combination thereof, and/or wherein the instructions are in a language selected from the group consisting of English, French, Spanish, Portuguese, Arabic, Chinese, and/or Hindi.

[0220] Detail 100: The kit of detail 97, further comprising a temperature monitoring device for ensuring that the malaria vaccine composition is stored and transported at the appropriate temperature range, wherein the temperature monitoring device is selected from the group consisting of a thermometer, a temperature data logger, and/or a vaccine vial monitor.

[0221] Any of the discussion points above can be inter-combined with any aspect(s) of the Examples presented further below or any other feature or aspect herein.

[0222] Example life-saving problem to be addressed by the technology herein: Insufficient malaria therapies and the parasite’s constant changes. Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions.

[0223] To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

EXAMPLES

[0224] Plasmodium falciparum malaria remains a significant cause of morbidity and mortality in developing countries with over 620,000 deaths in sub-Saharan Africa in 2020 (1). Highly effective vaccines are urgently needed, yet the vaccine development pipeline is limited with most vaccine candidates in development targeting only four parasite antigens (2, 3). This situation mandates novel strategies to identify new candidate antigens.

[0225] Before this disclosure, the most advanced malaria vaccine, RTS,S, targets the circumsporozoite protein (CSP) expressed by sporozoites and generates antibodies that block sporozoite invasion of hepatocytes. While the potential role for cell-mediated immune responses has been sought, to date, these responses have not been reliably implicated in RTS,S-mediated protection (4). Critical challenges to targeting CSP include the following: 1) the short duration of exposure of CSP to circulating vaccine- induced antibodies (sporozoites invade hepatocytes within 20 min for IV injected and 120 min for ID-injected parasites (5)), thus requiring very high levels of specific antibody for vaccine efficacy (>100 EU/ml to prevent 50% of infections (6)), and 2) lack of expression of CSP on blood stage parasites, such that if a single sporozoite escapes vaccine-induced antibodies, an un-checked, blood stage infection can ensue. Notably, anti-CSP responses do not contribute to naturally acquired resistance, which is mediated by anti-blood- stage antibodies and provides broad protection to adults in holo-endemic areas. In the definitive Phase III trial of RTS,S, these challenges resulted in a vaccine efficacy of RTS,S against severe malaria in 17.3% of infants if given as four doses, which declined to only 10.3% if given as three doses — neither of these comparisons was statistically significant (7).

[0226] Recently, vaccination with R21, a second formulation targeting the CSP protein, significantly reduced the incidence of clinical malaria (parasitemia with fever) by 74%-77% in a phase lib trial conducted in a high, seasonal transmission setting (8, 9). Currently, the efficacy of R21 against severe malaria remains unknown. [0227] Urgency: Because parasites, which escape pre-erythrocytic vaccines can result in potentially fulminant blood-stage infections, there is an urgent need to develop blood-stage vaccines, which will attenuate clinical disease (10). Despite this need, the blood stage vaccine development pipeline is extremely limited, with only six actively recruiting clinical trials for P. falciparum blood-stage vaccines registered on clinicaltrials.gov. Of these active trials, five are targeting the Rh5 antigen, while one targets MSP-1. Both vaccine candidates are designed to attenuate parasite replication by generating humoral responses, which block merozoite invasion of erythrocytes. Previous studies have demonstrated significant antigenic variation in MSP-1 (11- 13), and despite strong immunogenicity, this antigen failed to generate protection in Phase lib studies (14). Rh5 has limited polymorphism in field isolates (15, 16), is essential for erythrocyte invasion (17), anti-Rh5 blocks erythrocyte invasion (18), vaccination with Rh5 protects against P. falciparum challenge in non-human primates (19), and vaccination of humans results in a modestly reduced P. falciparum replication rate in controlled human challenge studies, but only at high anti-Rh5 concentrations (20).

EXAMPLE 1. WHOLE PROTEOME DIFFERENTIAL SCREENING

[0228] To identify new blood-stage vaccine candidate antigens, we applied our whole proteome differential screening method and identified PfGBP130 as a target of antibodies expressed by children who had acquired resistance to P. falciparum infection (21-23).

[0229] Materials and methods: Ethical approval; Protocols for the original longitudinal cohort study were approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health. Ethical clearance was obtained from the Institutional Review Boards of Seattle Biomedical Research Institute and the National Institute for Medical Research in Tanzania (Study No 1059357). Protocols for the use of animals were approved by the IACUC committee of Rhode Island Hospital (Study No 1758696).

[0230] Study population: Subjects participated in the Mother-Offspring Malaria Studies (MOMS) project as described (24, 25).

[0231] Inclusion criteria and clinical monitoring: We monitored N = 785 children for P. falciparum infection from birth up to 3.5 years of age as described (21). Briefly, blood smears were obtained every 2 weeks from birth to 1 year of age, and monthly thereafter. Routine blood samples were collected once every 6 months from 1.5 to 3.5 years of life.

[0232] Blood collection and malaria assessment: Venous blood was collected every 6 months and stored at 4°C until processing as described (21).

[0233] Selection of resistant and susceptible children: The selection was performed based on blood films collected between the ages of 2 and 3.5 years as described (21).

[0234] Library construction and differential screening: Phage display library construction and screening were performed as described (21). In our previous publication (21), we performed four rounds of positive selection followed by five rounds of negative selection, and this screen resulted in marked enrichment for high-affinity clones (44% of clones encoded PfGARP). To attenuate the strong enrichment observed after four rounds of positive selection and allow identification of phage clones with lower affinity or with slower growth characteristics, we sequenced phage that were isolated after three rounds of positive selection on plasma from resistant children and five rounds of negative selection on plasma from susceptible children.

EXAMPLE 2. EXPRESSION AND PURIFICATION OF RECOMBINANTS AND CODING NUCLEIC ACIDS [0235] Expression and purification of recombinant PfGBP130-A: PfGBP130-A (aa 111-374) was codon optimized and cloned into the plasmid pJ411 (Atum) with N-terminal StrepTagll (8 aa) and C-terminal 10xHIS tags. Expression and purification were performed as described (21), except final purification was achieved by chromatography on a 5-ml Strep-TactinXT SuperFlow affinity column according to manufacturer’s instructions (IBA-Lifesciences). Purified recombinant protein, designated PfGBP130-A, was buffer exchanged into 10 mmol/L of sodium phosphate, 0.05% Tween 20, 3% sucrose, concentrated to 500 pg/ml using tangential flow ultrafiltration (filter area 50 cm (2), pore size 5 kDa, Pall), and lyophilized and stoppered under nitrogen.

[0236] Expression and purification of recombinant PfGBP130-ecto and PfGARP-ecto: PfGBP130-ecto (aa 89-824) or PfGARP-ecto (aa 51-673) was codon optimized and cloned into the plasmid pD2610-v6 (Atum) with the N-terminal secretion signal from pHLsec (26) and a C- terminal 10xHIS tag. Endotoxin-free plasmid was transfected into HEK293 cells with lipofectamine (Invitrogen) according to manufacturer’s instructions. Culture supernatant was harvested on day 5 after transfection, and PfGBP130-ecto or PfGARP-ecto was purified by nickel affinity column chromatography. For PfGARP-ecto, we further purified the recombinant protein using hydrophobic and anion exchange chromatography as described (21). For both constructs, purified recombinant protein was buffer exchanged into 10 mmol/L of sodium phosphate, 0.05% Tween 20, 3% sucrose, concentrated to 500 pg/ml using tangential flow ultrafiltration (filter area 50 cm (2), pore size 5 kDa, Pall), and lyophilized and stoppered under nitrogen.

[0237] Production of PfGBP130-A mRNA: mRNAs were produced as previously described (27) using T7 RNA polymerase (Megascript, Ambion) on a linearized plasmid encoding codon- optimized (28) PfGBP130-A or PfGBP130-ecto.

[0238] Encapsulation of mRNA in LNPs: mRNAs were encapsulated in LNPs as previously described (21). mRNA-LNP formulations were stored at -80°C at a concentration of mRNA of ~1 pg/pl.

[0239] Parasite strains and culture: P. falciparum strains (3D7, Dd2, D10, W2, and INDO) were obtained from MR4. Two parasite isolates (one from a child, NIH 04122821, and one from an adult, NIH 0710) were collected from our Tanzanian field site and culture adapted. The parasites were cultured in vitro according to the methods of Trager and Jensen with minor modifications (29). Briefly, parasites were maintained in RPMI 1640 medium containing 25 mmol/L of HEPES, 5% human O+ erythrocytes, 5% Albumax II (Invitrogen), 24 mmol/L of sodium bicarbonate, and 10 pg/ml of gentamycin at 37°C with 5% CO₂, 1% O₂, and 94% N₂.

[0240] Anti-PfGBP130 antisera: Mouse anti-PfGBP130-A antisera were produced by either DNA-, recombinant protein-, or mRNA- based immunization as described (21). For DNA immunization, we subcloned the open reading frame encoding PfGBP130-A (amino acids H I- 374) into VR2001 , transformed this into the host Escherichia coli NovaBlue (Novagen), and purified endotoxin-free plasmid (Endofree Giga, Qiagen).

[0241] All anti-sera were generated in BALB/cJ mice. For DNA immunization, mice were immunized at baseline with 100 pg of plasmid (25-pg intramuscular injection in each hind leg and 50-pg intradermal injection at the base of the tail) followed by 50-pg intradermal injections at the base of the tail every 2 weeks for a total of four doses. For protein immunization, recombinant PfGBP130-A was emulsified in an equal volume of TiterMax adjuvant (CytRx Corporation) and 50 pg was injected intraperitoneally at 2-week intervals for a total of three doses. For mRNA- based immunization, mice were immunized intradermally with 10 pg of lipid-encapsulated mRNA (see below) encoding PfGBP130-A (amino acids 111-374) or PfGBP130-ecto (amino acids SI- 673) every 3 weeks for a total of three doses.

[0242] Anti-PfGBP130 antibody assays: Bead-based anti-PfGBP130 antibody assays were performed according to our published methods (30) as described (21) using PfGBP130-A or PfGBP130-ecto as target antigens and PfGARP-ecto as a negative control protein.

[0243] Growth inhibition assays: Growth inhibition assays (GIA) were carried out with anti- PfGBP130 polyclonal serum generated by immunization with DNA-, recombinant protein-, or mRNA- based constructs (31-33) as described (21). Controls included no sera, normal mouse sera, sera generated by immunization with empty plasmid vector, and sera generated by immunization with LNPs containing mRNA encoding poly C. All sera were used at 10% final concentration, except as noted in the serial dilution experiment presented in FIG. 1B.

[0244] FIGs. 1A-1C illustrate anti-PfGBP130-A generated by DNA, but not recombinant protein immunization markedly attenuates parasite replication in multiple parasite strains. FIG. 1A shows polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 79-89% in multiple parasite strains in vitro. Ring stage parasites at 0.3% parsitemia were cultured in the presence of anti-PfGBP130 mouse sera at 1 :10 dilution. Negative controls included no anti-sera and normal mouse sera. FIG. 1 B shows a parasite growth assay performed as in FIG. 1A, but with dilution series of anti-PfGBP130-A generated by DNA immunization. ICso for inhibition of parasite growth was 0.6% serum. FIG. 1C shows polyclonal anti-PfGBP130-A antibodies generated by recombinant protein immunization in mice resulted in no or modest (22-29%) growth inhibition in multiple parasite strains in vitro. For FIG. 1A and for FIG. 1C, bars represent means, circles represent values from replicate wells, and error bars represent SEM. For FIG. 1 B, circles represent means, error bars represent SEM. Results in FIG. 1A and in FIG. 1C are representative of 5 independent experiments.

[0245] Merozoite invasion assays: To assess the ability of anti-PfGBP130 antisera to block merozoite invasion of erythrocytes, we incubated Percoll-/sorbitol-purified schizonts with fresh erythrocytes (final parasitemia 1%) in the presence of media control, normal mouse sera, or anti- PfGBP130-A sera generated by DNA immunization. Cultures were incubated for 12 h, blood films were prepared, stained with Giemsa, and newly invaded ring stage parasites were enumerated.

[0246] Western blot: Parasite pellets were prepared using in vitro cultured 3D7 strain of P. falciparum. Parasite cultures at 10% parasitemia were treated with 0.15% saponin in PBS, pH 7.4 on ice for 10 min, followed by centrifugation (3,000g, 5 min). Parasite pellets were resuspended in cold PBS and centrifuged (3,000g, 5 min). Parasite pellets were dissolved in SDS sampleloading buffer with reducing agent (Bio-Rad), heated to 95°C for 10 min, and proteins were separated in 4%-12% gradient SDS-PAGE gels. Separated proteins were transferred to nitrocellulose membranes, and membranes were blocked in 5% BSA in 1X PBS (pH 7.4) and 0.05% Tween 20 for 1 h at 25°C. Membranes were probed with polyclonal anti-PfGBP130 generated by DNA-, mRNA- or recombinant protein-based immunization or normal mouse serum at 1 :1 ,000 dilution in 1X PBS (pH 7.4), 0.05% Tween 20 overnight at 4°C. Rabbit polyclonal antiactin diluted 1 :3,000 was added as a loading control. Membranes were washed in 1X PBS (pH 7.4) and 0.05% Tween 20, and bound antibody was detected with anti-mouse IgG antibody conjugated to IRDye (1 :3,000) and imaged on an LI-COR (Odyssey Imaging Systems).

[0247] Immunofluorescence assays: Blood smears of asynchronous 3D7 strain parasite cultures were prepared, fixed in cold methanol for 15 min and probed with anti-PfGBP130-A generated by DNA, mRNA or recombinant protein-based immunization (dilutions tested from 1 :50 to 1:200), and rabbit anti-PfMSP4 (obtained from MR4) diluted 1:500 in PBS, 5% BSA, pH 7.4. Blood smears were incubated with primary antibodies for 1 h at 25°C, washed three times in PBS, 0.05% Tween 20, and incubated with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular Probes) and goat anti-rabbit IgG conjugated with Alexa Fluor 594 (Molecular Probes). Blood smears were incubated for 10 min in 1 pg/ml of DAPI (Sigma-Aldrich) to label nuclei and cover slipped with Prolong Gold anti-fade reagent (Invitrogen). Blood smears were imaged using a confocal microscope (ZEISS LSM 900 Airyscan) equipped with a *63 oil-immersion objective.

EXAMPLE 3. ELUCIDATION/DISCUSSION OF RESULTS

[0248] Results: To identify novel vaccine candidates for P falciparum infection, we pooled plasma collected from 2-year old children living in a holoendemic region of Tanzania who participated in a longitudinal cohort study (24). We pooled plasma from children who were highly resistant or highly susceptible to infection as assessed with monthly blood films from ages 2 to 3.5 years (FIG. 6). Using these pooled plasmas, we performed differential biopanning using a P. falciparum blood-stage cDNA library constructed in the T7 bacteriophage using mRNA extracted from parasites freshly collected from our Tanzanian field site as described (21). Following three rounds of differential biopanning, we sequenced 100 clones (FIG. 7) and identified PfGBP130 as a target of antibodies expressed by resistant, but not susceptible, children. After removal of ribosomal-related genes, PfGBP130 accounted for 10% of all clones sequenced.

[0249] FIG. 6 shows a table illustrating epidemiologic characteristics of resistant and susceptible individuals used in differential screening assays. FIG. 7 shows a table illustrating Parasite genes identified following three rounds of differential bio-panning of P. falciparum phage display library.

[0250] PfGBP130 encodes an N-terminal PEXEL sequence (aa 84-88) followed by an ectodomain (PfGBP130-ecto, aa 89-824), which contains a charged 137-aa domain followed by 12 copies of a 50-aa repeat (see FIG. 2). The PfGBP130 clone we identified (PfGBP130-A) encoded aa 111-374, which comprises the majority of the N terminal charged domain and three copies of the 50-aa repeat region.

[0251] FIG. 2 shows an example domain structure of PfGBP130. PfGBP130 is an invariant, PEXEL containing merozoite surface antigen comprised of an N-terminal charged 225 aa domain followed by twelve copies of a 50 aa repeat domain. The PfGBP130 clone identified by differential screening (PfGBP130-A) encoded aa 111-374 which comprises the majority of the N terminal charged domain and three copies of the 50 aa repeat region.

[0252] Because of the proposed role for PfGBP130 in merozoite invasion of erythrocytes (34- 39), we evaluated the ability of anti-PfGBP130 to attenuate parasite growth in vitro. We vaccinated mice with E. co//-expressed PfGBP130-A formulated in TiterMax® (CytRx Corporation) adjuvant or as a codon-optimized DNA vaccine construct. Both vaccination regimens generated antigen-specific antibody responses as measured by a bead-based antibody detection assay against PfGBP130-A, PfGBP130-ecto, or the negative control protein PfGARP-ecto (FIGs. 8A-8C) with a titer of 1 :8,000 for DNA and 1 :512,000 for recombinant protein-based vaccination (FIG. 3). In parasite growth inhibition assays, anti-PfGBP130-A antisera generated by DNA vaccination, significantly attenuated parasite growth by 79%-89% compared to controls, and this activity was consistent across multiple parasite strains including freshly isolated parasites (all P < 0.0001 , FIG. 1A). This high degree of attenuation of parasite growth was confirmed using a second, independent lot of sera (titer = 1 :64,000), which attenuated parasite growth by 95% (FIG. 9A, FIG. 9B). Sera generated by immunization with empty DNA vector or DNA vector encoding an irrelevant P. falciparum gene (PfHISTc) had no impact on parasite growth (FIG. 9A, FIG. 9B). In serial dilution experiments, the ICso for parasite growth inhibition was 0.6% anti- PfGBP130-A sera, and at 10% serum, the parasite growth was inhibited by 100% (FIG. 1B). In contrast, anti-PfGBP130-A antisera generated by recombinant protein-based immunization had no impact on parasite growth for three strains (3D7, INDO, and a freshly collected isolate, NIH 710) and had statistically significant, but very modest (22%— 29%), attenuation of parasite growth for a further three strains (Dd2, W2, and a freshly collected field isolate, NIH 04122821 , FIG. 1C).

[0253] FIGs. 8A-8C show expression and purification of recombinant PfGBP130-A, PfGBP130-ecto and PfGARP-ecto. FIG. 8A shows expression of PfGBP130-A in E. coli. SDS- PAGE gel showing chromatographic purification of PfGBP130-A (aa 111-374) with an 8 amino acid Strep Tag II on the N terminal and a 10xHis tag on the C terminal. Lane 1) induced E. coli lysate, Lane 2) post NiNTA, Lane 3) post hydrophobic Interaction, Lane 4) post anion exchange, Lane 5) post Strep-Tactin . FIG. 8B shows expression of PfGBP130-ecto in HEK293 cells. SDS- PAGE gel showing chromatographic purification of PfGBP130-ecto (aa 89-824) with a 10xHis tag on the C terminal. Lane 1) supernatant from HEK293 cells transfected with PfGBP130-ecto encoding plasmid, Lane 2) post NiNTA purification. FIG. 8C shows expression of PfGARP-ecto in HEK293 cells. SDS-PAGE gel showing chromatographic purification of PfGARP-ecto (aa SI- 673) with a 10xHis tag on the C terminal. Lane 1) supernatant from HEK293 cells transfected with PfGARP-ecto encoding plasmid Lane 2) post NiNTA purification, Lane 3) post hydrophobic Interaction, Lane 4) post anion exchange.

[0254] FIGs. 9A-9B show anti-PfGBP130-A generated by DNA, but not recombinant protein immunization markedly attenuates parasite replication in P. falciparum 3D7 parasites. FIG. 9A shows polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 81-95% while no significant growth inhibition was observed for anti PfGBP130- A antibodies generated by recombinant protein immunization or with antisera raised against a negative control DNA vaccine construct, PfPHISTc. Ring stage parasites were cultured in the presence of mouse anti-sera at 1 :10 dilution. Negative controls included media alone, normal mouse sera, and a DNA plasmid construct encoding an irrelevant malaria gene (PfHISTc). FIG. 9B shows polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 94% compared to antisera from mice immunized with the empty plasmid vector (anti pVR2001). No significant growth inhibition was observed for antisera prepared from mice immunized with LNPs containing poly C mRNA or empty plasmid vector (anti-pVR2001) compared to media alone or normal mouse sera controls. Ring stage parasites were cultured in the presence of mouse anti-sera at 1 :10 dilution. In both A and B, bars represent means, circles represent values from replicate wells, and error bars represent SEM.

[0255] FIGs. 3A-3B show PfGBP130-A formulated as a DNA plasmid or recombinant protein is immunogenic in mice. FIG. 3A shows mice immunized with a DNA plasmid encoding PfGBP130-A generated low titer (1 :8,000) antibodies against PfGBP130-A and PfGBP130-ecto coated beads with no reactivity against negative control protein (PfGARP-ecto) coated beads. FIG. 3B shows mice immunized with recombinant protein encoding PfGBP130-A generated high titer (1 :512,000) antibodies against PfGBP130-A and PfGBP130-ecto coated beads with negligible binding to a negative control protein (PfGARP-ecto) coated beads.

[0256] To assess whether anti-PfGBP130 blocked merozoite invasion of RBCs, we incubated Percoll-/sorbitol-purified schizonts with fresh RBC in the presence of media control, normal mouse sera, oranti-PfGBP130-A sera generated by DNA immunization. Parasite cultures were incubated for 12 h, and newly invaded ring-stage parasites were enumerated. Anti-PfGBP130-A inhibited merozoite invasion by 88% compared to normal mouse sera (FIG. 4).

[0257] FIG. 4 shows a plot illustrating how antibodies to PfGBP130-A inhibit merozoite invasion. Polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit merozoite invasion by 88% in vitro. Schizont stage parasites were cultured in the presence of anti-PfGBP130-A mouse sera at 1 :10 dilution for 12 hours and newly invaded ring- stage parasites were enumerated. Negative controls included media alone and normal mouse sera. Bars represent means, circles represent values from replicate wells, and error bars represent SEM.

[0258] Because PfGBP130 formulated as a recombinant protein did not generate antibodies with high growth inhibitory activity, we evaluated LNP-encapsulated mRNA based on our prior experience generating functional anti-malarial antibodies (21), as well as the recent success of mRNA as a delivery vehicle for SARS-CoV2 vaccines (40). We synthesized PfGBP130-A and PfGBP130-ecto as lipid-encapsulated mRNA formulations, and both constructs were highly immunogenic in mice, with both generating titers of 1 :512,000 against PfGBP130-A- and PfGBP130-ecto-coated beads. Antibody titers following mRNA-based immunization were 8-64 times higher than titers generated by DNA immunization and were identical to titers following recombinant protein immunization (FIG. 5A, FIG. 5B). In /n vitro assays, polyclonal antibodies generated by both mRNA constructs inhibited parasite growth by 80% (FIG. 5C), while LNP- encapsulated mRNA encoding poly C had no impact on parasite growth (FIG. 9A, FIG. 9B).

[0259] FIGs. 5A-5C show immunization with PfGBP130-A or PfGBP130-ecto formulated as lipid encapsulated mRNA generates antibodies that markedly attenuate parasite replication. Immunization with FIG. 5A showing lipid encapsulated mRNA encoding PfGBP130-A or in FIG. 5B, which shows PfGBP130-ecto generates high titer (both 1 :512,000), specific antibodies against PfGBP130-A and PfGBP130-ecto coated beads with no reactivity against negative control protein (PfGARP-ecto) coated beads. FIG. 5C shows polyclonal murine anti-PfGBP130 antibodies generated by immunization with LNPs containing mRNA encoding PfGBP130-A or PfGBP130-ecto inhibit parasite growth by 80% in vitro. Ring stage parasites were cultured in the presence of anti-PfGBP130 mouse sera at 1 :10 dilution. Negative controls included no anti-sera and normal mouse sera. For FIG. 5A and FIG. 5B, circles represent means, error bars represent SEM. For FIG. 5C, bars represent means, circles represent values from replicate wells, and error bars represent SEM. Results in FIG. 5C are representative of 3 independent experiments.

[0260] To explore the discrepancy between antibody titer and parasite growth inhibition across the DNA-, mRNA-, and recombinant protein-generated anti-sera, we performed western blot analysis on extracts of schizont-stage 3D7 strain P. falciparum parasites. All three anti- PfGBP130-A antisera recognized a dominant protein migrating at 110 kDa in parasite-infected, but not uninfected, erythrocytes (FIG. 10). In immunofluorescence assays, none of the three anti- PfGBP130-A antisera generated a specific binding pattern when tested against infected erythrocytes (data not shown).

[0261] FIG. 10 shows characterization of anti-PfGBP130-A antisera by western blot. Lysates prepared from infected (lanes 1 , 3, 5, and 7) and uninfected (lanes 2, 4, 6, 8) erythrocytes were separated by SDS-PAGE, transferred to nitrocellulose and probed with murine anti-PfGBP130-A antisera generated by immunization with LNPs containing mRNA encoding PfGBP130-A (lanes 1 and 2), DNA plasmid encoding PfGBP130-A (lanes 3 and 4), recombinant PfGBP130-A protein (lanes 5 and 6), or normal mouse sera (lanes 7 and 8). Anti-actin served as a loading control.

[0262] Discussion: In the current report, we identified PfGBP130 as a target of antibodies expressed by children who had acquired resistance to, but not by children who remained susceptible to, P. falciparum infection.

[0263] Previously, we developed a whole proteome differential screening (WPDS) strategy, which identifies the subset of parasite antigens that are recognized by antibodies expressed by resistant individuals but not susceptible individuals (21-23). When employed with four rounds of positive selection, our WPDS method identified differentially recognized phage that were markedly oligoclonal with 44% of the clones encoding PfGARP (21). In the present study, to identify a broader clonal repertoire, including lower-affinity, slower-growing, and sub-dominant clones, we sequenced differentially recognized clones following only three rounds of positive selection. Using this approach, we identified PfGBP130 as a target of antibodies that block merozoite invasion resulting in arrested parasite replication.

[0264] PfGBP130 is an invariant merozoite surface antigen comprised of an N-terminal charged 225-aa domain followed by 12 copies of a 50-aa repeat domain (41). PfGBP130 is synthesized by trophozoite- and schizont-stage parasites and becomes associated with the surface of merozoites prior to egress (34). PfGBP130 interacts with the exofacial surface of erythrocytes (34) and appears to bind specifically to glycophorin A (35), though this interaction is disputed in some (36), but not other, reports (37). Rabbit polyclonal antibodies generated by immunization with an E. co//-expressed construct encoding 4.5 repeats of PfGPB130 inhibit merozoite invasion of erythrocytes (37), while a 19-amino acid synthetic peptides sequence derived from the PfGBP130 repeat can also inhibit merozoite invasion (39).

[0265] Vaccination of non-human primates (Aotus) with a recombinant PfGBP130 polypeptide produced in E. coli that encoded three copies of the 50 amino acid repeat did not protect against challenge infection (42). These results suggest that potentially protective epitopes within PfGBP130 may not be contained in the repeat regions, but rather may be in the N-terminal- charged domain (aa 1-225), which overlaps with the region identified on our differential screen (aa 111-374).

[0266] In the current study, we evaluated the impact of three PfGBP130 vaccine delivery platforms on both immunogenicity as well as in vitro parasite growth arrest. Initially, we formulated PfGBP130-A as a codon-optimized DNA vaccine construct in a mammalian expression plasmid and as a recombinant protein expressed in E. coli. While immunization with recombinant protein generated higher antibody titers compared to DNA-based immunization (FIG. 3A, FIG. 3B), only antibodies generated by DNA-based vaccination significantly arrested parasite replication in vitro (e.g., see FIG. 9A, FIG. 9B, FIGs. 1A-1C), and this parasite growth arrest was mediated by blocking merozoite invasion of new erythrocytes (FIG. 4). While the discordant results between DNA and recombinant protein immunization may have resulted from a misfolded E. coli- expressed protein, we have been unable to verify this assertion because we have not been successful in expressing PfGBP130-A in mammalian expression systems. Therefore, it remains unclear whether mammalian expression and/or the N-terminal region is required for generating antibodies that inhibit parasite growth.

[0267] To formulate PfGBP130 on a deployable and scalable delivery vehicle, we constructed lipid nanoparticle-encapsulated mRNA encoding PfGBP130-A (aa 111-374) or PfGBP130-ecto (aa 89-824). Both PfGBP130-A and PfGBP130-ecto, delivered as LNP-encapsulated mRNA- based vaccines, were highly immunogenic in mice (FIG. 5A, FIG. 5B) and generated antibodies that significantly inhibited parasite replication in vitro (FIG. 5C).

[0268] We did not find concordance between anti-PfGBP130-A antibody levels and in vitro parasite growth inhibition across the three delivery platforms evaluated. DNA-based immunization generated low-titer antibody levels, but these antibodies demonstrated high- parasite growth inhibition. mRNA-based immunization generated both high-titer antibody levels and high-parasite growth inhibition while protein-based immunization generated high titer antibody levels, but little to no parasite growth inhibition. The reactivity of anti-sera to native PfGBP130 in parasite extracts was similar in both specificity and intensity across the three delivery platforms (FIG. 10), and none of the antisera reacted with parasites in immunofluorescence assays. We are currently conducting linear epitope-mapping studies to further explore the discordance between anti-PfGBP130 antibody level and functional activity across the three delivery platforms.

[0269] The COVID-19 pandemic brought the benefits of lipid-encapsulated mRNA as a vaccine delivery platform into sharp focus. These benefits include rapid production of GMP-grade vaccine, remarkably high antibody titers following one or two doses, and the ability to easily alter the delivery payload with new variant sequences. The vaccine mobilization effort for COVID-19 also addressed several significant hurdles for mRNA-based vaccines, including the lack of global GMP production facilities, the absence of regulatory approval pathways, and the absence of significant safety data in large-scale trials in humans.

[0270] The current malaria vaccine landscape includes two approved, modestly effective pre- erythrocytic vaccines (RTS,S and R21). Because pre-erythrocytic vaccines do not confer sterile protection, there is an urgent need to address the blood-stage infections which will develop from parasites that escape these vaccines and infect hepatocytes with the goal of incorporating effective blood stage components into current pre-erythrocytic vaccines.

[0271] Based on our data demonstrating anti-PfGBP130-A antibodies generated by lipid- encapsulated mRNA-based vaccination attenuate parasite growth, we are now advancing this antigen/delivery platform combination toward vaccine trials in non-human primates.

[0272] Ethics statement: The studies involving humans were approved by The International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Boards of Seattle Biomedical Research Institute and the National Institute for Medical Research in Tanzania. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. The animal study was approved by the IACUC committee of Rhode Island Hospital. The study was conducted in accordance with the local legislation and institutional requirements.

Table of Example Sequences:

SEQ ID NO : 1 :

CGCACAGUUGCUACUCAAACAAAGAAAGAUGAAGAAAAUAAAUCAGUUGUUACCGAAGAACAAA

AAGUAGAAAGUGAUUCCGAAAAACAAAAAAGAACCAAAAAAGUAGUAAAAAAGCAAAUUAAUAU

AGGAGAUACAGAAAAUCAAAAAGAAGGAAAAAACGUUAAAAAAGUUAUAAAGAAAGAAAAGAAA

AAAGAAGAAUCUGGAAAACCAGAAGAAAAUAAACAUGCAAACGAAGCUUCAAAAAAACAGGAAC

CUAAAGCCUCAAAAGUAUCUCAAAAACCAUCAACUAGCACACGUUCAAAUAAUGAAGUAAAAAU

ACGAGCUGCUUCUAAUCAAGAAACAUUAACUAGUGCCGAUCCAGAAGGACAAAUAAUGAGAGAA

UAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAAUAUUUUAUAAAAUAUUAACUAACACCG

AUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACUAGUGCCGAUCC

AGAAGGUCAAAUAAUGAGAGAAUAUGCUUCCGAUCCAGAAUACCGUAAACACUUAGAAAUAUUU

UAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGACGUAGAAAGAAGAAAUGCCGAUAAUAAAG AAGAUUUAACCAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCAGAAUA UCGUAAACAUUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAGUAGAA

AGAAGAAAUGCCGAUAAUAAAGAA

SEQ ID NO : 2

GGAGAAGAUACGUGUGCACGAAAAGAAAAGACUACAUUAAGAAAAAGUAAGCAGAAAACAUCUA

CACGCACAGUUGCUACUCAAACAAAGAAAGAUGAAGAAAAUAAAUCAGUUGUUACCGAAGAACA

AAAAGUAGAAAGUGAUUCCGAAAAACAAAAAAGAACCAAAAAAGUAGUAAAAAAGCAAAUUAAU

AUAGGAGAUACAGAAAAUCAAAAAGAAGGAAAAAACGUUAAAAAAGUUAUAAAGAAAGAAAAGA

AAAAAGAAGAAUCUGGAAAACCAGAAGAAAAUAAACAUGCAAACGAAGCUUCAAAAAAACAGGA

ACCUAAAGCCUCAAAAGUAUCUCAAAAACCAUCAACUAGCACACGUUCAAAUAAUGAAGUAAAA

AUACGAGCUGCUUCUAAUCAAGAAACAUUAACUAGUGCCGAUCCAGAAGGACAAAUAAUGAGAG

AAUAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAAUAUUUUAUAAAAUAUUAACUAACAC

CGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACUAGUGCCGAU

CCAGAAGGUCAAAUAAUGAGAGAAUAUGCUUCCGAUCCAGAAUACCGUAAACACUUAGAAAUAU

UUUAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGACGUAGAAAGAAGAAAUGCCGAUAAUAA

AGAAGAUUUAACCAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCAGAA

UAUCGUAAACAUUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAGUAG

AAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACCAGUGCCGAUCCAGAAGGUCAAAUAAUGAG

AGAAUAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAGUAUUUCAUAAAAUAUUGACUAAU

ACCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAACAAAGAAUUAACCAGCUCAGACC

CAGAAGGUCAAAUAAUGAGAGAAUAUGCCGCUGAUCCAGAAUAUCGUAAACACUUAGAAGUAUU UCAUAAAAUAUUGACCAAUACCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAACAAA GAAGAUUUAACUAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCAGAAU AUCGUAAACACUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAGUAGA AAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACUAGUGCCGAUCCAGAAGGACAAAUAAUGAGA GAAUAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAAUAUUUCAUAAAAUAUUAACUAACA CCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGACUUAACUAGUGCCGA UCCAGAAGGUCAAAUAAUGAGAGAAUAUGCCGCUGAUCCAGAAUACCGUAAACACUUAGAAAUA UUUUAUAAAAUAUUAACUAACACAGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUA AAGAAGAAUUAACCAGCUCAGACCCAGAAGGUCAAAUAAUGAGAGAAUAUGCCGCUGAUCCAGA GUAUCGUAAACACUUAGAAAUAUUUCAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGAAGUA GAAAGAAGAAAUGCCGAUAAUAAAGAAGACUUAACUAGUGCCGAUCCAGAAGGUCAAAUAAUGA GAGAAUAUGCCGCUGAUCCAGAAUACCGUAAACACUUAGAAAUAUUUUAUAAAAUAUUAACUAA CACCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACCAGUGCC GAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUUCCGAUCCAGAAUACCGUAAACACUUAGAAA UAUUUUAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGACGUAGAAAGAAGAAAUGCCGAUAA CAAAGAAGAUUUAACUAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCA GAAUAUCGUAAACACUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAG UAGAAAGACAAAAUGCUGAUAAUAACGAAGCA

SEQ ID NO : 3

MRLSKVSDIKSTGVSNYKNFNSKNSSKYSLMEVSKKNEKKNSLGAFHSKKILLIFGI IYWLLN AY I CGDKYE KAVD YGFRE SRI IAE GED TCARKE KT TLRKSKQKTS TRTVATQTKKDE ENKSWT E E QKVE SD SE KQKRTKKWKKQ IN I GD TENQKE GKNVKKVI KKE KKKE E SGKPE ENKHANE ASK KQEPKASKVSQKPSTSTRSNNEVKIRAASNQETLTSADPEGQIMREYAADPEYRKHLE IFYKIL TNTDPNDEVERRNADNKEDLTSADPEGQIMREYASDPEYRKHLEIFYKILTNTDPNDDVERRNA DNKEDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVERRNADNKEDLTSADPEGQ IMREYAADPEYRKHLEVFHKILTNTDPNDEVERRNADNKELTSSDPEGQIMREYAADPEYRKHL EVFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPND EVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLE IFHKILTNTDPNDEVERRNADNKEDLT SADPEGQIMREYAADPEYRKHLE IFYKILTNTDPNDEVERRNADNKEELTSSDPEGQIMREYAA DPEYRKHLE IFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLE IFYKI LTNTDPNDEVERRNADNKEDLTSADPEGQIMREYASDPEYRKHLE IFYKILTNTDPNDDVERRN ADNKEDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVERQNADNNEA

SEQ ID NO : 4

RTVATQ TKKDE E NKSWTE E QKVE SD SE KQKRTKKWKKQ I N I GD TE NQKE GKNVKK I KKE KK KEESGKPEENKHANEASKKQEPKASKVSQKPSTSTRSNNEVKIRAASNQETLTSADPEGQIMRE YAADPE YRKHLE I FYKILTNTDPNDEVERRNADNKEDLTSADPEGQIMRE YASDPE YRKHLE I F YKILTNTDPNDDVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVE RRNADNKE

SEQ ID NO : 5

ATCCGGTGAGAATGGCAAAAGT T TATGCAT T TCT T TCCAGAC T TGT TCAACAGGCCAGCCAT TA CGC TCGTCATCAAAATCAC TCGCATCAACCAAACCGT TAT TCATTCGTGAT TGCGCC TGAGCGA GGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAGTGCAACCGGCGCAG GAACAC TGCCAGCGCATCAACAATAT TT TCACCTGAATCAGGATAT TC T TC TAATACC TGGAAC GC TGT T T T TCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC T TGATGGTCGGAAGTGGCATAAAT TCCGTCAGCCAGT T TAGTC TGACCATCTCATCTGTAACATC AT TGGCAACGC TACC TT TGCCATGT T TCAGAAACAAC TC TGGCGCATCGGGC T TCCCATACAAG CGATAGAT TGTCGCACC TGATTGCCCGACAT TATCGCGAGCCCAT T TATACCCATATAAATCAG CATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGGATATGGCTCATTTTTTACTT CO TCACC T TGTCGTATTATACTATGCCGATATAC TATGCCGATGAT TAATTGTCGACAC TGCGG GGGCTCTGGAGACGACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGT TCCCATAGTAACGCCAATAGGGAC T T TCCAT TGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCG CCCCC TAT TGACGTCAATGACGGTAAATGGCCCGCC TGGCAT TATGCCCAGTACATGACC TTAC GGGAC T T TCCTAC T TGGCAGTACATC TACG TAT TAG T CATC GC TAT TACCATGC TGATGCGGT T T TGGCAGTACACCAATGGGCGTGGATAGCGGT TTGAC TCACGGGGAT T TCCAAGTC TCCACCCC AT TGACGTCAATGGGAGT T TGT T T TGGCACCAAAATCAAC GGGAC T T TCCAAAATGTCGTAATA ACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGC TCGTTTAGTGAACCGTCAGATCCGTCTCAGGGGAGAGACCACACCCAAGCTGTCTAGAGCCGCC ACCATGGGCATTCTGCCCTCCCCTGGAATGCCCGCCCTGCTTTCACTGGTGTCCCTGCTGTCCG TGC TGC TGATGGGT TGTGTCGCCAGAAC TGTGGC TACCCAAACCAAGAAGGATGAGGAAAACAA GTCCGTGGTGACGGAGGAACAGAAGGTTGAGTCGGACTCCGAAAAGCAGAAGCGGACCAAGAAG GTGGTCAAGAAGCAGATTAACATCGGCGACACCGAAAACCAGAAGGAGGGAAAGAACGTGAAAA AGGTCATCAAGAAGGAGAAGAAGAAGGAGGAATCCGGAAAGCCAGAGGAGAACAAGCACGCCAA CGAAGCATCGAAGAAGCAGGAGCCCAAGGCC TCCAAAGTGTCCCAGAAGCCCAGCAC T TCGACC CGGTCCAACAACGAGGTCAAGAT TCGGGCCGCCAGCAATCAGGAAAC TC TGAC T TCGGCCGACC CAGAAGGACAAATCATGCGGGAGTACGCGGC TGACCCGGAATACCGGAAACACC TGGAGATC T T CTATAAGATCTTGACAAATACCGATCCCAACGATGAGGTAGAAAGACGCAATGCCGACAACAAA GAGGATC T TACCAGCGCAGATCCGGAGGGCCAAATAATGCGAGAGTACGCT TCCGATCCCGAAT ACCGCAAGCAC T TGGAGAT T TTC TACAAGAT TCTGACCAACACCGATCC TAACGATGACGTCGA ACGGAGAAACGCCGACAATAAGGAAGATC T TACC TCGGC TGATCCCGAGGGCCAGATCATGCGC GAATACGCGGCCGATCCGGAATACAGAAAGCATC TCGAGGTGT TCCACAAGAT TCT TAC TAACA CCGACCCGAATGATGAGGTGGAAAGGCGCAACGCTGACAATAAGGAACATCACCATCATCACCA CCACCATCACCAT TGATAAGGT TGAGGTC TC TAAAAC TGTC T TCCCAAC TTGT T TAT TGCAGC T TATAATGGT TACAAATAAAGCAATAGCATCACAAAT T TCACAAATAAAGCAT T T TT T TCACTGC AT TCTAGT TGTGGT T TGTCCAAAC TCATCAATGTATC TTATCATGTCGAAGACAGTCAGCCT TG AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT T T T TCCATAGGC TCCGCCCCCC TGAC GAGCAT CACAAAAATCGACGC TCAAGTCAGAGGTGGCG AAACCCGACAGGAC TATAAAGATACCAGGCGT TTCCCCC TGGAAGC TCCCTCGTGCGC TC TCC T GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGC TACAGAGTTC T TGAAGTGGTGGGC TAAC TACGGC TACAC TAGAAGAACAGT AT T TGGTATCTGCGC TC TGC TGAAGCCAGT TACC T TCGGAAAAAGAGT TGGTAGCTC T TGATCC GGCAAACAAACCACCGC TGGTAGCGGTGGT T T TT T TGTT TGCAAGCAGCAGAT TACGCGCAGAA AAAAAGGATCTCAAGAAGATCC T T TGATC T T T TC TACGGGGTC TGACGC TCAGTGGAACGACGC GCGCGTAAC TCACGT TAAGGGAT T T TGGTCATGAGT TAGAAAAAC TCATCGAGCATCAAATGAA AC TGCAAT T TAT TCATATCAGGAT TATCAATACCATATT T T TGAAAAAGCCGT T TC TGTAATGA AGGAGAAAACTCACCGAGGCAGT TCCATAGGATGGCAAGATCC TGGTATCGGTC TGCGAT TCCG AC TCGTCCAACATCAATACAACC TAT TAAT T TCCCC TCGTCAAAAATAAGGT TATCAAGTGAGA AATCACCATGAGTGACGACTGA

SEQ ID NO : 6

RTVATQ TKKDE E NKSWTE E QKVE SD SE KQKRTKKWKKQ I N I GD TE NQKE GKNVKKVI KKE KK KEESGKPEENKHANEASKKQEPKASKVSQKPSTSTRSNNEVKIRAASNQETLTSADPEGQIMRE YAADPE YRKHLE I FYKILTNTDPNDEVERRNADNKEDLTSADPEGQIMRE YASDPE YRKHLE I F YKILTNTDPNDDVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVE RRNADNKE

SEQ ID NO : 7

GE D TCARKE KT TLRKSKQKT S TRTVATQ TKKDE E NKSWTE E QKVE SD SE KQKRTKKWKKQ I N I GD TENQKE GKNVKKVI KKE KKKE E SGKPE ENKHANE ASKKQE PKASKVSQKPS TS TRSNNE VK IRAASNQETLTSADPEGQIMREYAAD PE YRKHLE IFYKILTNTDPNDEVERRNADNKEDLTSAD PEGQIMREYASDPEYRKHLE IFYKILTNTDPNDDVERRNADNKEDLTSADPEGQIMREYAADPE YRKHLE VFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYAAD PE YRKHLE VFHKILTN TDPNDEVERRNADNKELTSSDPEGQIMREYAAD PE YRKHLE VFHKILTNTDPNDEVERRNADNK EDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMR EYAADPEYRKHLE IFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLE I FYKILTNTDPNDEVERRNADNKEELTSSDPEGQIMREYAADPEYRKHLEIFHKILTNTDPNDEV ERRNADNKEDLTSADPEGQIMREYAADPEYRKHLE IFYKILTNTDPNDEVERRNADNKEDLTSA DPEGQIMREYASDPEYRKHLEIFYKILTNTDPNDDVERRNADNKEDLTSADPEGQIMREYAADP EYRKHLEVFHKILTNTDPNDEVERQNADNNEA

SEQ ID NO : 8

GGAGAAGAUACGUGUGCACGAAAAGAAAAGACUACAUUAAGAAAAAGUAAGCAGAAAACAUCUA CACGCACAGUUGCUACUCAAACAAAGAAAGAUGAAGAAAAUAAAUCAGUUGUUACCGAAGAACA AAAAGUAGAAAGUGAUUCCGAAAAACAAAAAAGAACCAAAAAAGUAGUAAAAAAGCAAAUUAAU AUAGGAGAUACAGAAAAUCAAAAAGAAGGAAAAAACGUUAAAAAAGUUAUAAAGAAAGAAAAGA AAAAAGAAGAAUCUGGAAAACCAGAAGAAAAUAAACAUGCAAACGAAGCUUCAAAAAAACAGGA ACCUAAAGCCUCAAAAGUAUCUCAAAAACCAUCAACUAGCACACGUUCAAAUAAUGAAGUAAAA AUACGAGCUGCUUCUAAUCAAGAAACAUUAACUAGUGCCGAUCCAGAAGGACAAAUAAUGAGAG AAUAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAAUAUUUUAUAAAAUAUUAACUAACAC CGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACUAGUGCCGAU CCAGAAGGUCAAAUAAUGAGAGAAUAUGCUUCCGAUCCAGAAUACCGUAAACACUUAGAAAUAU UUUAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGACGUAGAAAGAAGAAAUGCCGAUAAUAA AGAAGAUUUAACCAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCAGAA UAUCGUAAACAUUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAGUAG AAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACCAGUGCCGAUCCAGAAGGUCAAAUAAUGAG AGAAUAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAGUAUUUCAUAAAAUAUUGACUAAU ACCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAACAAAGAAUUAACCAGCUCAGACC CAGAAGGUCAAAUAAUGAGAGAAUAUGCCGCUGAUCCAGAAUAUCGUAAACACUUAGAAGUAUU UCAUAAAAUAUUGACCAAUACCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAACAAA GAAGAUUUAACUAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCAGAAU AUCGUAAACACUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAGUAGA AAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACUAGUGCCGAUCCAGAAGGACAAAUAAUGAGA GAAUAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAAUAUUUCAUAAAAUAUUAACUAACA CCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGACUUAACUAGUGCCGA UCCAGAAGGUCAAAUAAUGAGAGAAUAUGCCGCUGAUCCAGAAUACCGUAAACACUUAGAAAUA UUUUAUAAAAUAUUAACUAACACAGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUA AAGAAGAAUUAACCAGCUCAGACCCAGAAGGUCAAAUAAUGAGAGAAUAUGCCGCUGAUCCAGA GUAUCGUAAACACUUAGAAAUAUUUCAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGAAGUA GAAAGAAGAAAUGCCGAUAAUAAAGAAGACUUAACUAGUGCCGAUCCAGAAGGUCAAAUAAUGA GAGAAUAUGCCGCUGAUCCAGAAUACCGUAAACACUUAGAAAUAUUUUAUAAAAUAUUAACUAA CACCGAUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACCAGUGCC GAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUUCCGAUCCAGAAUACCGUAAACACUUAGAAA UAUUUUAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGACGUAGAAAGAAGAAAUGCCGAUAA CAAAGAAGAUUUAACUAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCA GAAUAUCGUAAACACUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAG UAGAAAGACAAAAUGCUGAUAAUAACGAAGCA

SEQ ID NO : 9

GGAGAAGATACGTGTGCACGAAAAGAAAAGAC TACAT TAAGAAAAAGTAAGCAGAAAACATC TA CACGCACAGTTGCTACTCAAACAAAGAAAGATGAAGAAAATAAATCAGTTGTTACCGAAGAACA AAAAGTAGAAAGTGATTCCGAAAAACAAAAAAGAACCAAAAAAGTAGTAAAAAAGCAAATTAAT ATAGGAGATACAGAAAATCAAAAAGAAGGAAAAAACGTTAAAAAAGTTATAAAGAAAGAAAAGA AAAAAGAAGAATCTGGAAAACCAGAAGAAAATAAACATGCAAACGAAGCTTCAAAAAAACAGGA ACCTAAAGCCTCAAAAGTATCTCAAAAACCATCAACTAGCACACGTTCAAATAATGAAGTAAAA ATACGAGC TGC T TC TAATCAAGAAACAT TAAC TAGTGCCGATCCAGAAGGACAAATAATGAGAG AATATGC TGCTGATCCAGAATATCGTAAACAC TTAGAAATAT T TTATAAAATAT TAAC TAACAC CGATCCAAATGATGAAGTAGAAAGAAGAAATGCCGATAATAAAGAAGATTTAACTAGTGCCGAT CCAGAAGGTCAAATAATGAGAGAATATGC T TCCGATCCAGAATACCGTAAACAC TTAGAAATAT T T TATAAAATAT TAACTAACACCGATCCAAATGATGACGTAGAAAGAAGAAATGCCGATAATAA AGAAGAT T TAACCAGTGCC GAT CCAGAAGGTCAAATAATGAGAGAATATGC TGC TGATCCAGAA TATCGTAAACAT T TAGAAGTAT T TCATAAAATAT TGACTAATACCGATCCAAATGATGAAGTAG AAAGAAGAAATGCCGATAATAAAGAAGAT T TAACCAGTGCCGATCCAGAAGGTCAAATAATGAG AGAATATGC TGCTGATCCAGAATATCGTAAACAC T TAGAAG TAT T TCATAAAATAT TGAC TAAT ACCGATCCAAATGATGAAGTAGAAAGAAGAAATGCCGATAACAAAGAAT TAACCAGC TCAGACC CAGAAGGTCAAATAATGAGAGAATATGCCGC TGATCCAGAATATCGTAAACAC T TAGAAGTAT T TCATAAAATATTGACCAATACCGATCCAAATGATGAAGTAGAAAGAAGAAATGCCGATAACAAA GAAGAT T TAAC TAGTGCCGATCCAGAAGGTCAAATAATGAGAGAATATGCTGC TGATCCAGAAT ATCGTAAACAC T TAGAAG TAT T TCATAAAATAT TGAC TAATACCGATCCAAATGATGAAGTAGA AAGAAGAAATGCCGATAATAAAGAAGATTTAACTAGTGCCGATCCAGAAGGACAAATAATGAGA GAATATGC TGC TGATCCAGAATATCGTAAACACT TAGAAATAT T TCATAAAATAT TAAC TAACA CCGATCCAAATGATGAAGTAGAAAGAAGAAATGCCGATAATAAAGAAGACTTAACTAGTGCCGA TCCAGAAGGTCAAATAATGAGAGAATATGCCGCTGATCCAGAATACCGTAAACACTTAGAAATA T T T TATAAAATAT TAAC TAACACAGATCCAAATGATGAAGTAGAAAGAAGAAATGCCGATAATA AAGAAGAATTAACCAGCTCAGACCCAGAAGGTCAAATAATGAGAGAATATGCCGCTGATCCAGA GTATCGTAAACAC T TAGAAATAT T TCATAAAATAT TAAC TAACACCGATCCAAATGATGAAGTA GAAAGAAGAAATGCCGATAATAAAGAAGAC T TAAC TAGTGCCGATCCAGAAGGTCAAATAATGA GAGAATATGCCGC TGATCCAGAATACCGTAAACAC T TAGAAATAT T T TATAAAATAT TAACTAA CACCGATCCAAATGATGAAGTAGAAAGAAGAAATGCCGATAATAAAGAAGAT T TAACCAGTGCC GATCCAGAAGGTCAAATAATGAGAGAATATGC TTCCGATCCAGAATACCGTAAACAC T TAGAAA TAT TT TATAAAATAT TAAC TAACACCGATCCAAATGATGACGTAGAAAGAAGAAATGCCGATAA CAAAGAAGATT TAAC TAGTGCCGATCCAGAAGGTCAAATAATGAGAGAATATGC TGC TGATCCA GAATATCGTAAACAC TTAGAAGTAT T TCATAAAATAT TGAC TAATACCGATCCAAATGATGAAG TAGAAAGACAAAATGCTGATAATAACGAAGCA

SEQ ID NO : 10

GE D TCARKE KT TLRKSKQKT S TRTVATQ TKKDE E NKSWTE E QKVE SD SE KQKRTKKWKKQ I N I GD TENQKE GKNVKKVI KKE KKKE E SGKPE ENKHANE ASKKQE PKASKVSQKPS TS TRSNNE VK IRAASNQETLTSADPEGQIMREYAADPEYRKHLE IFYKILTNTDPNDEVERRNADNKEDLTSAD PEGQIMREYASDPEYRKHLE IFYKILTNTDPNDDVERRNADNKEDLTSADPEGQIMREYAADPE YRKHLEVFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLEVFHKILTN TDPNDEVERRNADNKELTSSDPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVERRNADNK EDLTSADPEGQIMREYAADPEYRKHLEVFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMR EYAADPEYRKHLE IFHKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYAADPEYRKHLE I FYKILTNTDPNDEVERRNADNKEELTSSDPEGQIMREYAADPEYRKHLEIFHKILTNTDPNDEV ERRNADNKEDLTSADPEGQIMREYAADPEYRKHLE IFYKILTNTDPNDEVERRNADNKEDLTSA DPEGQIMREYASDPEYRKHLEIFYKILTNTDPNDDVERRNADNKEDLTSADPEGQIMREYAADP EYRKHLEVFHKILTNTDPNDEVERQNADNNEA

SEQ ID NO : 11

CGCACAGUUGCUACUCAAACAAAGAAAGAUGAAGAAAAUAAAUCAGUUGUUACCGAAGAACAAA AAGUAGAAAGUGAUUCCGAAAAACAAAAAAGAACCAAAAAAGUAGUAAAAAAGCAAAUUAAUAU AGGAGAUACAGAAAAUCAAAAAGAAGGAAAAAACGUUAAAAAAGUUAUAAAGAAAGAAAAGAAA AAAGAAGAAUCUGGAAAACCAGAAGAAAAUAAACAUGCAAACGAAGCUUCAAAAAAACAGGAAC CUAAAGCCUCAAAAGUAUCUCAAAAACCAUCAACUAGCACACGUUCAAAUAAUGAAGUAAAAAU ACGAGCUGCUUCUAAUCAAGAAACAUUAACUAGUGCCGAUCCAGAAGGACAAAUAAUGAGAGAA UAUGCUGCUGAUCCAGAAUAUCGUAAACACUUAGAAAUAUUUUAUAAAAUAUUAACUAACACCG AUCCAAAUGAUGAAGUAGAAAGAAGAAAUGCCGAUAAUAAAGAAGAUUUAACUAGUGCCGAUCC AGAAGGUCAAAUAAUGAGAGAAUAUGCUUCCGAUCCAGAAUACCGUAAACACUUAGAAAUAUUU UAUAAAAUAUUAACUAACACCGAUCCAAAUGAUGACGUAGAAAGAAGAAAUGCCGAUAAUAAAG AAGAUUUAACCAGUGCCGAUCCAGAAGGUCAAAUAAUGAGAGAAUAUGCUGCUGAUCCAGAAUA UCGUAAACAUUUAGAAGUAUUUCAUAAAAUAUUGACUAAUACCGAUCCAAAUGAUGAAGUAGAA AGAAGAAAUGCCGAUAAUAAAGAA

SEQ ID NO : 12

ATCCGGTGAGAATGGCAAAAGT T TATGCAT T TCT T TCCAGAC T TGT TCAACAGGCCAGCCAT TA CGC TCGTCATCAAAATCAC TCGCATCAACCAAACCGT TAT TCATTCGTGAT TGCGCC TGAGCGA GGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAGTGCAACCGGCGCAG GAACAC TGCCAGCGCATCAACAATAT TT TCACCTGAATCAGGATAT TC T TO TAATACC TGGAAC GC TGT T T T TCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC T TGATGGTCGGAAGTGGCATAAAT TCCGTCAGCCAGT T TAGTC TGACCATCTCATCTGTAACATC AT TGGCAACGC TACC TT TGCCATGT T TCAGAAACAAC TC TGGCGCATCGGGC T TCCCATACAAG CGATAGAT TGTCGCACC TGATTGCCCGACAT TATCGCGAGCCCAT T TATACCCATATAAATCAG CATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGGATATGGCTCATTTTTTACTT CC TCACC T TGTCGTATTATACTATGCCGATATAC TATGCCGATGAT TAATTGTCGACAC TGCGG GGGCTCTGGAGACGACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGT TCCCATAGTAACGCCAATAGGGAC T T TCCAT TGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCG CCCCC TAT TGACGTCAATGACGGTAAATGGCCCGCC TGGCAT TATGCCCAGTACATGACC TTAC GGGAC T T TCCTAC T TGGCAGTACATC TACGTATTAGTCATCGC TAT TACCATGC TGATGCGGT T T TGGCAGTACACCAATGGGCGTGGATAGCGGT TTGAC TCACGGGGAT T TCCAAGTC TCCACCCC ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATA ACCCCGCCCCGTTGACGCAATAAGGTTGAGGTCTCTAAAACTGTCTTCCCAACTTGTTTATTGC AGC TTATAATGGT TACAAATAAAGCAATAGCATCACAAAT T TCACAAATAAAGCAT T T T T TTCA CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCGAAGACAGTCAGC C T TGAGCGGTATCAGCTCAC TCAAAGGCGGTAATACGGT TATCCACAGAATCAGGGGATAACGC AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT TGC TG GCGTT T T TCCATAGGCTCCGCCCCCC TGACGAGCATCACAAAAATCGACGC TCAAGTCAGAGGT GGCGAAACCCGACAGGAC TATAAAGATACCAGGCGT T TCCCCC TGGAAGCTCCC TCGTGCGC TC TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTC CAACCCGGTAAGACACGAC T TATCGCCAC TGGCAGCAGCCAC TGGTAACAGGAT TAGCAGAGCG AGGTATGTAGGCGGTGC TACAGAGT TCT TGAAGTGGTGGGC TAAC TACGGC TACAC TAGAAGAA CAGTAT T TGGTATC TGCGC TCTGC TGAAGCCAGT TACCT TCGGAAAAAGAGT TGGTAGC TCT TG ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT TTTTTTGTT TGCAAGCAGCAGAT TACGCGC AGAAAAAAAGGATC TCAAGAAGATCC TT TGATCT T T TCTACGGGGTC TGACGC TCAGTGGAACG ACGCGCGCGTAAC TCACGT TAAGGGATT T TGGTCATGAGT TAGAAAAAC T CATC GAGCAT CAAA TGAAAC TGCAAT T TATTCATATCAGGAT TATCAATACCATAT T TT TGAAAAAGCCGT T TC TGTA ATGAAGGAGAAAAC TCACCGAGGCAGTTCCATAGGATGGCAAGATCC TGGTATCGGTC TGCGAT TCCGAC TCGTCCAACATCAATACAACCTAT TAAT T TCCCC TCGTCAAAAATAAGGT TATCAAGT GAGAAATCACCATGAGTGACGAC TGA

[0273] The invention contemplates that any of the above-described sequences, methods, devices or combinations can be derivatized or can be structurally altered to further save lives, for example, by addition or substitution of one or more atoms using a radioisotope or using a different element (e.g., B or boron in place of C or carbon), by removal of an ester or by addition of a salt form, an amino acid, a sugar, or a peptide. In addition, hydrates and/or solvates can be formed by 1) dissolving the vaccine in water and/or solvent and slowly drying, whereby water and/or solvent remain hydrogen bonded with OH groups in the molecule or associated with the sequences. A formation of a hydrate or solvate can typically be confirmed by an attenuated total reflection (ATR) infrared spectrum acquired from the solid-state sample. The ATR spectrum of a hydrate or solvate will typically show increased broad bands (indicating hydrogen bonding) above about 3200cm'¹, as compared to the non-hydrate or non-solvate solid sample. In some embodiments, the above-described vaccines are attached to or associated with a targeting moiety. In some embodiments, the targeting moiety is a particle or an antibody with affinity for a specific type of cell. While various crystal structures are contemplated, these investigations will also be initiated.

REFERENCES:

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Claims

CLAIMS l/l/e claim:

1. A malaria vaccine composition configured to provide Plasmodium falciparum proteins or amino acid sequences including PfGBP130 or PfGBP130-A surface antigen sequences, wherein when the malaria vaccine composition is administered to a human subject specific anti-malaria antibodies are produced; the malaria vaccine composition being configured to provide a production of antibodies within the human subject that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites, thereby preventing and/or treating malaria in the human subject.

2. The malaria vaccine composition of claim 1 , wherein the Plasmodium falciparum proteins or amino acid sequences are derived from PfGBP130 or PfGBP130-A and/or wherein the vaccine includes sequences from PfGBP130 or PfGBP130-A formulated as a recombinant protein.

3. The malaria vaccine composition of claim 1 , wherein the Plasmodium falciparum proteins or amino acid sequences are derived from a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences and/or wherein the vaccine includes mRNA encoding for PfGBP130-A (SEQ ID NO: 1) or a PfGBP130-ecto (SEQ ID NO: 2).

4. The malaria vaccine composition of claim 1 , wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130 surface antigen sequences (SEQ ID NO: 3).

5. The malaria vaccine composition of claim 1 , wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130-A surface antigen sequences (SEQ ID NO: 4).

6. The malaria vaccine composition of claim 1 , wherein the antibodies generated within the human subject are specific to the Plasmodium falciparum proteins or amino acid sequences.

7. The malaria vaccine composition of claim 1 , wherein the antibodies generated within the human subject are specific to the PfGBP130 or PfGBP130-A surface antigen sequences and/or wherein the vaccine includes a DNA plasmid (SEQ ID NO: 5) encoding PfGBP130-A.

8. The malaria vaccine composition of claim 1, wherein the antibodies inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites.

9. The malaria vaccine composition of claim 1 , wherein the antibodies inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites.

10. The malaria vaccine composition of claim 1 , wherein the malaria vaccine composition includes amino acids (aa) 111-374 of PfGBP130-A (SEQ ID NO: 6) and/or includes aa 89-824 of PfGBP130 (SEQ ID NO: 7).

11. The malaria vaccine of claim 1 , wherein the vaccine includes a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences or includes mRNA (SEQ ID NO: 8) and/or DNA (SEQ ID NO: 9) that encodes for aa 89-824 of PfGBP130- ecto (SEQ ID NO: 10) and/or encodes for aa 111-374 of PfGBP130-A (SEQ ID NO: 11).

12. The malaria vaccine composition of claim 1, wherein the malaria vaccine composition treats malaria in the human subject and/or wherein the malaria vaccine is formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector including SEQ ID NO: 12, or a combination thereof.

13. A method of preventing and/or treating malaria in a human subject, the method comprising administering to the human subject a malaria vaccine composition configured to provide Plasmodium falciparum proteins or amino acid sequences including PfGBP130 or PfGBP130-A surface antigen sequences, wherein when the malaria vaccine composition is administered to the human subject specific anti-malaria antibodies are produced; the malaria vaccine composition being configured to provide a production of antibodies within the human subject that inhibit red blood cell invasion by Plasmodium falciparum malaria parasites.

14. The method of claim 13, wherein the Plasmodium falciparum proteins or amino acid sequences are derived from PfGBP130 or PfGBP130-A formulated as a recombinant protein.

15. The method of claim 13, wherein the Plasmodium falciparum proteins or amino acid sequences are derived from a nucleic acid coding sequence including at least one of all possible coding sequences for these amino acid sequences or wherein the vaccine includes mRNA encoding for PfGBP130-A (SEQ ID NO: 1) or a PfGBP130-ecto (SEQ ID NO: 2).

16. The method of claim 13, wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130 surface antigen sequences (SEQ ID NO: 3).

17. The method of claim 13, wherein the Plasmodium falciparum proteins or amino acid sequences comprise PfGBP130-A surface antigen sequences (SEQ ID NO: 4).

18. The method of claim 13, wherein the antibodies generated within the human subject are specific to the Plasmodium falciparum proteins or amino acid sequences.

19. The method of claim 13, wherein the antibodies generated within the human subject are specific to the PfGBP130 or PfGBP130-A surface antigen sequences and/or wherein the vaccine includes a DNA plasmid (SEQ ID NO: 5) encoding PfGBP130-A.

20. The method of claim 13, wherein the antibodies inhibit red blood cell invasion by binding to Plasmodium falciparum malaria parasites.

21. The method of claim 13, wherein the antibodies inhibit red blood cell invasion by neutralizing Plasmodium falciparum malaria parasites.

22. The method of claim 13, wherein the malaria vaccine composition includes amino acids (aa) 111-374 of PfGBP130-A (SEQ ID NO: 6) and/or includes aa 89-824 of PfGBP130 (SEQ ID NO: 7).

23. The method of claim 13, wherein the vaccine includes mRNA (SEQ ID NO: 8) and/or DNA (SEQ ID NO: 9) that encodes for aa 89-824 of PfGBP130-ecto (SEQ ID NO: 10) and/or encodes for aa 111-374 of PfGBP130-A (SEQ ID NO: 11).

24. The method of claim 13, wherein the malaria vaccine composition treats malaria in the human subject and/or wherein the malaria vaccine is formulated in a lipid nanoparticle encapsulation, a virus-like particle, a nanoparticle, a conjugate, a plasmid vector including SEQ ID NO: 12, or a combination thereof.