RESPONSE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §

119 of U.S. Provisional Application Serial No. 63/369909 filed on July 29, 2022, the contents of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH &

DEVELOPMENT

[0002] This invention was made with government support under Intramural

Research Grant Number IZIAAI001253, awarded by the National Institute of Allergy and

Infectious Diseases (NIAID), an institute of the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The immune response to an infecting microorganism is a complex process involving both innate and adaptive immunity. Adaptive immunity comprises both humoral immunity’ pathways and cell-mediated immunity pathways that work together to produce molecules that recognize antigens present in the infecting microorganism. Humoral immunity involves the stimulation of B-cells, which produce antibodies that recognize and bind short amino acid sequences, called epitopes, in proteins produced by the infecting microorganism. Some of the antibodies are neutralizing antibodies, which means that binding of the antibody to an epitope in a protein of the microorganism prevents the microorganism from causing further disease. The neutralizing antibody may “neutralize” the infecting microorganism by binding to a site on a protein necessary for the microorganism to bind a cell receptor and enter the cell. Alternatively, the neutralizing antibody may prevent a protein of the microorganism from adopting a conformational change necessary for attachment or entry of the microorganism into the cell, or necessary for functions once the microorganism has entered the cell. Not all antibodies are neutralizing. Some antibodies are capable of binding to proteins from microorganism, but such binding has little or no effect on the ability of the microorganism to replicate and infect cells within an individual. Such antibodies are referred to as non -neutralizing antibodies. Thus, an effective immunogen is one that preferentially elicits neutralizing antibodies rather than non- neutralizing antibodies.

[0004] Vaccines work by stimulating the immune system to produce antibodies against proteins present on infecting microorganisms. Preferably, a vaccine elicits the production of neutralizing antibodies. However, not all microbial antigens produce proteins that elicit neutralizing antibodies, making the production of vaccines for certain microorganisms difficult. One such infectious microorganism is Plasmodium falciparum, a causative agent of malaria. Progress in reducing malaria morbidity and mortality has stalled, and emerging parasite resistance against existing drugs has intensified the need for alternative treatment strategies and preventive measures. A vaccine that targets malaria merozoites (or blood-stage parasites) would directly prevent parasite infection of red cells and clinical symptoms. To achieve a broadly protective blood-stage vaccine, it is crucial to identify essential and strain-transcending vaccine immunogens, the key epitopes that elicit potent neutralizing antibody responses, and the immune evasion mechanisms employed by the parasite to circumvent protection.

[0005] Merozoite surface proteins are high-priority candidate vaccine antigens as they are prime targets of the humoral immune response. Of all such proteins, Merozoite Surface Protein 1 (MSP-1) is the most abundant, is essential for Plasmodium development and is proposed to have a role in early erythrocyte attachment, invasion and cgrcss. MSP- l interactions with red cell proteins to facilitate these roles have been well- characterized. MSP-1 is known to undergo two distinct proteolytic processing steps (FIG. 1) to first form 83 kDa, 30 kDa, 38 kDa and 42 kDa fragments, followed by cleavage of the 42 kDa fragment into 33 kDa and 19 kDa fragments. The C-terminal p19 is attached to the merozoite surface through a glycosylphosphatidylinositol (GPI) anchor and the remaining fragments are shed upon formation of a tight junction with the red blood cell (RBC). The structure of the ectodomain of MSP-1 lacking p19 revealed a concentration-dependent monomer-dimer equilibrium affected by the presence of red cell proteins which may compete for the dimerization interface, p19 is maintained on the merozoite surface after invasion and thought to have a role in intraerythrocytic parasite development. p19 consists of two epidermal growth factor (EGF)-like domains. EGF-like domains are found in the extracellular domain of membrane-bound or secreted proteins and serve a variety of functional roles including mediation of protein/protein interactions. The EGF-like domain includes six disulfide-bonded cysteine residues that stabilize a two-stranded beta-sheet connected to a second short, two-stranded sheet.

[0006] Antibodies targeting all MSP-1 subunits inhibit parasite growth to varying degrees and antibodies targeting p19 appear to be most potent. Naturally acquired antibodies targeting p19 prevent merozoite invasion of RBCs and are associated with protection from clinical malaria and protection appears to be FcyRI-mediated in a transgenic rodent malaria model for MSP-1. Monoclonal antibodies (mAbs) to MSP-1 isolated from rodents and more recently from individuals with naturally acquired immunity have been characterized. The murine mAb G17.12 binds the first EGF-like domain of MSP-1 p19 and does not inhibit erythrocyte invasion. In contrast, murine mAbs 12.8 and 12.10 recognize overlapping epitopes on EGF domain 1 and inhibit erythrocyte invasion by preventing secondary proteolytic processing of MSP-1. Recently, isolation of human IgG mAbs from individuals living in malaria-endemic areas with naturally acquired immunity identified three hmAbs, one of which, 42D6, showed potent activity in inhibiting parasite growth. A separate study of human IgG identified the hmAb MaliM03 that binds a similar epitope as murine mAb G17. 12 and likewise does not show any parasite growth inhibitory activity as an IgG. Interestingly, forced multimerization of MaliM03 by incorporation into an IgM backbone achieved strong parasite binding and inhibited merozoite invasion of RBCs. Immunization with p19 has been investigated in both animal and clinical studies as an approach induce protection. While p19-specific neutralizing antibodies were induced by a chimeric MSP vaccine in rabbits, phase 1 clinical trials of p19-based vaccines met with limited success. Additionally, various clinical studies have tested MSP-1 -based vaccines, which were safe and elicited a humoral immune response. However, limited effects on parasite growth rates in the blood and limited efficacy were observed.

SUMMARY

[0007] The present disclosure generally relates to methods of improving vaccines. More specifically, the present disclosure relates to methods of modifying the amino acid sequence of a protein having neutralizing epitopes and interfering, non-neutralizing epitopes, to direct the immune system to a specific region of the protein. The disclosure also relates to proteins made using the disclosed methods, as well as methods of using such proteins to detect neutralizing antibodies, and to vaccinate individuals against infectious microorganisms.

[0008] One aspect of the disclosure is a method of immunofocusing an immune response to a neutralizing epitope in an initial protein, the initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more non- neutralizing interfering epitopes, the method comprising modifying the initial amino acid sequence to produce a modified amino acid sequence comprising the neutralizing epitope such that the immune response is preferentially, or entirely, directed towards the neutralizing epitope in a modified protein comprising the modified amino acid sequence. In certain aspects, the immunogenicity of the neutralizing epitope in the initial protein may not be significantly greater than the immunogenicity of the one or more interfering, non- neutralizing epitopes in the initial protein, wherein the immunogenicity of the neutralizing epitope in the modified protein may be significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the modified protein.

[0009] One aspect of the disclosure is a method of improving the ability of an initial protein to elicit a neutralizing immune response, the initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more non-neutralizing interfering epitopes, the method comprising modifying the initial amino acid sequence to produce a modified amino acid sequence such that the immunogenicity of the neutralizing epitope in a modified protein comprising the modified amino acid sequence is significantly greater than the immunogenicity of the one or more non-neutralizing epitopes in the modified protein.

[0010] One aspect of the disclosure is a method of producing a vaccine candidate using an initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes, the method comprising modifying the initial amino acid sequence to produce a modified amino acid sequence, such that the immunogenicity of the neutralizing epitope in a modified protein comprising the modified amino acid sequence is significantly greater than the immunogenicity of the one or more non-neutralizing epitopes in the modified protein, and producing a modified protein that comprises the modified amino acid sequence, thereby producing a vaccine candidate. [0011] One aspect of the disclosure is a method of identifying a vaccine candidate using an initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes, the method comprising: modifying the initial amino acid sequence to produce a modified amino acid sequence, such that the immunogenicity of the neutralizing epitope in a modified protein comprising the modified amino acid sequence is significantly greater than the immunogenicity of the one or more non-neutralizing epitopes in the modified protein; producing the modified protein; and, assaying a neutralizing antibody and a non-neutralizing antibody for their abilities to bind the modified protein. In certain aspects, if ability of the neutralizing antibody to bind the modified protein is not significantly less than the ability of the neutralizing antibody to bind the initial protein, and if the ability of the non-neutralizing antibody to bind the modified protein is significantly less than the ability of the non- neutralizing antibody to bind the initial protein, the modified protein is identified as a vaccine candidate.

[0012] In these aspects, the immunogenicity of the neutralizing epitope in the modified protein may not be reduced relative to the immunogenicity of the neutralizing epitope in the initial protein, modifying the initial amino acid sequence reduces the immunogenicity of the one or more non-neutralizing epitope in the modified protein relative to the immunogenicity of the neutralizing epitope in the initial protein. In these aspects, modifying the initial amino acid sequence may increase the immunogenicity of the neutralizing epitope in the modified protein relative to the immunogenicity of the neutralizing epitope in the initial protein. In these aspects, modifying the initial amino acid sequence may reduce the affinity of a non-neutralizing antibody for the one or more non- neutralizing epitopes in the modified protein relative to the affinity of the non-neutralizing antibody for the one or more non-neutralizing epitopes in the initial protein. In these aspects, modifying the initial amino acid sequence may increase the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein. In these aspects, the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein may be significantly greater than the affinity of a non-neutralizing antibody for the one or more non- neutralizing epitopes in the modified protein. [0013] In these aspects, modifying may comprise introducing a mutation at one or more amino acid positions within the initial amino acid sequence to produce the modified amino acid sequence, and the mutation may comprise a substitution mutation, a deletion mutation, or an insertion mutation. The one or more amino acid positions may not comprise amino acid residues within the neutralizing epitope. The one or more amino acid positions may comprise amino acid residues within the non-neutralizing epitope, which may be contact residues within the non-neutralizing epitope. In these aspects, the location of the one or more amino acid positions and what mutation is made at each of the one or more amino acid positions may be determined by subjecting the initial amino acid sequence to computational design to produce a heterogeneous pool of modified amino acid sequences, each of which comprises a mutation at one or more amino acid positions, and each of which has an associated stability score; clustering high scoring, modified amino acid sequences based on sequence similarity; and, selecting at least one modified amino acid sequence from each cluster and using the sequence of the selected at least one modified amino acid sequence to determine the location(s) of the one or more amino acid position(s) and which mutation is made at each of the one or more amino acid positions. In such aspects, the mutation may be a substitution mutation, and the computational design may comprise assigning a computational search depth to each amino acid position in the initial amino acid sequence, the search depth being one of fixed, intermediate, or deep, wherein a fixed search depth is defined as leaving a residue at the assigned position unchanged, an intermediate search depth is defined as allowing sampling from a pool of evolutionarily constrained amino acids, and, a deep search depth is defined as allowing sampling from a pool of all amino acids; at each position assigned an intermediate or deep search depth, substituting an amino acid in the initial amino acid sequence with an amino acid sampled from the pool of amino acids, the breadth of the pool at each position being defined by the assigned search depth at that position, to produce modified amino acid sequences; and for each modified amino acid sequence, determining the thermodynamic potential of the sequence in a folded state, and assigning a stability score based thereupon, thereby producing the heterogenous pool of modified amino acid sequences each of which comprises amino acid substitutions at one or more amino acid positions, and each of which has an associated stability score. In such aspects, the pool of evolutionarily constrained amino acids for each amino acid position assigned an intermediate search depth may consist of those amino acids present at the corresponding amino acid position in proteins related to the initial protein, related proteins comprising those proteins determined to be related to the initial protein using a position- specific iterative local alignment process such as psiBLAST. In these aspects, the initial protein may be from an infectious microorganism, which may be from the genus Plasmodium. In these aspects, the initial protein may be a Plasmodium falciparum (P. falciparum) surface protein, which may be selected from the group consisting of merozoite surface proteins (MSPs) MSP1, MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10, MSP12, p19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PfRH2a, PfRH2b, PfRH4, PfRH5, RAMA, and RALP1. In these aspects, the initial protein may be P. falciparum surface protein p19 (MSP1-19). In these aspects, the initial protein may comprise an amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 97% identical or 100% identical to SEQ ID NO:2, and the neutralizing epitope may comprise Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2. The one or more non-neutralizing, interfering epitopes may be selected from the group consisting of an epitope comprising Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and, an epitope comprising Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Ser69, Arg71, Phe87 and Asp88 of SEQ ID NO:2. In these aspects, the computational design may be conducted in silico and may comprises the use of protein modeling software for structure-based design, such as the Rosetta software suite.

[0014] One aspect of the disclosure is a modified protein produced using a method of the disclosure. A modified protein produced using the method of any one of claims 1-30. The modified protein may comprise a modified amino acid sequence derived from an initial protein, the initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes; wherein the modified amino acid sequence is at least 50% identical to the entire length of the initial amino acid sequence; wherein the difference between the initial and modified amino acid sequences is due a mutation at one or more positions of the initial amino acid sequence; wherein the one or more amino acid positions do not comprise contact residues of the neutralizing epitope; and, wherein the immunogenicity of the neutralizing epitope in the modified protein is significantly greater than the immunogenicity of the one or more non-neutralizing epitopes in the modified protein; or wherein the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of a non-neutralizing antibody for the one or more non-neutralizing epitopes in the modified protein. In these aspects, the immunogenicity of the neutralizing epitope in the initial protein may not be significantly greater than the immunogenicity of the one or more non-neutralizing epitopes in the initial protein; and/or the affinity of a neutralizing antibody for the neutralizing epitope in the initial protein may not be significantly greater than the affinity of a non-neutralizing antibody for the one or more non-neutralizing epitopes in the initial protein. In these aspects, the mutation at one or more amino acid positions comprises a substitution mutation, a deletion mutation, or an insertion mutation.

[0015] One aspect of the disclosure is a modified P. falciparum p19 protein derived from a wild- type (wt) P . falciparum p19 protein, the wt P. falciparum p19 protein comprising a neutralizing epitope, a first interfering non-neutralizing epitope, and a second interfering non-neutralizing epitope, wherein the first and second non-neutralizing epitopes each, individually, mask the neutralizing epitope, wherein the neutralizing epitope in the wt p19 protein comprises Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2; wherein the amino acid sequence of the modified P falciparum p19 protein is at least 50% identical to the amino acid sequence of the wt P. falciparum p19 protein; wherein the difference between the wt and modified amino acid sequences are due to an mutation at one or more amino acid positions; and, wherein the immunogenicity of the neutralizing epitope in the modified protein is significantly greater than the immunogenicity of the one or more non-neutralizing epitopes in the modified protein; or wherein the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of a non-neutralizing antibody for the one or more non-neutralizing epitopes in the modified protein. In these aspects, the immunogenicity of the neutralizing epitope in the modified protein may not be reduced relative to the immunogenicity of the neutralizing epitope in the wt p19 protein. The immunogenicity of the first and/or second non-neutralizing interfering epitopes in the modified protein may not be reduced relative to the immunogenicity of the first and/or second non-neutralizing interfering epitopes in the wt protein. In these aspects, the wt p19 protein may SEQ ID NO:2, the first non-neutralizing interfering epitope in the initial protein may comprise Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and the second non-neutralizing interfering epitope comprises Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu38, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, V142, Glu43, Pro45, Ser69, Arg71, Phe87 and Asp88 of SEQ ID NO:2.

[0016] One aspect of the disclosure is a protein comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises: a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20.

[0017] One aspect of the disclosure is a fusion protein comprising at least a portion of a self-assembling subunit protein joined to a modified protein of the disclosure, wherein the at least a portion of a self-assembling subunit directs self-assembly of the fusion protein into nanoparticles. In certain aspects, the modified protein may be joined to the carboxyl terminal end of the at least a portion of a self-assembling subunit protein. In certain aspects, the modified protein may be joined to the amino-terminal end of the at least a portion of a self-assembling subunit protein. The self-assembling subunit protein may be lumazine synthase (LS), the E2p protein of G. stearothermophilus (N-terminus of dihydrolipoyl acetyltransferase) (E2p), the EncA protein of Myxococcus xanthus (C-terminus of encapsulin protein) (EncA) or an apoferritin (ApoF) protein. In certain aspects, the ferritin protein may be from Heliobacter pylori. The fusion protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92.

[0018] One aspect of the disclosure is a nanoparticle comprising a fusion protein of the disclosure.

[0019] One aspect of the disclosure is an isolated nucleic acid molecule comprising a nucleotide sequence encoding a modified protein of the disclosure or a fusion protein of the disclosure. The isolated nucleotide sequence may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91. One aspect of the disclosure is a plasmid or a viral vector comprising an isolated nucleic acid molecule of the disclosure.

[0020] One aspect of the disclosure is a composition, which may be a therapeutic composition, comprising a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, or a viral vector, of the disclosure. The therapeutic composition may comprise a pharmaceutically acceptable excipient.

[0021] One aspect of the disclosure is a vaccine comprising a therapeutic composition of the disclosure.

[0022] One aspect of the disclosure is a kit comprising a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, a viral vector, or a vaccine, of the disclosure.

[0023] One aspect of the disclosure is a method of vaccinating an individual against infection by P. falciparum, comprising administering to the individual a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, a viral vector, or a vaccine, of the disclosure

[0024] One aspect of the disclosure is a method of protecting an individual against infection by a microorganism of the genus Plasmodium, comprising administering to the individual a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, a viral vector, or a vaccine, of the disclosure.

[0025] One aspect of the disclosure is a method of protecting an individual against malaria, comprising administering to the individual a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, a viral vector, or a vaccine, of the disclosure. [0026] One aspect of the disclosure is a method of treating a malaria patient, comprising administering to the patient a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, a viral vector, or a vaccine, of the disclosure.

[0027] One aspect of the disclosure is a modified protein, a fusion protein, a nanoparticle a nucleic acid molecule, a plasmid, a viral vector, or a vaccine for use in vaccinating an individual against infection by P. falciparum, protecting an individual against infection by a microorganism of the genus Plasmodium, protecting an individual against malaria, or treating a malaria patient.

[0028] One aspect of the disclosure is a method of detecting the presence of a neutralizing antibody in a sample, wherein the neutralizing antibody specifically recognizes the neutralizing epitope in a modified protein of the disclosure, comprising contacting the sample with the modified protein, and detecting binding of a neutralizing antibody in the sample, if any, with the modified protein, thereby detecting the presence of a neutralizing antibody in the sample.

[0029] One aspect of the disclosure is a method of determining the efficacy of a vaccine comprising a modified protein of the disclosure to induce a neutralizing antibody that binds the neutralizing epitope in the modified protein, comprising: determining the level of the neutralizing antibody in a first sample from a vaccinated individual using a method comprising contacting the first sample with the modified protein, and detecting binding of a neutralizing antibody in the first sample, if any, with the modified protein; and comparing the level of neutralizing antibody with the level of neutralizing antibody in a second sample from an unvaccinated individual, thereby determining the efficacy of the vaccine. The second sample may be from the vaccinated individual at a time prior to vaccination. Detecting binding of the neutralizing antibody to the modified protein may comprise detecting a complex between the neutralizing antibody and the modified protein, which may comprise a detectable label. [0030] One aspect of the disclosure is a method of increasing the affinity of an initial neutralizing antibody comprising an antigen-binding site that specifically binds a neutralizing epitope in a protein, the protein comprising the neutralizing epitope and one or more interfering non-neutralizing epitopes, the method comprising: modifying the initial amino acid sequence of the neutralizing antibody to produce a modified sequence such that the affinity of an antibody comprising the modified amino acid sequence for the neutralizing epitope is increased relative to the affinity of the initial neutralizing antibody for the neutralizing epitope; and, wherein the affinity of the antibody comprising the modified amino acid sequence for the neutralizing epitope is significantly greater than the affinity of a non- neutralizing antibody for the one or more interfering non-neutralizing epitopes. Modifying may comprise introducing a mutation, which may be an insertion, a deletion, or a substitution, at one or more amino acid positions in the initial amino acid sequence to produce the modified amino acid sequence. The one or more amino acid positions may comprise contact residues in the antigenic binding site of the neutralizing antibody. The location of the one or more amino acid positions, and what mutation is made at each of the one or more amino acid positions, may be determined by: subjecting the initial amino acid sequence to computational design to produce a heterogeneous pool of modified amino acid sequences, each of which comprises a mutation at one or more amino acid positions, and each of which has an associated stability score; clustering high scoring, modified amino acid sequences based on sequence similarity; and, selecting at least one modified amino acid sequence from each cluster and using the sequence of the selected at least one modified amino acid sequence to determine the location(s) of the one or more amino acid position(s) and which mutation is made at the one or more amino acid positions. In these aspects, the mutation may be a substitution mutation, and the computational design may comprise: assigning a computational search depth to each amino acid position in the initial amino acid sequence, the search depth being one of fixed, intermediate, or deep, wherein a fixed search depth is defined as leaving a residue at the assigned position unchanged, an intermediate search depth is defined as allowing sampling from a pool of evolutionarily constrained amino acids, and, a deep search depth is defined as allowing sampling from a pool of all amino acids; at each position assigned an intermediate or deep search depth, substituting an amino acid in the initial amino acid sequence with an amino acid sampled from a pool of amino acids, the breadth of the pool at each position being defined by the assigned search depth at that position, to produce modified amino acid sequences; and for each modified amino acid sequence, determining the thermodynamic potential of the sequence in a folded state, and assigning a stability score based thereupon, thereby producing the heterogenous pool of modified amino acid sequences each of which comprises amino acid substitutions at one or more amino acid positions, and each of which has an associated stability score. The pool of evolutionarily constrained amino acids for each amino acid position assigned an intermediate search depth may consist of those amino acids present at the corresponding amino acid position proteins related to the initial protein. Related proteins may comprise those proteins determined to be related to the initial protein using a position-specific iterative local alignment process. In these aspects, the protein may be a Plasmodium falciparum (P. falciparum) surface protein, which may be selected from the group consisting of merozoite surface proteins (MSPs) MSP1, MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10, MSP12, P19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PfRH2a, PfRH2b, PfRH4, PfRH5, RAMA, and RALP1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG.1 illustrates assembly and processing of the MSP-1.

[0032] FIGS. 2A & 2B show purification of and characterization of hmAbs. FIG. 2A is a reducing SDS-PAGE profile of expressed and purified hmAbs. FIG. 2B is an ELISA showing binding of eight different hmAbs to recombinantly expressed full-length MSP-1 and p19 from Plasmodium falciparum 3D7 from 5 independent assays.

[0033] FIGS. 3A & 3B show expression and purification of recombinant antigens. FIG. 3A shows size exclusion chromatography (SEC) profile of recombinantly expressed p19 on Superdex 75 Increase 10/300 GL at 0.80 ml/min. FIG. 3B shows SEC profile of recombinantly expressed full-length MSP-1 on Superdex 200 Increase 10/300 GL at 0.75 ml/min. The inset in FIGS. 3 A & 3B are SDS-PAGE gels stained with Coomassie Blue showing the purity of the monomeric p19 (14.339 kDa) and full-length MSP-1 (193 kDa), respectively.

[0034] FIG. 4 shows in vitro growth inhibition assay (GIA) of each hmAb tested at 1.0 mg/ml against the Plasmodium falciparum 3D7 blood stage in five independent assays. The individual biological replicates from each assay and mean (bars) are shown. [0035] FIGS. 5A-5C illustrate that binding kinetics and GIA do not show correlation. FIGS. 5A-5C show the dissociation constant (K _D ) (5 A), association rate constant (k _a ) (5B), and dissociation rate constant (k _dis ) (5C) plotted against % GIA for hmAbs. Kinetic data are as in Table 1 and % GIA value represents an average for each hmAb tested at 1.0 mg/ml against the P. falciparum 3D7 blood stage in 5 independent assays.

[0036] FIG. 6 illustrates epitope binning for p19-specific human mAbs. Primary saturating antibodies tested are listed in the left column, while secondary competing antibodies are listed at the top in rows. Data indicate the percent of competing antibody binding compared to the maximum competing antibody response in the absence of the primary antibody. Boxes are colored according to competition status. Antibodies that displayed < 50% maximal binding are colored light red and are considered “competing”. Negative values were normalized to 0.

[0037] FIG. 7 shows the crystal structures of p19 bound to the Fab fragment of 42D6 (left), 42C11 (center) and 42C3 (right). All crystal structures are shown according to the same p19 orientation, p19 is represented as white surface.

[0038] FIGS.8A-8F shows the representative electron density of the 42D6, 42C11, and 42C3 epitopes on p19. Electron density for the 42D6 (FIG. 8A), 42C11 (FIG. 8C), and 42C3 (FIG. 8E) epitope residues from a 2Fo-Fc map (gray mesh) contoured at 1.0 σ level (1.43 rmsd). Electron density for the 42D6 (FIG. 8B), 42C11 (FIG. 8D), and 42C3 (FIG. 8F) epitope residues from a 2mFo-DFc composite omit map (pink mesh) contoured at 1.0 σ level (1.43 rmsd). Epitope residues are depicted as a stick model.

[0039] FIGS. 9A-9C show a detailed view of the p19 epitopes for 42D6 (FIG.9A), 42C3 (FIG.9B) and 42C11 (FIG. 9C) mAbs, respectively. Grey, p19; orange, p19 residues contacted by 42D6 heavy chain CDRs; blue, p19 residues contacted by 42C11 heavy and light chain CDRs; green, p19 residues contacted by 42C3 heavy and light chain CDRs.

[0040] FIG. 10 shows the structural basis for the inhibition of parasite growth by p19-specific hmAb 42D6. Superposition of p19-Fab co-complex crystal structures, p19 is represented as white surface and Fabs are shown as cartoon representation and colored. The epitope for non-neutralizing hmAb MaliM03 (PDB ID: 6XQW) and murine mAb G17.12 (PDB ID: 1OB1) is colored in magenta and Fab fragment of MaliM03 and G17. 12 are colored in light magenta and light pink, respectively. The potent neutralizing hmAb 42D6 is shown to recognize a novel epitope on p19.

[0041] FIGS. 11A & 11B show that hmAbs 42C11, 42C5, 42C3, 42D7, and 42A9 were isolated from the same individual and are likely clonally related. Amino acid sequence alignment of the variable region of heavy (FIG. 11A) and light (FIG. 11B) chains of hmAbs. CDRs are shown at the bottom of the alignment.

[0042] FIG. 12 shows p19 sequence polymorphisms. Polymorphism mapped onto the p19 surface. 42D6 epitope is colored in orange. Polymorphic residues are shown in red and the observed substitutions as well as the percent minor allele frequency (MAF) of substitutions are indicated. p19 is represented as white surface and 42D6 Fab fragment as bright orange cartoon.

[0043] FIGS. 13A-13C show the binding affinity of 42D6 Fab to p19 constructs with point mutations representative of sequence polymorphisms in the 42D6 epitope as measured by bio-layer interferometry (BLI).

[0044] FIG. 14 shows a sequence alignment of p19 from various strains of P. falciparum. Polymorphic residues between strains indicated by black arrows. Interface residues of p19 with 42D6 Fab are indicated by black ovals.

[0045] FIG. 15 shows cross-neutralization potential of 42D6. In vitro GIA dilution series against the Pf3D7 reference strain, PfFVO, and PfDd2 strains. IC ₅₀ values were determined by interpolation after fitting data to a four-parameter dose-response curve.

[0046] FIGS. 16A & 16B show GIA of neutralizing hmAb 42D6 (1.0 mg/ml) in the presence of an increasing concentration of non-neutralizing hmAbs 42C3 and 42C11 that block its binding to p19.

[0047] FIG. 17 shows superposition of p19-Fab co-complex crystal structures, p19 is represented as white surface and Fabs are shown as cartoon representation and colored as in FIG. 7.

[0048] FIG. 18 illustrates high affinity interfering/blocking hmAb block the binding of potent neutralizing hmAb proving the concept of antigenic diversion. [0049] FIGS.19A-D show that native MSP1-19 displayed on self- assemblgin nanoparticle platforms improve the antibody response. FIG. 19A. shows the immunization and blood draw schedule for rats. FIGS. 20B-D show serum IgG titers against MSP1-19 on days 21 (FIG.20B), day 35 (FIG.20C), and day 56 (FIG. 20D). Dashed line indicates detection limit of assay and bars represent GMT. MSP1-19 WT =MSP 1 monomer; MSP1-19 WT LuS = Fusion of MSP1-19 to the C-terminus of A. aeolicus lumazine synthase (LS) nanoparticle; MSP1-19 WT E2p = Fusion of MSP1-19 to the N-terminus of G. stearothermophilus dihydrolipoyl acetyltransferase; ApoF = Fusion of MSP1-19 to the N- terminus of H. pylori ferritin nanoparticle; MSP1-19 WT Encapsuli = Fusion of MSP1-19 to the C-terminus of Myxococcus xanthus encapsulin protein; Adjuvant only- Adjuvant only immunized rats. (The study was consisted of six groups and six rats per each group were immunized with 20 μg antigen each. Antigen was formulated as a 1 :1 ratio in AddaS03™ Adjuvant (InvivoGen) on day 0 (Vacl), day 21 (Vac2) and day 42 (Vac3), and 100 μl formulated antigen was delivered by subcutaneous injection.)

[0050] FIGS.20A-C show that MSP1-19 display on lumazine synthase nanoparticle improves the neutralizing antibody response. FIGS. 20A-C shows the results from in vitro growth inhibition assays (GIA) using 10.0 mg/ml (FIG. 20A), 5.0 mg/ml (FIG.20B), and 2.5 mg/ml (FIG. 20C) total IgG purified from pooled serum against the Plasmodium falciparum 3D7 strain. Significance determined using a Kruskal- Wallis analysis followed by Dunn’s test for multiple comparisons to the MSP1-19 monomer. MSP1-19 ApoF = Fusion of MSP1-19 to the N-terminus of H. pylori ferritin nanoparticle. MSP1-19 LuS = Fusion of MSP1-19 to the C-terminus of A. aeolicus lumazine synthase nanoparticle. MSP1- 19 E2P = Fusion of MSP1-19 to the N-terminus of G. stearothermophilus dihydrolipoyl acetyltransferase. 75B10 Ctrl = control monoclonal antibody with high GIA activity. PBS - PBS immunized rats. The study was consisted of six groups and six rats per each group were immunized with 10 μg antigen each. Antigen was formulated as a 1 :1 ratio in AddaS03™ Adjuvant (InvivoGen) on day 0 (Vacl, 04/27/22) and day 21 (Vac2, 05/18/22), and 100 μl formulated antigen was delivered by subcutaneous injection.

[0051] FIGS.21A-D show an overview of the SPEEDesign pipeline used to create MSP-1-19 immunogens. FIG. 21A illustrates full-length MSP-1 structure highlighting the carboxy-terminal fragment MSP1-19. FIG. 21B indicates the design process for MSP 1- 19 preserved neutralizing epitopes while focusing on extensively designing residues at non- neutralizing epitopes. All other residues were designed conservatively. FIG.21C illustrates that two computational design strategies were employed to generate 80,000 decoys, each with specific amino acid changes (indicated in red) from the native MSP1-19 sequence. FIG.21D illustrates that in vitro screening of forty-eight sequences selected from the top scoring decoys led to the identification of eight promising lead candidates (highlighted in boxes).

[0052] FIG. 22 shows an alignment of the sequence of wild-type (WT) MSP1-19 protein with the sequence of several modified MSP1-19 proteins. The black bar to the left indicates modified proteins in which the 42D6 epitope residues were fixed. The grey bar indicates modified proteins in which the MaliM03 epitope residues were fixed. Positions that were mutated are underlined. Grey boxed residues in the WT sequence indicate the MaliMO epitope. Clear boxed residues in the WT sequence indicate the 42D6 epitope.

[0053] FIG.23 demonstrates that modified MSP1-19 immunogens retain neutralizing epitopes. Binding of neutralizing and non-neutralizing antibodies was assessed using an enzyme-linked immunosorbent assay (ELISA). The broadly neutralizing monoclonal antibody 42D6 demonstrated similar binding affinity to the native MSP1-19 antigen across all eight immunogens tested. Conversely, the non-neutralizing monoclonal antibodies 42C3 and 42C11 exhibited minimal binding to the immunogens. For each immunogen, the bars, left to right, correspond to 42D6, 42C11, and 42C3.

[0054] FIGS.24A-D show antibody levels in rats immunized with self- assembling nanoparticle platform lumazine synthase displaying immunogens are similar to those of lumazine synthase displaying Native MSP1-19. FIG.24A shows the immunization and blood draw schedule for rats. FIGS.24B-D show serum IgG titers against MSP1-19. Dashed line indicates detection limit of assay and bars represent GMT. (The study was consisted of six groups and six rats per each group were immunized with 20 μg antigen each. Antigen was formulated as a 1 : 1 ratio in AddaS03™ Adjuvant (InvivoGen) on day 0 (Vacl), day 21 (Vac2) and day 42 (Vac3), and 100 μl formulated antigen was delivered by subcutaneous injection.)

[0055] FIGS.25A-F show that structure-based design MSPl-19-19-LuS elicits significantly potent strain-transcending antibodies relative to native MSP1-19 displayed on lumazine synthase. FIG.25A shows the results of in vitro GIA of pooled purified IgG from each group at day 56 tested at 5.0 mg/ml against the Plasmodium falciparum 3D7 blood stage. In vitro GIA dilution series of pooled purified IgG from each group at day 56 against Plasmodium falciparum 3D7 (FIG.25B), Plasmodium falciparum FVO (FIG.25C), Plasmodium falciparum Dd2 (FIG.25D). The data are plotted as median with 95 % CI and arise from three independent biological replicates. FIGS. 25E-G show the concentration (mg/ml) of pooled purified IgG required to demonstrate 50% inhibition (IC50) against Plasmodium falciparum E 3D7 (FIG.25E), Plasmodium falciparum FVO (FIG.25F), and Plasmodium falciparum Dd2 (FIG.25G) were determined by interpolation after fitting data globally to a four-parameter dose-response curve. The global fit for IC50 with 95 % CI is represented as black bars and individual IC50 values from independent assays presented as points. Statistical comparisons were made using an extra sum-of-squares F-test with Bonferroni correction.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present disclosure relates to vaccines, therapeutic antibodies and methods of making such vaccines and antibodies. More specifically, the disclosure relates to methods of improving the immunogenicity of a microbial protein having both neutralizing epitopes and non-neutralizing epitopes. It is understood in the relevant art that an antigenic protein comprising both neutralizing and non-neutralizing epitopes may elicit both neutralizing antibodies and non-neutralizing antibodies, and that the affinities of these antibodies for their respective epitopes may differ. In certain instances, the affinity of the non-neutralizing antibody may be greater than the affinity of the neutralizing antibody for the neutralizing epitope. In such instances, if the non-neutralizing epitope is near, or overlapping with, the neutralizing epitope in the folded protein, binding of the non- neutralizing antibody to the non-neutralizing epitope can interfere with binding to the neutralizing antibody to the neutralizing epitope. Moreover, because the affinity of the non- neutralizing antibody is greater than the affinity of the neutralizing antibody for the neutralizing epitope, the non-neutralizing antibody will be bound to the protein more often, effectively preventing the neutralizing antibody from binding the neutralizing epitope. The result of such an arrangement is that the neutralizing epitope is not seen by the immune system, and an individual infected by a microorganism expressing such a protein would fail to mount an effective neutralizing immune response against the microorganism. Similarly, such a protein would not be useful as a vaccine against the originating microorganism since it would not elicit a neutralizing immune response.

[0057] One way to address the aforementioned situation is to modify the antigenic protein so that the immune response elicited by the modified protein in an individual preferentially, or entirely, recognizes a neutralizing epitope in the antigenic protein. Thus, a method of the disclosure may generally be practiced by modifying the amino acid sequence of an antigenic protein having a neutralizing epitope and an interfering, non- neutralizing epitope, so that the immune response elicited by a protein comprising the modified amino acid sequence preferentially, or entirely, recognizes of the neutralizing epitope. Such modifications may reduce the immunogenicity of the interfering, non- neutralizing epitope in the protein, eliminate the interfering, non-neutralizing, epitopes in the protein, or significantly increase the affinity of the neutralizing antibody for the neutralizing epitope. Methods of the disclosure may also be practiced by modifying a neutralizing antibody that binds a neutralizing epitope in a microbial protein, such that the affinity of the neutralizing antibody for a neutralizing epitope is significantly increased. Also disclosed herein are therapeutic compositions and vaccines produced using the disclosed methods.

[0058] Before the present disclosure is further described, it is to be understood that the disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the claims.

[0059] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, a compound refers to one or more compound molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. [0060] Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. Terms and phrases, which are common to the various aspects disclosed herein, are defined below.

[0062] One aspect of the disclosure is a method of immunofocusing an immune response to a neutralizing epitope present in an initial protein, the initial protein having an initial amino acid sequence and comprising one or more interfering, non- neutralizing epitopes, the method comprising modifying the amino acid sequence of the initial protein to produce a modified protein comprising the neutralizing epitope, such that the immune response is preferentially, or entirely, directed towards the neutralizing epitope in the modified protein.

[0063] In certain aspects, the immunogenicity of the neutralizing epitope in the modified protein is not reduced relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0064] In certain aspects, the immunogenicity of the neutralizing epitope in the initial protein is not significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein, whereas the immunogenicity of the neutralizing epitope in the modified protein is significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the modified protein. [0065] In certain aspects, modifying the initial amino acid sequence reduces the immunogenicity of the one or more interfering, non-neutralizing epitopes in the modified protein relative to the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0066] In certain aspects, modifying the initial amino acid sequence increases the immunogenicity of the neutralizing epitope in the modified protein relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0067] In certain aspects, the affinity of a neutralizing antibody for the neutralizing epitope in the initial protein is not significantly greater than the affinity of a non- neutralizing antibody for the one or more interfering non-neutralizing epitopes in the initial protein, whereas the affinity of the neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of the non-neutralizing antibody for the one or more interfering non-neutralizing epitopes in the modified protein.

[0068] In certain aspects, modifying the initial amino acid sequence reduces the affinity of a non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the modified protein relative to the affinity of the non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the initial protein.

[0069] In certain aspects, modifying the initial amino acid sequence increases the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein.

[0070] In certain aspects, modifying the initial amino acid sequence comprises inserting one or more glycosylation sites into the initial protein such that a glycan added at the glycosylation site shields the one or more interfering, non-neutralizing epitopes from the immune system. [0071] In certain aspects, modifying the initial amino acid sequence comprises inserting a peptide sequence into the initial amino acid sequence, wherein in the finally folded protein, the inserted peptide sequence masks at least one of the one or more interfering, non-neutralizing epitopes. In certain aspects, the method comprises producing a modified protein comprising the modified amino acid sequence.

[0072] In certain aspects, modifying the amino acid sequence of the initial protein comprises introducing a mutation at one or more amino acid positions in the initial amino acid sequence. In certain aspects, the one or more amino acid positions may not comprise amino acid positions in the neutralizing epitope. In aspects in which the one or more positions may comprise an amino acid position in the neutralizing epitope, mutations introduced at such amino acid position should increase the immunogenicity and/or affinity of the neutralizing epitope. In certain aspects, the one or more amino acid positions may comprise at least one amino acid position in the one or more interfering non-neutralizing epitope, such that the modified protein comprises at least one modified interfering non- neutralizing epitope. Preferably, mutations introduced at amino acid positions in the one or more interfering non-neutralizing epitope reduce the immunogenicity of the interfering non- neutralizing epitope, or the affinity of a non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes. In certain aspects, introduced mutations may comprise deletion mutations, insertion mutations, and/or substitution mutation. It should be understood that in all methods of the disclosure, mutations made at one amino acid position need not be the same type of mutation made at a different amino acid position. For example, a modified protein may comprise a deletion mutation at one amino acid position and an amino acid substitution at another amino acid position. Moreover, in modified proteins comprising more than one amino acid substitution, the amino acid residue substituted in at one amino acid positions may differ from the amino acid residue substituted in at any other amino acid position. In certain aspects, the step of modifying may comprise modifying the initial amino acid sequence so that the affinity of a neutralizing antibody for the modified protein relative to the affinity of the neutralizing antibody for the initial protein is not reduced, wherein the affinity of a non-neutralizing antibody for the modified protein relative to the affinity of the non-neutralizing antibody for the initial protein is reduced. In certain aspects, the step of modifying may comprise modifying the initial amino acid sequence to produce a modified protein comprising one or more modified interfering non-neutralizing epitopes, wherein the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein is not reduced, and wherein the affinity of a non-neutralizing antibody for the one or more modified interfering non-neutralizing epitopes in the modified protein is reduced. In certain aspects, the step of modifying may comprise modifying the initial amino acid sequence to produce a modified protein comprising one or more modified interfering non-neutralizing epitopes, wherein the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of a non-neutralizing antibody for the one or more modified interfering non-neutralizing epitopes in the modified protein. In certain aspects, the method may comprise producing a modified protein comprising the modified amino acid sequence.

[0073] As used herein, “immunofocusing”, (a.k.a. “antigen engineering”), refers to a process in which the three-dimensional (3D) structure of an antigen (e.g., protein) is altered in order to influence an immune response to the antigen, such that the immune response is preferentially, or completely, directed towards, or away from, a particular portion of the antigen. For instance, if the antigen is a protein, the 3D-structure of the protein may be altered in such a way that when the altered protein is administered to an individual, it preferentially elicits antibodies to a particular epitope, such as a neutralizing epitope. In certain aspects, preferentially directing the immune response to the neutralizing epitope may comprise enhancing the immunogenicity of the neutralizing epitope. In certain aspects, preferentially directing the immune response to the neutralizing epitope may comprise reducing the immunogenicity of, or completely removing, non-neutralizing epitopes present in the protein. In certain aspects, preferentially directing the immune response to the neutralizing epitope may comprise shielding the interfering, non-neutralizing epitopes, and optionally other regions of the initial protein, by introducing one or more glycosylation sites into the initial protein such that a glycan added at the glycosylation site shields the one or more non-neutralizing epitopes from the immune system. Such non-neutralizing epitopes may be interfering non-neutralizing epitopes that interfere with (e.g., masks) the ability of the immune system to “see” the neutralizing epitope or interfere with the ability of a neutralizing antibody to bind the neutralizing epitope. Thus, in certain aspects, the protein may be altered in such a way that when the altered protein is administered to an individual, it does not elicit an immune response to a particular epitope, such as an interfering, non- neutralizing epitope. In such instances, the 3D-shape of the protein may be altered by modifying the amino acid sequence of the protein. Using such a process, the immune response to an antigen may be “tuned” so that the antigen elicits a desired immune response (e.g., a neutralizing immune response), which may be a humoral immune response (e.g., an antibody response), or a cellular immune response (e.g., a T cell response).

[0074] It should be understood that, in the present disclosure, influencing an immune response does not necessarily mean altering an antigen so that no non- neutralizing antibodies are produced. In certain aspects, influencing an immune response may refer to altering an antigen such that it elicits an immune response comprising a desired population of antibodies. For example, an immune response elicited to a modified protein of the disclosure may comprise antibodies that recognize neutralizing epitopes (i.e., neutralizing antibodies) and antibodies that recognize non-neutralizing (including interfering, non-neutralizing) epitopes (i.e., non-neutralizing antibodies). However, in such an immune response, preferably the affinity of the elicited neutralizing antibodies for neutralizing epitopes is significantly greater than the affinity of the elicited non-neutralizing antibodies for interfering, non-neutralizing epitopes, such that a non-neutralizing antibody is not able to block a neutralizing antibody from binding to the neutralizing epitope.

[0075] The term “epitope” refers to the portion of a peptide or protein which is specifically bound by an antigen-binding region of an antibody. Epitopes comprise contact residues. A contact residue is an amino acid residue that forms a bond (e.g., ionic bond, hydrophobic bond, etc.) with an amino acid residue in another protein. For example, a contact residue in an epitope forms a bond with a contact residue in the antigen binding region of an antibody. Contact residues in an epitope are not necessarily close to other contact residues in the epitope but are usually brought into spatial proximity in the finally folded protein.

[0076] As used herein, the phrase “neutralizing immune response” refers to an immune response that renders a microorganism against which the immune response is elicited, non- pathogenic (i.e., no longer able to cause disease). One example of a neutralizing immune response is a neutralizing antibody response in which an individual infected by a microorganism produces antibodies that bind one or more antigens (e.g., proteins) in the microorganism, thereby preventing the microorganism from infecting cells, producing or releasing progeny microorganism, or conducting functions necessary for the survival and/or replication of the microorganism. Such antibodies are referred to as “neutralizing antibodies”. A “neutralizing epitope” is an epitope that elicits, and/or is recognized by, a neutralizing antibody.

[0077] As used herein, “non-neutralizing antibodies” are antibodies that bind to epitopes on an antigen in a microorganism, but which do not render the microorganism non-pathogenic. Thus, non-neutralizing antibodies do not prevent the microorganism from infecting cells, producing or releasing progeny microorganism, or conducting any functions necessary for the survival and/or replication of the microorganism. A “non-neutralizing epitope” is an epitope that elicits, and is recognized by, a non- neutralizing antibody. An “interfering non-neutralizing, epitope” refers to a non-neutralizing epitope in an antigen that, when bound by a non-neutralizing antibody, prevents a neutralizing antibody from binding to a neutralizing epitope in the antigen. Without being bound by theory, binding of the non-neutralizing, interfering epitope by the non-neutralizing antibody may interfere with, or prevent, binding of the neutralizing antibody to a neutralizing epitope by physically blocking the neutralizing antibody from accessing the neutralizing epitope, or by altering the structure of an antigen (e.g., the conformation of the antigen) such that the neutralizing antibody no longer recognizes the neutralizing epitope in the antigen. It should be understood that, unless specified otherwise, the phrase “non-neutralizing epitopes” includes “interfering non-neutralizing epitopes”. A “modified interfering non-neutralizing epitope” is an epitope derived from an “interfering non-neutralizing epitope”, wherein the sequence of the “modified interfering non-neutralizing epitope” is derived by introducing at least one mutation into the sequence of the “interfering non-neutralizing epitope”. Because the sequence of the “modified interfering non-neutralizing epitope” has been modified, its immunogenicity and/or affinity may be, and preferably is, reduced and thus the “modified interfering non-neutralizing epitope” may no longer interfere with binding of a neutralizing antibody to a neutralizing epitope. However, for clarity and consistency, term “interfering” is still used with regard to such a non-neutralizing epitope to connect the epitope to its origin.

[0078] The phrase “recognize an epitope”, and the like, with regard to an antibody means the antibody specifically binds an epitope. “Specifically binds” means that an antibody binds an epitope (e.g., a neutralizing epitope) in a target protein with an affinity significantly greater than its affinity for an epitope in an unrelated epitope in an unrelated protein. “Affinity” is a measure of the strength of the binding interaction between an antibody and the epitope. Affinity is determined by the sum of the closeness of the stereochemical fit between the antibody and epitope, the size of the area of contact between them (i.e., the interface), and the number and distribution of charged and hydrophobic groups in the interface. Without being bound by theory, it is understood that binding of an antibody to an epitope is reversible, meaning that antibody binding consists of binding to the epitope (“on”) and release from the epitope (“off’). Each of these events comprises a certain association constant, which represent how fast an antibody binds the epitope and how fast the antibody dissociates from the epitope, respectively. The ratio of the disassociation rate constant (k _dis ) to the association rate constant (k _a ) (k _dis /k _a ) is referred to as the equilibrium dissociation constant (K _D ), which is inversely proportional to the affinity of the antibody. Thus, the smaller the K _D , the greater the affinity of the antibody for an epitope. According to the present disclosure, an antibody that specifically binds a epitope has a K _D of at least 10 ^-6 M. An antibody that has high affinity for an epitope has a K _D of at least 10 ^-9 M. An antibody that has high affinity for an epitope has a K _D of at least 10 ^-12 M.

[0079] As used herein, “immunogenicity” means the ability of a protein, or an epitope within the protein, to elicit a specific immune response. One method of measuring immunogenicity is by administering a protein comprising an epitope to a subject and measuring the type and/or strength of the immune response generated against the protein and/or epitope. The immune response that is measured may be a T-cell immune response or it may be a B-cell immune response. In one aspect, measuring immunogenicity may comprise determining the level and/or affinity of antibodies elicited against a protein and/or an epitope.

[0080] As used herein, the term immunogenic refers to the ability of a specific protein, or a specific region thereof, to elicit an immune response to the specific protein, or to proteins comprising an amino acid sequence having a high degree of identity with the specific protein. According to the present invention, two proteins having a high degree of identity have amino acid sequences at least 80% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical. Methods of determining the percent identity between two amino acid or nucleic acid sequence are known in the art.

[0081] As used herein, “significant”, when used with “greater” or “less”, and the like, indicates a change in value of at least 25%, at least 50%, at least 0.5 logs, or at least 1 log.

[0082] One aspect of the disclosure is a method of improving the ability of an initial protein to elicit a neutralizing immune response, the initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more interfering, non- neutralizing epitopes, the method comprising modifying the initial amino acid sequence to produce a modified amino acid sequence such that the immunogenicity of the neutralizing epitope in a modified protein comprising the modified amino acid sequence is significantly greater than the immunogenicity of the one or more interfering, interfering, non-neutralizing epitopes in the modified protein.

[0083] In certain aspects, the immunogenicity of the neutralizing epitope in the modified protein is not reduced relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0084] In certain aspects, the immunogenicity of the neutralizing epitope in the initial protein is not significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0085] In certain aspects, modifying the initial amino acid sequence reduces the immunogenicity of the one or more interfering, non-neutralizing epitopes in a modified protein relative to the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0086] In certain aspects, modifying the initial amino acid sequence increases the immunogenicity of the neutralizing epitope in the modified protein relative to the immunogenicity of the neutralizing epitope in the initial protein. [0087] In certain aspects, the affinity of a neutralizing antibody for the neutralizing epitope in the initial protein is not significantly greater than the affinity of a non- neutralizing antibody for the one or more interfering non-neutralizing epitopes in the initial protein, whereas the affinity of the neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of the non-neutralizing antibody for the one or more interfering non-neutralizing epitopes in the modified protein.

[0088] In certain aspects, modifying the initial amino acid sequence reduces the affinity of a non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the modified protein relative to the affinity of the non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the initial protein.

[0089] In certain aspects, modifying the initial amino acid sequence increases the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein.

[0090] In certain aspects, modifying the initial amino acid sequence comprises inserting one or more glycosylation sites into the initial protein such that a glycan added at the glycosylation site shields the one or more non-neutralizing epitopes from the immune system.

[0091] In certain aspects, modifying the initial amino acid sequence comprises inserting a peptide sequence into the initial amino acid sequence, wherein in the finally folded protein, the inserted peptide sequence masks at least one of the one or more non-neutralizing epitopes. In certain aspects, the method comprises producing a modified protein comprising the modified amino acid sequence.

[0092] One aspect of the disclosure is a method of producing a vaccine candidate using an initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes, the method comprising: modifying the initial amino acid sequence to produce a modified amino acid sequence such that the immunogenicity of the neutralizing epitope in a modified protein comprising the modified amino acid sequence is significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the modified protein, and producing a modified protein that comprises the modified amino acid sequence, thereby producing a vaccine candidate. In certain aspects, the immunogenicity of the neutralizing epitope in the modified protein is not reduced relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0093] In certain aspects, the immunogenicity of the neutralizing epitope in the initial protein is not significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0094] In certain aspects, modifying the initial amino acid sequence reduces the immunogenicity of the one or more interfering, non-neutralizing epitopes in a modified protein relative to the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0095] In certain aspects, modifying the initial amino acid sequence increases the immunogenicity of the neutralizing epitope in the modified protein relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0096] In certain aspects, the affinity of a neutralizing antibody for the neutralizing epitope in the initial protein is not significantly greater than the affinity of a non- neutralizing antibody for the one or more interfering non-neutralizing epitopes in the initial protein, whereas the affinity of the neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of the non-neutralizing antibody for the one or more interfering non-neutralizing epitopes in the modified protein.

[0097] In certain aspects, modifying the initial amino acid sequence reduces the affinity of a non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the modified protein relative to the affinity of the non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the initial protein.

[0098] In certain aspects, modifying the initial amino acid sequence increases the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein.

[0099] In certain aspects, modifying the initial amino acid sequence comprises inserting one or more glycosylation sites into the initial protein such that a glycan added at the glycosylation site shields the one or more interfering, non-neutralizing epitopes from the immune system.

[0100] In certain aspects, modifying the initial amino acid sequence comprises inserting a peptide sequence into the initial amino acid sequence, wherein in the finally folded protein, the inserted peptide sequence masks at least one of the one or more interfering, non-neutralizing epitopes. In certain aspects, the method comprises producing a modified protein comprising the modified amino acid sequence.

[0101] In certain aspects, the method may comprise assaying a neutralizing antibody and a non-neutralizing antibody for their abilities to bind the modified protein. In certain aspects, if the ability of the neutralizing antibody to bind the modified protein is not significantly less than the ability of the neutralizing antibody to bind the initial protein, and if the ability of the non-neutralizing antibody to bind the modified protein is significantly less than the ability of the non-neutralizing antibody to bind the initial protein, the modified protein may be identified as a vaccine candidate. In certain aspects, if the affinity of the neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of a non-neutralizing antibody for the one or more interfering, non- neutralizing epitopes in the modified protein, the modified protein may be identified as a vaccine candidate.

[0102] One aspect of the disclosure is a method of identifying a vaccine candidate using an initial protein having an initial amino acid sequence and comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes, the method comprising: modifying the initial amino acid sequence to produce a modified amino acid sequence such that the immunogenicity of the neutralizing epitope in a modified protein comprising the modified amino acid sequence is significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the modified protein; producing the modified protein, and, assaying a neutralizing antibody and a non- neutralizing antibody for their abilities to bind the modified protein. In certain aspects, the immunogenicity of the neutralizing epitope in the modified protein is not reduced relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0103] In certain aspects, the immunogenicity of the neutralizing epitope in the initial protein is not significantly greater than the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0104] In certain aspects, modifying the initial amino acid sequence reduces the immunogenicity of the one or more interfering, non-neutralizing epitopes in a modified protein relative to the immunogenicity of the one or more interfering, non-neutralizing epitopes in the initial protein.

[0105] In certain aspects, modifying the initial amino acid sequence increases the immunogenicity of the neutralizing epitope in the modified protein relative to the immunogenicity of the neutralizing epitope in the initial protein.

[0106] In certain aspects, the affinity of a neutralizing antibody for the neutralizing epitope in the initial protein is not significantly greater than the affinity of a non- neutralizing antibody for the one or more interfering non-neutralizing epitopes in the initial protein, whereas the affinity of the neutralizing antibody for the neutralizing epitope in the modified protein is significantly greater than the affinity of the non-neutralizing antibody for the one or more interfering non-neutralizing epitopes in the modified protein.

[0107] In certain aspects, modifying the initial amino acid sequence reduces the affinity of a non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the modified protein relative to the affinity of the non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the initial protein.

[0108] In certain aspects, modifying the initial amino acid sequence increases the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein. [0109] In certain aspects, modifying the initial amino acid sequence comprises inserting one or more glycosylation sites into the initial protein such that a glycan added at the glycosylation site shields the one or more interfering, non-neutralizing epitopes from the immune system.

[0110] In certain aspects, modifying the initial amino acid sequence comprises inserting a peptide sequence into the initial amino acid sequence, wherein in the finally folded protein, the inserted peptide sequence masks at least one of the one or more interfering, non-neutralizing epitopes. In certain aspects, the method comprises producing a modified protein comprising the modified amino acid sequence.

[0111] In certain aspects, if the ability of the neutralizing antibody to bind the modified protein is not significantly less than the ability of the neutralizing antibody to bind the initial protein, and if the ability of the non-neutralizing antibody to bind the modified protein is significantly less than the ability of the non-neutralizing antibody to bind the initial protein, the modified protein may be identified as a vaccine candidate. In certain aspects, if the affinity of the neutralizing antibody for the neutralizing epitope in the modified is significantly greater than the affinity of a non-neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the modified protein, the modified protein may be identified as a vaccine candidate.

[0112] The modified protein may be produced using any known method to produce a specific protein. In certain aspects, the modified protein is produced synthetically (e.g., comprising chemical synthesis). In certain aspects, a nucleic acid molecule comprising a nucleic acid sequence encoding the modified amino acid sequence is produced and the nucleic acid expressed recombinantly to produce the modified protein.

[0113] In certain aspects of the disclosure, modifying the amino acid sequence of the initial protein so that the immune response is preferentially, or entirely, directed towards the neutralizing epitope in the modified protein may comprise identifying one or more amino acid positions in the amino acid sequence at which introducing a specific mutation would result in a modified protein that elicits an immune response preferentially, or entirely, directed towards the neutralizing epitope in the modified protein. In certain aspects, the specific mutation may comprise deleting an amino acid residue. In certain aspects, the specific mutation may comprise inserting an amino acid residue or substituting an amino acid residue. In these latter aspects (inserting or substituting), the method may also comprise determining which of the known 22 amino acid residues should be inserted at the one or more amino acid positions. In certain aspects, the one or more amino acid positions comprise all the amino acid positions in the amino acid sequence. In certain aspects, the one or more positions are from a portion of the amino acid sequence. In certain aspects, the portion consists of an epitope which maybe the neutralizing epitope or the one or more non- neutralizing epitope (including an interfering non-neutralizing epitope). In certain aspects, modifying the amino acid sequence may comprise deleting or substituting contact residues in an epitope. In certain aspects, modifying the amino acid sequence does not alter contact residues in the neutralizing epitope. In certain aspects, introducing a specific mutation at the one or more amino acid positions may significantly increase the immunogenicity of the neutralizing epitope. In certain aspects, introducing a specific mutation at the one or more amino acid positions may significantly decrease the immunogenicity of at least one of the one or more interfering, non-neutralizing epitopes. In certain aspects, introducing a specific mutation at the one or more amino acid positions, may significantly increase the affinity of a neutralizing antibody for the neutralizing epitope. In certain aspects, introducing a specific mutation at the one or more amino acid positions, may significantly decrease the affinity of a non-neutralizing antibody for the at least one of the one or more interfering non- neutralizing epitope.

[0114] In these methods, identifying one or more amino acid positions in the amino acid sequence at which a specific mutation would yield a modified protein of the disclosure may comprise using any method suitable for identifying the relative positions of amino acid residues in a protein. For example, protein crystallography is a well-established method for determining the 3D-structure of a protein, or protein complex, and thus, the relative positions of amino acid residues within a protein. A more recently developed technique comprises using computational design. “Computational design” comprises a method of performing structure guided design using protein modeling computer programs (e.g., ROSETTA) to predict the structure of proteins and/or to identify amino acid sequence variants that fold into a particular shape. Such modeling uses the thermodynamic potential of an amino acid sequence to identify folded conformations having the greatest stability. As applied to the present disclosure, computational design may be used to determine which modified amino acid sequences would produce a modified protein in which the affinity of a neutralizing antibody for the neutralizing epitope is significantly greater than the affinity of a non-neutralizing antibody for a non-neutralizing epitope (including an interfering, non- neutralizing antibody). The identified modified amino acid sequences, each having a mutation at the one or more amino acid positions, may then be used to produce proteins having such sequences, and/or combinations thereof, which may then be tested using in vitro assays and/or tested in vivo for their ability to elicit an immune response. One example of a computational design process useful for practicing methods of the present disclosure is SPEEDesign, the details of which are disclosed in International Patent Application No: PCT/US2022/070744, which is incorporated herein in its entirety by reference.

[0115] In these methods, identifying the one or more amino acid positions in the amino acid sequence at which a specific mutation would result in a modified protein of the disclosure may comprise using computational design. In certain aspects, the computational design may comprise substituting amino acid residues at the one or more amino acid position in the initial amino acid sequence to produce a heterogenous pool of modified amino acid sequences, each of which comprises an amino acid substitution at one or more amino acid positions, and each of which has an associated stability score. In certain aspects, at least one high scoring, modified amino acid sequence, may be selected from the heterogenous pool of modified amino acid sequences and the selected modified sequences used to identify the one or more amino acid position and which amino acid residues are substituted at the one or more amino acid positions. In certain aspects, high scoring modified amino acid sequence from the heterogenous pool of modified amino acid sequences may be clustered based on sequence similarity, and at least one high scoring, modified amino acid sequence selected from each cluster. In such aspects, at least one of the selected sequences may be used to identify the one or more amino acid position and which amino acid residues are substituted at the one or more amino acid positions.

[0116] In these methods, the step of substituting amino acid residues in the initial amino acid sequence may be performed computationally and may comprise keeping amino acid residues at certain amino acid positions fixed, while substituting amino acid residues at other locations to produce the modified amino acid sequence, and calculating the effect of the substitution on the stability of the amino acid sequence in a folded state. At amino acid positions at which substitutions are made, the range of amino acids that may be substituted in at each position may differ at each position. For example, at some positions, any known amino acid residues may be substituted in, while at other positions, only amino acid residues from a constrained pool of amino acid residues may be substituted in. In certain aspects, the range of amino acid residues in the constrained pool may be evolutionarily defined. A pool of amino acid residues that is evolutionarily defined means that, for a given position in the amino acid sequence of the initial protein, the pool contains only those amino acid residues present at the corresponding position of a protein identified as related to the initial protein. “Corresponding position” means the amino acid position in a related protein that aligns with a specific amino acid residue in the initial amino acid sequence, when the initial amino acid sequence is aligned with the related protein. In certain aspects, proteins related to the initial protein are defined as those proteins deemed related using a position specific iterative local alignment process. In one example of such process, a database containing a large number of amino acid sequences (e.g., the BLAST non-redundant protein sequences database at the National Center for Biotechnology Information, the RefSeq select protein database, the UniProtSwissKB/SwissProt protein database, etc.), preferably all known protein sequence, is searched using the initial amino acid sequence to identify proteins having a high degree of local alignment, and a profile or a position-specific score matrix (PSSM) is calculated from the multiple alignment. The PSSM captures the conservation pattern in alignment and stores it as a matrix of scores for each position in the alignment-highly conserved positions receive high scores and weakly conserved positions receive scores near zero. This profile is used in place of the original substitution matrix for a further search of the database to detect sequences that match the conservation pattern specified by the PSSM. The newly detected sequences from this second round of the search, which are above the specified score (e- value) threshold are again added to alignment and the profile is refined for another round of searching. This process is iteratively continued until desired or until convergence, i.e., the state where no new sequences are detected above the defined threshold. One example of such an iterative local alignment process is the psiBLAST program (described in Nucleic Acids Research 1996, 25:3389-3402, and available at the National Center for Biotechnology Information, Bethesda, MD). [0117] Thus, in these methods, computational design may comprise assigning a computational search depth to each amino acid position in the initial amino acid sequence, the search depth being one of fixed, intermediate, or deep, wherein a fixed search depth is defined as leaving a residue at the assigned position unchanged, an intermediate search depth is defined as allowing sampling from an evolutionarily constrained pool of amino acids, and a deep search depth is defined as allowing sampling from all amino acids, and at each position assigned an intermediate or deep search depth, substituting the amino acid residue at each position assigned an intermediate or deep search depth with an amino acid sampled from a pool of amino acids, the breadth of the pool at each position being defined by the assigned search depth at that position, to produce modified amino acid sequences. The result of this process is a heterogenous population of modified amino acid sequences. The thermodynamic potential of each modified sequence in a folded state may then be calculated, and each modified sequence assigned a stability score based on the calculated thermodynamic potential. The result of this process is the heterogenous pool of modified amino acid sequences, each of which comprises an amino acid substitution at one or more amino acid positions, and each of which has an associated stability score.

[0118] In these methods, the initial protein may be from an infectious microorganism. In these methods, the infectious microorganism may be of the genus Plasmodium. In these methods, the infectious microorganism may be selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In these methods, the initial protein may be selected from the group consisting of merozoite surface proteins (MSPs) MSP1 , MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10, MSP12, P19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PfRH2a, PfRH2b, PfRH4, PfRH5, RAMA, and RALP 1. In these methods, the initial protein is Plasmodium falciparum surface protein MSP1-19). In these methods, the initial protein may comprise an amino acid sequence at least 90% identical, at least 95%identical, at least 97%identical, or 100% identical to SEQ ID NO:2. In these methods, the initial protein may comprise an amino acid sequence at least 90% identical, at least 95%identical, at least 97%identical, or 100% identical to SEQ ID NO:2, wherein the neutralizing epitope comprises Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2. In these methods, the initial protein may comprise an amino acid sequence at least 90% identical, at least 95%identical, at least 97%identical, or 100% identical to SEQ ID NO:2, wherein the one or more non- neutralizing, interfering epitopes are selected from the group consisting of: a) an epitope comprising Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and b) an epitope comprising Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Ser69, Arg71, Phe87 and Asp88 of SEQ ID NO:2.

[0119] One aspect of the disclosure is a modified protein produced using a method of the disclosure. In one aspect, the protein is produced using a method comprising modifying the initial amino acid sequence of an initial protein comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes, to produce a modified amino acid sequence, such that the immunogenicity of the one or more non-neutralizing interfering epitopes in a modified protein comprising the modified amino acid sequence is reduced, wherein the immunogenicity of the neutralizing epitope in the protein comprising the modified amino acid sequence is not reduced, and producing a modified protein that comprises the modified amino acid sequence. In certain aspects, the step of modifying comprises modifying the initial amino acid sequence to produce a modified amino acid sequence in which the affinity of a neutralizing antibody for the neutralizing epitope in a modified protein comprising the modified sequence is not reduced, wherein the affinity of a non-neutralizing antibody for the one or more interfering non-neutralizing epitopes in the modified protein is reduced. In certain aspects, the step of modifying comprises modifying the initial amino acid sequence to produce a modified amino acid sequence in which the affinity of a neutralizing antibody for the neutralizing epitope in a modified protein comprising the modified sequence is greater than the affinity of a non-neutralizing antibody for the one or more interfering non-neutralizing epitopes in the modified protein. In certain aspects, the method may comprise assaying a neutralizing antibody and a non-neutralizing antibody for their abilities to bind the modified protein. In certain aspects, if the ability of the neutralizing antibody to bind the modified protein is not significantly less than the ability of the neutralizing antibody to bind the initial protein, and if the ability of the non- neutralizing antibody to bind the modified protein is significantly less than the ability of the non-neutralizing antibody to bind the initial protein, the modified protein may be identified as a vaccine candidate. In certain aspects, if the affinity of the neutralizing antibody for the neutralizing epitope in the modified is significantly greater than the affinity of a non- neutralizing antibody for the one or more interfering, non-neutralizing epitopes in the modified protein, the modified protein may be identified as a vaccine candidate. In certain aspects, the method comprises modifying the initial amino acid sequence to include one or more glycosylation sites into the initial protein such that a glycan added at the glycosylation site shields the one or more interfering, non-neutralizing epitopes from the immune system.

[0120] One aspect of the disclosure is a modified protein comprising a modified amino acid sequence, wherein the modified sequence is derived from an initial amino acid sequence of an initial protein comprising a neutralizing epitope and one or more interfering non-neutralizing epitopes, wherein the difference between the modified amino acid sequence and the initial amino acid sequence comprises, or consists of, amino acid substitutions at one or more amino acid positions, wherein the modified protein comprises the neutralizing epitope, wherein the amino acid substitutions at the one or more amino acid position cause an immune response to the modified protein to be immunfocused to the neutralizing epitope in the modified protein. In certain aspects, the modified amino acid sequence is at least 50% identical to the initial amino acid sequence, wherein the percent identify is measured over the entire length of the initial amino acid sequence. In certain aspects, the one or more amino acid positions do not comprise amino acid position in the neutralizing epitope. In certain aspects, the one or more amino acid positions comprise amino acid position in the neutralizing epitope, wherein the immunogenicity of the neutralizing epitope is significantly increased relative to the immunogenicity of the neutralizing epitope in the initial protein. In certain aspects, the one or more amino acid positions comprise amino acid position in the neutralizing epitope, wherein the affinity of a neutralizing antibody for neutralizing epitope is significantly increased relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein. In certain aspects, the one or more amino acid positions comprise at least one amino acid position in the one or more interfering non-neutralizing epitopes such that the modified protein comprise at least one modified interfering, non-neutralizing epitope, wherein the immunogenicity of the neutralizing epitope in the modified protein is not reduced relative to the immunogenicity of the neutralizing epitope in the initial protein, and wherein the immunogenicity of the at least one modified interfering, non-neutralizing epitopes in the modified protein is reduced relative to the immunogenicity of the one or more non-neutralizing interfering epitopes in the initial protein. In certain aspects, the one or more amino acid positions comprise at least one amino acid position in the one or more interfering non-neutralizing epitopes such that the modified protein comprise at least one modified interfering non-neutralizing epitope, wherein the affinity of a neutralizing antibody for the neutralizing epitope in the modified protein is not reduced relative to the affinity of the neutralizing antibody for the neutralizing epitope in the initial protein, and wherein the affinity of a non-neutralizing antibody for the modified interfering non-neutralizing epitope in the modified protein is reduced relative to the affinity of the non-neutralizing antibody for the interfering non-neutralizing epitope in the initial protein. In certain aspects, the modified protein comprises one or more introduced glycosylation sites such that a glycan added at the glycosylation site shields the one or more interfering, non-neutralizing epitopes from the immune system. In certain aspects, the modified protein may lack glycosylation sites present in the initial protein. In certain aspects, the modified protein comprises an introduced peptide sequence, such that in the finally folded protein, the inserted peptide sequence masks at least one of the one or more interfering, non-neutralizing epitopes.

[0121] In certain aspects, the initial protein may be from an infectious microorganism, which may be from the genus Plasmodium. In certain aspects, the infectious microorganism may be selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In certain aspects, the initial protein may be selected from the group consisting of merozoite surface proteins (MSPs) MSP1, MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10, MSP12, P19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PlRH2a, PfRH2b, PfRH4, PfRH5, RAMA, and RALP 1. In certain aspects, the initial protein is Plasmodium falciparum surface protein MSP1-19, which is a carboxyl- terminal fragment of MSP1. In certain aspects, the initial protein may comprise an amino acid sequence at least 80% identical, at least 90% identical, at least 95%identical, at least 97%identical, or 100% identical to SEQ ID NO:2. In certain aspects, the initial protein may comprise an amino acid sequence at least 90% identical, at least 95%identical, at least 97%identical, or 100% identical to SEQ ID NO:2, wherein the neutralizing epitope comprises Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2. In certain aspects, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, wherein the one or more non-neutralizing, interfering epitopes are selected from the group consisting of: a) an epitope comprising, or consisting of, Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and b) an epitope comprising, or consisting of, Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Ser69, Arg71, Phe87 and Asp88 of SEQ ID NO:2.

[0122] In certain aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises at least 15, at least 16, or at least 17 amino acid residues selected from the group consisting of a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the modified protein may comprise, or consist of, an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20. In certain aspects, the modified protein may comprise a mutation at a position corresponding to Ser3 of SEQ ID NO:2 and/or Thr48 of SEQ ID NO:2. In certain aspects, the modified protein may comprise an alanine residue at a position corresponding to position 3 of SEQ ID NO:2 and/or an alanine residue at a position corresponding to position 48 of SEQ ID NO:2.

[0123] In certain asepcts, a modified protein of the disclosure may be encoded by a nucleic acid molecule comprising, or consisting of, a nucleotide sequence at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19.

[0124] One aspect of the disclosure is a fusion protein comprising a modified protein of the disclosure joined to at least a portion of a self-assembling subunit protein, wherein the at least a portion of a self-assembling subunit protein directs self- assembly of the fusion protein into nanoparticles. In certain aspects, the modified protein is joined to the carboxyl-terminal end of the at least a portion of a self-assembling subunit protein. In certain aspects, the modified protein is joined to the amino-terminal end of the at least a portion of a self-assembling subunit protein. In certain aspects, the modified protein is joined directly to the at least a portion of a self-assembling subunit protein. “Joined directly” means that the terminal amino acid of the modified protein is joined to the terminal end of the at least a portion of a self-assembling subunit protein, without any intervening amino acid residues. In certain aspects, the modified protein is joined to the at least a portion of a self-assembling subunit protein by a linker. A “linker” refers to a single amino acid residue or a short peptide (approximately 2-20 amino acids) that connects the modified peptide to the at last a portion of a self-assembling subunit protein, and which does not directly contribute to the activity (e.g., formation of a nanoparticle, elicitation of an immune response) of the fusion protein or a nanoparticle comprising the fusion protein. In certain aspects, the modified protein is produced using a method of the disclosure. In certain aspects, the modified protein is produced using an initial protein from an infectious microorganism, which may be from the genus Plasmodium. In certain aspects, the infectious microorganism may be selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In certain aspects, the initial protein may be selected from the group consisting of merozoite surface proteins (MSPs) MSP1, MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10, MSP12, P19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PfRH2a, PlRH2b, PfRH4, P1RH5, RAMA, and RALP1. In certain aspects, the initial protein is Plasmodium falciparum surface protein MSP1-19, which is a carboxyl- terminal fragment of MSP 1. In certain aspects, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2. In these methods, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, wherein the neutralizing epitope comprises Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2. In certain aspects, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, wherein the one or more non-neutralizing, interfering epitopes are selected from the group consisting of: a) an epitope comprising Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and b) an epitope comprising Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Ser69, Arg71, Phe87 and Asp88 of SEQ ID NO:2. In certain aspects, the modified protein comprises, or consists of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises at least 15, at least 16, or at least 17 amino acid residues selected from the group consisting of a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the modified protein comprises, or consists of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the modified protein may comprise, or consist of, an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20.

[0125] Examples of suitable self-assembling subunit proteins for producing fusion proteins of the disclosure include, but are not limited to, lumazine synthase (LS), the E2p protein of G. stearothermophilus (N-terminus of dihydrolipoyl acetyltransferase) (E2p), the EncA protein of Myxococcus xanthus (C-terminus of encapsulin protein) )EncA) or a ferritin protein. In certain aspects, the at least a portion of the self-assembling subunit protein may be from a LS protein, the G. stearothermophilus E2p protein, the Myxococcus xanthus, EncA protein or a ferritin protein. Thus, in certain aspects, the modified protein is joined to at last a portion of a self-assembling subunit protein, the self-assembling subunit protein being selected from the group consisting of a LS protein, the G. stearothermophilus E2p protein, the Myxococcus xanthus EncA protein or a ferritin protein.

[0126] In certain aspects, the at least a portion of a self-assembling subunit protein is from an LS protein. In certain aspects, the LS protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:93. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:38.

[0127] In certain aspects, the at least a portion of a self-assembling subunit protein may be from the E2p protein. In certain aspects, E2p protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:94. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, and SEQ ID NO:55. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:56.

[0128] In certain aspects, the at least a portion of a self-assembling subunit protein is from the EncA protein. In certain aspects, the EncA protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:95. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74.

[0129] In certain aspects, the at least a portion of a self-assembling subunit protein is from an ApoF protein. In certain aspects, the ApoF protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:96. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, and SEQ ID NO:92.One asepcts of the disclosure is a fusion protein comprising, or consisting of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92.

[0130] One aspect of the disclosure is a nanoparticle comprising a fusion protein of the disclosure. As used herein, a nanoparticle refers to a particle formed from self- assembling, monomeric subunit proteins, examples of which include a LS protein, the G. stearothermophilus E2p protein, the Myxococcus xanthus, EncA protein or a ferritin protein. In certain aspects, nanoparticles of the disclosure may assemble from fusion proteins of the disclosure such that the modified protein portion of the fusion protein is displayed on the surface of the nanoparticle. Nanoparticles of the present invention are generally spherical, or spheroid, in shape, although other shapes, for example, rod, cube, sheet, oblong, ovoid, and the like, are also useful for practicing the present invention. In certain asepcts, nanoparticles of the disclosure may be formed from two or more types of fusion proteins of the disclosure. That is, a nanoparticle may comprise a first fusion protein comprising a first modified protein comprising a first set of mutations, and a second fusion protein comprising a second modified protein comprising a second set of mutations where the first and second sets of mutations may overlap but are not identical.

[0131] One aspect of the disclosure is a nucleic acid molecule comprising a nucleotide sequence encoding a modified protein of the disclosure or a fusion protein of the disclosure. In certain aspects, the nucleic acid molecule encodes a modified protein comprising, or consisting of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20, wherein the amino acid sequence comprises at least 15, at least 16, or at least 17 amino acid residues selected from the group consisting of a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the nucleic acid molecule encodes a modified protein comprising, or consisting of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20, wherein the amino acid sequence comprises a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In certain aspects, the nucleic acid molecule encodes a modified protein comprising, or consisting of, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20. In certain aspects, the nucleic acid molecule may encode a fusion protein of the disclosure. In certain asepcts, the encoded fusion protein may comprise a modified protein of the disclosure joined to at least a portion of a self-assembling subunit protein, as disclosed herein. In certain aspects, the at least a portion of the self-assembling subunit protein may be from a LS protein, the G. stearothermophilus E2p protein, the Myxococcus xanthus, EncA protein or an apoferritin protein (ApoF), as disclosed herein. In certain aspects, the at least a portion of the self-assembling subunit protein may comprise a LS protein, the G. stearothermophilus E2p protein, the Myxococcus xanthus, EncA protein or an ApoF protein. In certain aspects, the LS protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:93. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37. In certain aspects the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:38. In certain aspects, the at least a portion of a self-assembling subunit protein may be from the E2p protein. In certain aspects, E2p protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:94. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:56. In certain aspects, the at least a portion of a self-assembling subunit protein may be from the EncA protein. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:95. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74. In certain aspects, the at least a portion of a self-assembling subunit protein may be from ApoF. In certain aspects, the ApoF protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to SEQ ID NO:96. In certain aspects the fusion protein may be encoded by a nucleic acid molecule comprising a nucleic acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91. In certain aspects, the fusion protein may comprise an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92.

[0132] In certain aspects, a nucleic acid molecule of the disclosure may comprise, or consist of, a nucleotide sequence at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91.

[0133] One aspect of the disclosure is a plasmid or a viral vector comprising a nucleic acid molecule of the disclosure. In certain aspects, the nucleic acid molecule may comprise a promoter operably linked to the nucleic acid sequence encoding modified protein or fusion protein of the disclosure. In certain aspects, the plasmid may be an expression plasmid. In certain aspects, the viral vector may be an expression vector. In certain aspects, the plasmid or the viral vector may be formulated for administration to an individual.

[0134] One aspect of the disclosure is a composition comprising a modified protein of the disclosure, a fusion protein of the disclosure, a nanoparticle of the disclosure, a nucleic acid molecule of the disclosure, a plasmid of the disclosure, or a viral vector of the disclosure. In certain aspects, the composition may comprise a solvent, which may be an aqueous solvent or an organic solvent.

[0135] One aspect of the disclosure is a therapeutic composition comprising a modified protein of the disclosure, a fusion protein of the disclosure, a nanoparticle of the disclosure, a nucleic acid molecule of the disclosure, a plasmid of the disclosure, or a viral vector of the disclosure, and pharmaceutically acceptable excipients. Examples of suitable physiologically acceptable excipients include, but are not limited to, buffers, such as Hanks' solution, Ringer's solution, or physiological saline buffer, stabilizers, amino acids, glycerin, ascorbic acid, sodium bicarbonate, sodium phosphate and anti-microbial agents (e.g., antibiotics).

[0136] One aspect of the disclosure is a vaccine comprising a fusion protein of the disclosure, a nanoparticle of the disclosure, a nucleic acid molecule of the disclosure, a plasmid of the disclosure, a viral vector of the disclosure, a composition of the disclosure, or a therapeutic composition of the disclosure. In certain aspects, the vaccine may comprise an adjuvant. Suitable adjuvants are known to those skilled in the art. [0137] One aspect of the disclosure is a kit comprising at least one component selected from the group consisting of a modified protein of the disclosure, a fusion protein of the disclosure, a nanoparticle of the disclosure, a nucleic acid molecule of the disclosure, a plasmid of the disclosure, a viral vector of the disclosure, a composition of the disclosure, a therapeutic composition of the disclosure, and a vaccine of the disclosure. The kit may also contain associated components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes and the like. The kit may also contain instructions for using components of the kit, such as instruction for producing a modified protein of the disclosure, producing a fusion protein of the disclosure, producing a nanoparticle of the disclosure, or producing a vaccine of the disclosure. The kit may also comprise instructions for vaccinating an individual against infection by a microorganism of the genus Plasmodium, protecting an individual against developing malaria, or treating malaria, using one or more components of the kit.

[0138] In these aspects, a modified protein in a kit of the disclosure may be produced using a method of the disclosure. In these aspects, the modified protein may be produced using an initial protein from an infectious microorganism, which may be from the genus Plasmodium. In these aspects, the infectious microorganism may be selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In these aspects, the initial protein may be selected from the group consisting of merozoite surface proteins (MSPs) MSP 1 , MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10, MSP12, P19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PfRH2a, PfRH2b, PfRH4, PfRH5, RAMA, and RALP1. In these aspects, the initial protein may be Plasmodium falciparum surface protein MSP1-19, which is a carboxyl-terminal fragment of MSP 1. In these aspects, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2. In these aspects, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, wherein the neutralizing epitope comprises Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2. In these aspects, the initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO:2, wherein the one or more non-neutralizing, interfering epitopes are selected from the group consisting of: a) an epitope comprising Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and b) an epitope comprising Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Ser69, Arg71, Phe87 and Asp88 of SEQ ID NO:2. In these aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises at least 15, at least 16, or at least 17 amino acid residues selected from the group consisting of a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In these aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In these aspects, a modified protein in a kit of the disclosure may comprise, or consist of, an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18 and SEQ ID NO:20. In certain aspects, the kit may comprise a nucleic acid molecule comprising or consisting of a nucleotide sequence at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO: 73.

[0139] One aspect of the disclosure is a method of vaccinating an individual against infection by a microorganism, comprising administering to the individual a modified protein of the disclosure, a fusion protein of the disclosure, a nanoparticle of the disclosure, a nucleic acid molecule of the disclosure, a plasmid of the disclosure, a viral vector of the disclosure, a composition of the disclosure, a therapeutic composition of the disclosure, or a vaccine of the disclosure.

[0140] One aspect of the disclosure is a method of vaccinating an individual against infection by a microorganism of the genus Plasmodium, comprising administering to the individual a modified protein of the disclosure, a fusion protein comprising a modified protein of the disclosure, a nanoparticle comprising a modified protein of the disclosure, a nucleic acid molecule encoding a modified protein of the disclosure, a plasmid encoding a modified protein of the disclosure, a viral vector encoding a modified protein of the disclosure, a composition comprising a modified protein of the disclosure, a therapeutic composition comprising a modified protein of the disclosure, or a vaccine comprising a modified protein of the disclosure, wherein the modified protein is produced using an initial protein from a microorganism of the genus Plasmodium.

[0141] One aspect of the disclosure is a method of protecting an individual against infection by a microorganism of the genus Plasmodium, comprising administering to the individual a modified protein of the disclosure, a fusion protein comprising a modified protein of the disclosure, a nanoparticle comprising a modified protein of the disclosure, a nucleic acid molecule encoding a modified protein of the disclosure, a plasmid encoding a modified protein of the disclosure, a viral vector encoding a modified protein of the disclosure, a composition comprising a modified protein of the disclosure, a therapeutic composition comprising a modified protein of the disclosure, or a vaccine comprising a modified protein of the disclosure, wherein the modified protein is produced using an initial protein from a microorganism of the genus Plasmodium.

[0142] One aspect of the disclosure is a method of protecting an individual against developing malaria, comprising administering to the individual a modified protein of the disclosure, a fusion protein comprising a modified protein of the disclosure, a nanoparticle comprising a modified protein of the disclosure, a nucleic acid molecule encoding a modified protein of the disclosure, a plasmid encoding a modified protein of the disclosure, a viral vector encoding a modified protein of the disclosure, a composition comprising a modified protein of the disclosure, a therapeutic composition comprising a modified protein of the disclosure, or a vaccine comprising a modified protein of the disclosure, wherein the modified protein is produced using an initial protein from a microorganism of the genus Plasmodium. In certain aspects, the individual is known to have been infected with a microorganism selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In certain aspects, the individual has not been infected with a microorganism selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi.

[0143] One aspect of the disclosure is a method of treating a malaria patient, comprising administering to the individual a modified protein of the disclosure, a fusion protein comprising a modified protein of the disclosure, a nanoparticle comprising a modified protein of the disclosure, a nucleic acid molecule encoding a modified protein of the disclosure, a plasmid encoding a modified protein of the disclosure, a viral vector encoding a modified protein of the disclosure, a composition comprising a modified protein of the disclosure, a therapeutic composition comprising a modified protein of the disclosure, or a vaccine comprising a modified protein of the disclosure, wherein the modified protein is produced using an initial protein from a microorganism of the genus Plasmodium. [0144] In these aspects, the microorganism of the genus Plasmodium may be selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In these aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises at least 15, at least 16, or at least 17 amino acid residues selected from the group consisting of a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In these aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 ofSEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. In these aspects, the modified protein may comprise, or consist of, an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20.

[0145] Administration of a modified protein of the disclosure, a fusion protein of the disclosure, a nanoparticle of the disclosure, a nucleic acid molecule of the disclosure, a plasmid of the disclosure, a viral vector of the disclosure, a composition of the disclosure, a therapeutic composition of the disclosure, or a vaccine of the disclosure may comprise any route of administration suitable for eliciting an immune response. Examples of suitable routes of administration include parenteral administration, oral administration, nasal administration. [0146] Because interfering, non-neutralizing epitopes in modified proteins of the disclosure have been modified or blocked, modified proteins of the disclosure may be used to detect or measure neutralizing antibodies in a sample. Thus, one aspect of the disclosure is a method of detecting a neutralizing antibody in a sample, wherein the neutralizing antibody specifically binds a neutralizing epitope in a modified protein of the disclosure, comprising contacting the sample with a modified protein of the disclosure, and detecting binding of a neutralizing antibody present in the sample to the modified protein, thereby detecting a neutralizing antibody in the sample. In certain aspects, the modified protein may be present in a nanoparticle of the disclosure, and the method may comprise contacting the sample with the nanoparticle of the disclosure. In certain aspects, the step of contacting comprises using conditions that allow any neutralizing antibody present in the sample to specifically bind a neutralizing epitope in the modified protein. In certain aspect, the method comprises detecting a complex between the neutralizing antibody present in the sample to the modified protein, or the nanoparticle comprising the modified protein. Any method of detecting binding of an antibody to an antigen may be used to detect binding of a neutralizing antibody present in the sample to the modified protein, or the nanoparticle comprising the modified protein. In certain aspects, detection of antibody binding may comprise the use of a label, such as a colorimetric label, a chemiluminescent label, or a fluorescent label. Examples of assays suitable to detecting antibody binding include, but are not limited to, an enzyme linked immunosorbent assay (ELISA), an immunoprecipitation assay, and a surface plasmon resonance assay. In certain aspects, detecting a neutralizing antibody in a sample may comprise measuring the amount of antibody (e.g., titer) of neutralizing antibody in the sample. In certain aspects, the sample may comprise tissue from the first individual. In certain aspects, the sample may comprise a biological liquid, which may comprise whole blood, plasma, serum, saliva, urine, mucous, tears, cerebral spinal fluid (CSF), lymph, or other bodily fluids.

[0147] One aspect of the disclosure is a method of diagnosing an individual as being protected from an infectious microorganism, comprising contacting a sample from the individual with a modified protein of the disclosure, wherein the modified protein is produced using an initial protein from the infectious microorganism, and detecting binding of neutralizing antibody present in the sample, if any, to the modified protein, thereby detecting neutralizing antibody in the sample, wherein if the individual has neutralizing antibodies, diagnosing the individual as being protected from malaria. In certain aspects, detecting a neutralizing antibody in a sample may comprise measuring the amount of antibody (e.g., titer) of neutralizing antibody in the sample. In certain aspects, the level of neutralizing antibody in the individual is determined, wherein if the individual has a high level of antibody, the individual is diagnosed as being protected from the infectious microorganism. In certain aspects, the level of neutralizing antibody is determined and may be compared to a level of neutralizing antibodies known to protect an individual from the infectious microorganism. In such aspects, the protective level of neutralizing antibodies may be determined by comparing levels of neutralizing antibodies present in individuals known to be protected from the infectious microorganism with levels of antibodies present in individuals known to be unprotected from the infectious microorganism. In certain aspects, the level of neutralizing antibody in the individual is determined and compared to the level of neutralizing antibodies present in individuals known to be protected from the infectious microorganism and/or with levels of antibodies present in individuals known to be unprotected from the infectious microorganism.

[0148] One aspect of the disclosure is a method of diagnosing an individual as being protected from malaria, comprising contacting a sample from the individual with a modified protein of the disclosure, and detecting binding of neutralizing antibody present in the sample, if any, to the modified protein, thereby detecting neutralizing antibody in the sample, wherein if the individual has neutralizing antibodies, diagnosing the individual as being protected from malaria. In certain aspects, detecting a neutralizing antibody in a sample may comprise measuring the amount of antibody (e.g., titer) of neutralizing antibody in the sample. In certain aspects, the level of neutralizing antibody in the individual is determined, wherein if the individual has a high level of antibody, the individual is diagnosed as being protected from malaria. In certain aspects, the level of neutralizing antibody is determined and may be compared to a level of neutralizing antibodies known to protect an individual from malaria. In such aspects, the protective level of neutralizing antibodies may be determined by comparing levels of neutralizing antibodies present in individuals known to be protected from malaria with levels of antibodies present in individuals known to be unprotected from malaria (i.e., susceptible to getting malaria). In certain aspects, the level of neutralizing antibody in the individual is determined and compared to the level of neutralizing antibodies present in individuals known to be protected from malaria and/or with levels of antibodies present in individuals known to be unprotected from malaria.

[0149] In these aspects, the modified protein may be present in a nanoparticle of the disclosure, and the method may comprise contacting the sample with the nanoparticle of the disclosure. In these aspects, the step of contacting comprises using conditions that allow any neutralizing antibody present in the sample to specifically bind a neutralizing epitope in the modified protein. In these aspects, the method comprises detecting a complex between the neutralizing antibody present in the sample to the modified protein, or the nanoparticle comprising the modified protein. Any method of detecting binding of an antibody to an antigen may be used to detect binding of a neutralizing antibody present in the sample to the modified protein, or the nanoparticle comprising the modified protein. In these aspects, detection of antibody binding may comprise the use of a label, such as a colorimetric label, a chemiluminescent label, or a fluorescent label. Examples of assays suitable to detecting antibody binding include, but are not limited to, an enzyme linked immunosorbent assay (ELISA), an immunoprecipitation assay, and a surface plasmon resonance assay. In these aspects, the sample may comprise tissue from the first individual. In these aspects, the sample may comprise a biological liquid, which may comprise whole blood, plasma, serum, saliva, urine, mucous, tears, cerebral spinal fluid (CSF), lymph, or other bodily fluids.

[0150] One aspect of the disclosure is a method of determining the efficacy of a vaccine to induce in a first individual a neutralizing antibody that specifically binds a neutralizing epitope in the vaccine, comprising vaccinating the first individual using the vaccine, and after a suitable period of time determining the level of the neutralizing antibody, if any, in a first sample from the first individual by contacting the sample with a modified protein of the disclosure, wherein the modified protein comprises the neutralizing epitope, and detecting binding of neutralizing antibody present in the first sample to the modified protein, thereby determining the efficacy of the vaccine to induce a neutralizing antibody in the first individual. In certain aspects, detecting binding of neutralizing antibody to the modified protein may comprise determining the level of neutralizing antibody present in the first sample. In certain aspects, the level of neutralizing antibody in the first sample may be compared to the level of neutralizing antibody in one or more samples from one or more individuals, which may be pooled. The level of neutralizing antibody in the one or more samples may be historic, meaning such level was determined at a time prior to vaccination of the first individual. In certain aspects, the level of neutralizing antibody in the first sample is compared to the level of neutralizing antibody in an initial sample obtained from the first individual prior to vaccination. In certain aspects, the sample may comprise tissue from the first individual. In certain aspects, the sample may comprise a biological liquid, which may comprise whole blood, plasma, serum, saliva, urine, mucous, tears, cerebral spinal fluid (CSF), lymph, or other bodily fluids. In certain aspects, the modified protein may be present in a nanoparticle of the disclosure, and the method may comprise contacting the sample with the nanoparticle of the disclosure. In certain aspects, the step of contacting may comprise using conditions that allow any neutralizing antibody present in the sample to specifically bind a neutralizing epitope in the modified protein. In certain aspect, the method comprises detecting a complex between the neutralizing antibody present in the sample to the modified protein, or the nanoparticle comprising the modified protein. Any method of detecting binding of an antibody to an antigen may be used to detect binding of a neutralizing antibody present in the sample to the modified protein, or the nanoparticle comprising the modified protein. In certain aspects, detection of antibody binding may comprise the use of a label, such as a colorimetric label, a chemiluminescent label, or a fluorescent label.

[0151] In these methods, the modified protein may be produced using a method of the disclosure. In these aspects, the modified protein may be produced using an initial protein from an infectious microorganism, which may be from the genus Plasmodium. The infectious microorganism may be selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. The initial protein may be selected from the group consisting of merozoite surface proteins (MSPs) MSP1, MSP2, MSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP 10, MSP12, P19, Pf38, PF41, Pf92, Pfl 13, GLURP, SERA3, SERA4, SERA5, SERA6, AMA1, EBA140, EBA175, EBA181, EBL1, MTRAP, PTRAMP, GAMA, CyRPA, PfRipr, PfRHl, PfRH2a, PfRH2b, PfRH4, PfRH5, RAMA, and RALP1. The initial protein may be Plasmodium falciparum surface protein MSP1-19, which is a carboxyl- terminal fragment of MSP1. The initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2. The initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, wherein the neutralizing epitope comprises Gln6, Asn15, Ser16, Leu31, Thr63, Glu65, Lys73, Thr75, Cys76, Glu77, Cys78, Thr79, Lys80, Pro81, Asp82, Ser83, Tyr84, Pro85, Leu86, Phe87, and Asp88 of SEQ ID NO:2. The initial protein may comprise an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, wherein the one or more non-neutralizing, interfering epitopes are selected from the group consisting of: a) an epitope comprising Ile2, Gln6, Lys10, Gln11, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Arg71, Lys73, Phe87 and Asp88 of SEQ ID NO:2; and b) an epitope comprising Cys7, Lys10, Cys12, Pro13, Gln14, Asn15, Ser16, Gly17, Cys18, Leu31, Leu32, Asn33, Tyr34, Glu37, Lys40, Cys41, Val42, Glu43, Pro45, Ser69, Arg71, Phe87 andAsp88 of SEQ ID NO:2. In these aspects, the modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises at least 15, at least 16, or at least 17 amino acid residues selected from the group consisting of a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. The modified protein may comprise, or consist of, an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:20, wherein the amino acid sequence comprises a glutamine residue at the position corresponding to position 6 of SEQ ID NO:2; an asparagine residue at the position corresponding to position 15 of SEQ ID NO:2; a serine residue at the position corresponding to position 16 of SEQ ID NO:2, a leucine residue at the position corresponding to position 31 of SEQ ID NO:2, a threonine residue at the position corresponding to position 63 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 65 of SEQ ID NO:2, a lysine residue at the position corresponding to position 73 of SEQ ID NO:2, a threonine residue at the position corresponding to position 75 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 76 of SEQ ID NO:2, a glutamic acid residue at the position corresponding to position 77 of SEQ ID NO:2, a cysteine residue at the position corresponding to position 78 of SEQ ID NO:2, a threonine residue at the position corresponding to position 79 of SEQ ID NO:2, a lysine residue at the position corresponding to position 80 of SEQ ID NO:2, a proline residue at the position corresponding to position 81 of SEQ ID NO:2, an asparagine residue at the position corresponding to position 82 of SEQ ID NO:2, a serine residue at the position corresponding to position 83 of SEQ ID NO:2, a tyrosine residue at the position corresponding to position 84 of SEQ ID NO:2, a proline residue at the position corresponding to position 85 of SEQ ID NO:2, a leucine residue at the position corresponding to position 86 of SEQ ID NO:2, a phenylalanine residue at the position corresponding to position 87 of SEQ ID NO:2, and, an aspartic acid residue at the position corresponding to position 88 of SEQ ID NO:2. The modified protein may comprise, or consist of, an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18 and SEQ ID NO:20.

[0152] This written description uses examples to disclose the invention, including the best mode, and to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0153] EXAMPLES

[0154] Example 1. Production of p19-specific human monoclonal antibodies.

[0155] A panel of MSP-1-specific IgG ⁺ B cell receptor sequences was generated from adult volunteers enrolled in an observational cohort study conducted in the malaria- endemic community of Kalifabougou, Mali. These sequences were cloned into human immunoglobulin G1 (IgG1), kappa (k) or lambda scaffolds to produce recombinant hmAbs. Paired heavy- and light-chain plasmids were co-expressed (FIG. 2A), and antigen specificity was confirmed by ELISA reactivity of purified recombinant hmAbs to p19 and full-length MSP-1 (FIG. 2B). The expressed non-glycosylated p19 and full-length MSP-1 were monomeric and monodisperse, as observed by size-exclusion chromatography and SDS-PAGE (FIGS. 3A & 3B). Eight of the MSP-1 -specific hmAbs generated bound to p19. Six of these eight antibodies were isolated from one individual and the other two from a second individual (Table 1).

[0156] Example 2. Binding kinetics characterization of isolated antibodies [0157] Antibody affinity and binding kinetics may be important determinants for protection and neutralization. Accordingly, the binding kinetics of eight antigen-binding fragments (Fabs) to p19 by were determined by biolayer interferometry (BLI) and a range of dissociation constants (K _D ) from 0.66 nM to 300 nM (Table 2) observed. hmAbs 42C3 and 42A9 bound to p19 with the strongest affinities and 42C11 bound ~2-3- fold weaker. hmAbs 42C5 and 42D6 bound with moderate affinity ~6-fold weaker than 42C3, and 42D7 bound ~13-fold weaker than 42C3. Finally, 75E9 and 75F4 bound more than 300-fold weaker than 42C3. These diverse binding affinities for p19 derive predominantly from varied dissociation rates ranging from 0.15 x 10 ^-3 to 62.21 x 10 ^-3 s ^-1 while association rates are relatively consistent between antibodies (Table 2).

[0158] Example 3. 42D6 potently neutralizes blood-stage parasites

[0159] The ability of hmAbs to block the entry of merozoites into erythrocytes was evaluated using the standardized growth inhibition activity (GIA) assay (FIG. 4). hmAb 42D6 inhibited parasite growth by >90 % (against Plasmodium falciparum 3D7) at 1.0 mg/ml, and had a binding affinity of 4.24 nM. 42C3 and 42A9 had stronger binding affinities than 42D6 but showed no inhibition of parasite growth at 1.0 mg/ml in GIA. These data establish that there is no correlation between binding kinetics and GIA (FIGS. 5A-5C), and that binding kinetics alone are insufficient to predict inhibition of erythrocyte invasion by merozoites.

[0160] Example 4. Epitopes for p19-specific hmAbs are overlapping

[0161] Epitope binning revealed that the panel of eight p19 hmAbs all compete with one another for binding by biolayer interferometry (BLI). Six of the eight Fabs had slow dissociation rates suitable for use as the primary antibody, and all eight Fabs were suitable for use as the secondary or competing antibody. Strikingly, all hmAbs competed with one another suggesting their epitopes are either adjacent or overlapping (FIG. 6).

[0162] Example 5. Structure of p19 in complex with Fab fragments 42D6, 42C11 and 42C3

[0163] The lack of correlation between GIA and antibody affinity or epitope-binning prompted a comprehensive structural analysis to map the epitopes of inhibitory and non-inhibitory antibodies. Co-complex crystal structures of p19 with 42D6, 42C11, and 42C3 were determined to resolutions of 2.0, 1.9 and 2.3 A°, respectively (FIG. 7 and FIGS. 8A-8F, Table 3). 42C11 and 42C3 were selected for further study due to their diverse growth inhibitory potential and diverse binding affinity. [0164] The structures revealed that 42D6 binds p19 via heavy-chain interactions with residues at the central β-sheets and C-terminal loop of EGF-like domain II and a few interactions with residues from the N-terminal loop and the loop C-terminal to the central β-sheets of EGF-like domain I (FIG. 9A). 42D6 has an interacting buried surface area (BSA) of 710.0 A ² with p19, and residues from all three CDR loops of the heavy chain contact twenty-one p19 residues (Table 4). The conformational epitope recognized by the hmAb 42D6 on p19 does not overlap with epitopes for non-neutralizing hmAb MaliM03 (PDB ID: 6XQW) ²⁹ or murine mAb G17.12 (PDB ID: 1OB1) ¹⁷ (FIG.10).

[0165] In contrast to the 42D6 epitope, co-crystal structures of non- neutralizing hmAbs 42C11 and 42C3 revealed that both hmAbs primarily recognize EGF- like domain I while making few contacts with EGF-like domain II (FIGS. 9B & 9C). The epitopes for 42C11 and 42C3 are largely overlapping (FIGS. 7 & 10) with large interacting BSAs of 876.8 and 902.7 A ² , respectively. In addition, both their heavy- and light-chains contributed almost equally to p19 contacts and BSA (Tables 5 and 6, respectively).

[0166] Example 6. 42C11 and 42C3 represent an immunodominant antibody lineage

[0167] The shared epitope of 42C11 and 42C3 is consistent with their high sequence similarity, including similar CDR3 sequences and shared heavy- and light-chain germlines. These antibodies, in addition to the similar 42C5, 42D7, and 42A9, were isolated from the same individual and are likely clonally related (FIG.11). Interestingly, 6/29 of the MSP-1 -specific heavy-chain IgG sequences isolated from this individual had highly similar sequences suggesting significant clonal expansion of this antibody lineage. All these hmAbs were poorly neutralizing and competed with all other hmAbs (FIG. 4 & FIG. 6). This lineage utilizes the IGHV3-30 germline, which is the most common germline across multiple individuals for MSP-1 /AMA 1 -specific B cells ²⁸ . Together, these observations suggest that 42C11 and 42C3 represent a clonally expanded immunodominant antibody lineage. [0168] Example 7. 42D6 targets a conserved epitope on p19

[0169] Antigen polymorphism can potentially limit strain-transcending protection by vaccine-induced or naturally acquired antibodies and should be evaluated in the context of hmAb binding and neutralization. Polymorphic residues within p19 identified from 3,488 amino acid sequences in the MalariaGEN Pf3k database (FIG. 12) (https://www.malariagen.net) were structurally mapped. Nineteen 42D6 epitope residues in p19 were invariant and two residues exhibited polymorphisms with varying frequencies [Glu65Lys (0.5%), and Leu86Phe (19.0%)]. Thr61, which is in the vicinity of the 42D6 epitope, also exhibited polymorphism (Thr61Lys) with an observed frequency of 76.6%. None of the polymorphisms had a major effect on binding with Thr61Lys and Leu86Phe decreasing affinity less than 3-fold, and the rare Glu65Lys polymorphism decreasing affinity 6-fold (FIGS. 13A-C and Table 7). These data suggest the 42D6 epitope is broadly conserved and 42D6 recognizes a wide array of p19 variants.

[0170] Example 8. 42D6 is a strain-transcending broadly-neutralizing human mAb

[0171] The strain-transcending neutralizing potential of 42D6 was further examined by performing GIA assays against three diverse strains of Plasmodium falciparum-. 3D7, Dd2 and FVO. These strains contain high frequency polymorphisms within p19 and form a strong foundation to evaluate the breadth of 42D6. p19 from Dd2 possesses three polymorphisms relative to 3D7: Thr61Lys, Ser70Asn and Arg71Gly; and p19 from FVO possesses four polymorphisms: GlylGln, Thr61Lys, Ser70Asn and Arg71Gly (FIG. 14). All strains were neutralized by 42D6 with half-maximum inhibitory concentration (IC ₅₀ ) values of 0.106 mg/ml, 0.259 mg/ml, and 0.317 mg/ml against P. falciparum 3D7, FVO and Dd2 strains, respectively (FIG. 15). These data indicate that 42D6 is a likely to be a broadly neutralizing human antibody.

[0172] Example 9. High affinity non-neutralizing antibodies against the adjacent or overlapping region interfere with the effect of neutralizing antibodies (antigenic diversion)

[0173] The aforementioned work establishes that 42D6 is a potently neutralizing p19-specific hmAb that targets a unique epitope. It also identified epitopes for two non-neutralizing hmAbs 42C3 and 42C11 , which partially overlap with the neutralizing epitope of 42D6 hmAb. These diverse hmAb parameters and functions prompted the question of how these hmAbs may interact or interfere with each other and modulate parasite survival. The interactions of these hmAbs were examined using combination GIA assay to evaluate potential effects. Strikingly, combining 42C3 with 42D6 completely abrogated the ability of 42D6 to neutralize parasites (FIG. 16A). Similarly, combining 42C11 with 42D6 reduced GIA inhibition to a level similar to 42C11 alone (FIG. 16A & 16B). These data are consistent with the non-neutralizing high-affinity antibodies 42C11 and 42C3 preventing binding of 42D6, thereby enabling parasite survival (FIGS. 17 & 18).

[0174] Example 10. MSP1-19 display on nanoparticles improves the antibody response. Nanoparticles were produced using fusion proteins containing MSP1-19 protein fused to either the C-terminus of A. aeolicus lumazine synthase (LS) (MSP1-19 WT LuS), the N-terminus of G. stearothermophilus dihydrolipoyl acetyltransferase (MSP1-19 WT E2p); the N-terminus of H. pylori ferritin nanoparticle (MSP1-19 WT ApoF ), or the C- terminus of Myxococcus xanthus encapsulin protein (MSP1-19 WT Encapsulin). FIG. 19A shows an immunization and blood draw schedule for rats. The study was consisted of six groups and six rats per each group were immunized with 20 μg antigen each. Antigen was formulated as a 1 : 1 ratio in AddaS03™ Adjuvant (InvivoGen), and 100 μl formulated antigen delivered by subcutaneous injection.on day 0 (Vac1), day 21 (Vac2) and day 42 (Vac3). Samples were taken on days 21, 35, and 56, and the titer of IgG in each sample determined. The results are shown in FIGS. 19B-D.

[0175] Example 11. MSP1-19 display on nanoparticles improves the neutralizing antibody response and demonstrates potent strain-transcending antibodies compared to native MSP1-19 monomer.

[0176] Nanoparticles were produced using fusion proteins containing MSP1-19 protein fused to either apoferritin from H. pylori, lumazine synthase LuS) from A aoelicus, dihydrolipoyl acetyltransferase from B stearothermophilus (E2P), or the C- terminus of Myxococcus xanthus encapsulin protein (EncA) and rats vaccinated using one class of nanoparticle. Each group of rats contained 6 rats and immunization was done using 20 μg of antigen. Antigen was formulated as a 1 : 1 ratio in AddaS03 Adjuvant. 100 μl formulated antigen was administered to each rat by subcutaneous injection. One group of rats received PBS and another received MSP1-19 monomer. Second and third dose of each nanoparticle and antigen was administered at 21 and 42 days post-vaccination. Blood samples were taken at 21 days, 35 days, and at 56 days, and the samples tested for neutralizing antibodies using an in vitro Plasmodium falciparum growth inhibition assay (GIA). The results of this study, which are shown in FIGS. 20A-C, show that displaying MSP1-19 on self-assembling nanoparticle platforms elicits potent strain-transcending antibodies compared to native MSP 1 - 19 monomer. The results also show (FGIS. 20D-F) that the use of nanoparticle platforms produces antibodies having a reduced IC ₅₀ .

[0177] Example 13 Designed MSP1-19 modified proteins

[0178] SPEEDesign was used to create MSP1-19 modified immunogens, as illustrated in FIGS. 21A-D. Forty-eight of the resulting sequences were produced and the proteins screened for stability resulting in eight promising candidates. An alignment of the eight candidates is shown in FIG. 22.

[0179] The eight candidates were tested for their ability to bind the 42D6 neutralizing antibody, and the 42C3 and42Cl 1 non-neutralizing monoclonal antibodies. The results, which are shown in FIG. 23 show that the modified MSP1-19 immunogens retained the 42D6 neutralizing epitope but exhibited minimal binding to the 42C3 and 42C11 non- neutralizing monoclonal antibodies.

[0180] Example 14. Modified MSP1-19 immunogens on nanoparticles.

[0181] Nanoparticles were created using fusion proteins comprising one of the eight modified MSP1-19 immunogens fused to lumazine synthase. The study was consisted of six groups and six rats per each group were immunized with 20 μg antigen each as shown in FIG.24A. Antigen was formulated as a 1 : 1 ratio in AddaS03™ Adjuvant (InvivoGen) and 100 μl formulated antigen was delivered by subcutaneous injection on day 0 (Vac1), day 21 (Vac2) and day 42 (Vac3). Blood samples were obtained on days 21, 35 and 56 post vaccination. FIGS. 24B-D show the titers obtained for each immunogen on days 21, 35, and 56. FIGS. 25A show the results of GIA’s demonstrating that three of the modified immunogens, and the native WT MSP1-19 inhibit the growth of P. falciparum 3D7 at high concentrations of IgG. [0182] Purified serum IgG was then tested for its ability to inhibit the growth of various P falciparum strains. The results, which are given in FIGS. 25B-D, show that lumazine synthase-based nanoparticles displaying the modified MSP1-19 immunogens elicit significantly greater, potent strain-transcending antibodies compared to native WT MSP1-19. Moreover, antibodies produced using the modified immunogens had a lower IC ₅₀ values compared to antibodies produced using native WT MSP1-19 (FIGS.25E-G).