Malaria protein nanoparticle vaccines and uses thereof

Abstract

The invention provides a composition comprising a Plasmodium species multimeric protein capable of forming a nanoparticle and at least one antigen of interest, wherein the multimeric protein comprises a monomer chosen from Plasmodium species pyridoxal 5'-phosphate synthase (PLP), chaperone 60 protein (Cpn60), and caseinolytic protease (Clp). In embodiments, the invention provides the composition wherein the monomers assemble to form a multimeric nanoparticle and / or which comprises an adjuvant. The invention further provides a nucleic acid encoding the multimeric protein and the at least one antigen of interest, E. coli comprising the nucleic acid, and the use of such in a method for producing the inventive multimeric protein and the at least one antigen of interest. Also provided are a method and use of the inventive multimeric protein and the at least one antigen of interest for immunizing a subject against Plasmodium species.

Description

Leydig 774102 NIH E-182-2024-0-PC-011MALARIA PROTEIN NANOPARTICLE VACCINES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 695,288, filed September 16, 2024, which is incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support by the National Institutes of Health, National Institute of Allergy and Infectious Diseases, under grant numbers: 1ZIAAI001253, 1ZIAAI001237, and 1ZIAAI001236. The Government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 43,077 Byte XML file named “774102. xml,” dated September 15, 2025.BACKGROUND OF THE INVENTION

[0004] Malaria remains a major threat to global health and to the survival of children under the age of five in endemic areas. The World Health Organization (WHO) continues to report more than 435,000 deaths and nearly 220 million cases annually. The development of a highly efficacious vaccine will be a central tool for the long-term goal of disease control and eradication.

[0005] Studies using irradiated sporozoites and animal models for pre-erythrocytic malarial immunity suggest that protection against malaria infection is mediated by parasite specific cytotoxic CD8+ T cells and by neutralizing antibodies against the repeat domain of circumsporozoite protein (CSP). Plasmodium falciparum circumsporozoite protein (CSP) is densely expressed on the surface of sporozoites and required for motility and hepatocyte invasion of sporozoites. In addition, CD4+ IFN-y-secreting T cells may also contribute toLeydig 774102 NIH E-182-2024-0-PC-012 protection through T-cell dependent antibody response. An effective malaria vaccine may therefore need to induce both cellular and humoral immune responses.

[0006] Subunit vaccines are prepared from purified antigens or parts of antigens that elicit a protective immune response. Subunit vaccines have fewer safety issues, lower cost, and are easier to produce and store compared with vaccines based on attenuated and inactivated pathogens. However, subunit antigens are often insufficiently immunogenic to elicit an adequate protective response. Subunit antigens are therefore displayed on self-assembling protein nanoparticles to enhance their immunogenic response.

[0007] Currently, about twenty vaccines are in clinical trials for malaria. Of these, the RTS,S / AS01 vaccine is the first vaccine to be approved for use in children in sub-Saharan Africa by the WHO. RTS,S has demonstrated efficacy of 30% after four injections in children under the age of five. The titers of protective antibodies appear to wane over time necessitating multiple boosters.

[0008] Accordingly, a clinical need persists for vaccine, e.g., against malaria, which can elicit a robust immune response against the pathogen without undesirable off-target immune response, and one which can be manufactured efficiently. The present invention addresses these concerns.BRIEF SUMMARY OF THE INVENTION

[0009] The invention provides a composition comprising a Plasmodium species multimeric protein capable of forming a nanoparticle and at least one antigen of interest, wherein the multimeric protein comprises a monomer chosen from Plasmodium species pyridoxal 5’- phosphate synthase (PLP), chaperone 60 protein (Cpn60), and caseinolytic protease (Clp). In embodiments, the invention provides the composition wherein the monomers assemble to form a multimeric nanoparticle and / or which comprises an adjuvant. The invention further provides a nucleic acid encoding the multimeric protein and the at least one antigen of interest, E. coli comprising the nucleic acid, and the use of such in a method for producing the inventive multimeric protein and the at least one antigen of interest. Also provided are a method and use of the inventive multimeric protein and the at least one antigen of interest for immunizing a subject against Plasmodium species.Leydig 774102 NIH E-182-2024-0-PC-013

[0010] The inventive nanoparticle platform, as it is based on proteins derived from Plasmodium species, as opposed to non -native (i.e., non-Plasmodium) sources such as viruses or bacteria, can avoid unwanted immune response, which could otherwise divert the immune response from the fused antigens. Also, the use of proteins from the malaria pathogen, as opposed to non-native sources, can induce cell-mediated immunity that could facilitate a synergistic immune response with any antigen presented. Additionally, the inventive nanoparticle platform presents advantages from a manufacturing standpoint, as the component monomers can be synthesized in E. coli. Also, the multimeric nanoparticle platform according to the present invention typically forms a ring structure and can present multiple copies of multiple antigens, eliciting a robust immune response when used as a vaccine.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0011] Figure A-l. Construction, expression, purification, and complex formation of engineered PLP synthase, a, Schematic diagram of the CSPj5c and PLP synthase fusion constructs, b, Size exclusion chromatograms showing the purified CSP-P1-CSP and CSP-P2- CSP components and the larger complex formed upon their combination, c, Reducing SDS- PAGE gel showing the proteins present in the indicated chromatogram peaks, d, Negative stain electron microscopy analysis of the CSP-P1-CSP + CSP-P2-CSP complex. The bottom panel represents the 2D classification. Scalebar = 50 nm.

[0012] Figure A-2. Binding of neutralizing CSP monoclonal antibodies, CIS43 and mAb317 to the CSP-P1-CSP+CSP-P2-CSP complex a, Size exclusion chromatograms showing that incubating CIS43 (upper panel) or mAb317 (lower panel) Fabs with CSP-P1- CSP+CSP-P2-CSP increases the molecular weight. Dashed line indicates elution volume of CSP-P1-CSP+CSP-P2-CSP complex without Fab. b, BLI binding data and curve fits for the CSP monoclonal antibody CIS43 (upper panel) and mAb317 IgGs (lower panel) to CSP-P1- CSP+CSP-P2-CSP, showing strong binding of these monoclonal antibodies to the CSP-P1- CSP+CSP-P2-CSP nanoparticles.

[0013] Figure A-3. Two-dose vaccination regimen elicits robust antibody titers and sterile protection against parasite challenge, a, Two-dose immunization regimen. Created with Biorender.com. b, CSPj5c-specific IgG titers after the first (day 21-bleed) and the second (dayLeydig 774102 NIH E-182-2024-0-PC-01435-bleed) immunization in C57BL / 6 mice. Bars indicate median values and circles represent individual animals, p-values determined by Mann-Whitney test, c, Sterile protection in C57BL / 6 mice after two immunizations followed by intradermal challenge with 500 PfCSP transgenic P. berghei parasites. The presence of infected RBCs was determined via thin blood smears prepared daily for 4-12 days post challenge. Statistically significant differences in survival analysis were calculated by the Kaplan-Meier method.

[0014] Figure A-4. Design of a combined CSP and CelTOS nanoparticle immunogen, a, FPLC chromatograms showing the complex formation of CSP-Pl-CSP+PvCelTOS-P2-CSP and CSP-Pl-CSP+CSP-P2-PvCelTOS. b, Reducing SDS-PAGE analysis showing that the complex consists of two components in approximately equal amounts, c, Negative stain electron microscopy analysis of CSP-P1-CSP + PvCelTOS-P2-CSP and CSP-P1-CSP+CSP-P2- PvCelTOS. The bottom panel represents the 2D classification. Scale bar = 50 nm.

[0015] Figure A-5. CSPj5c- and PvCelTOS-specific antibody titers after immunization and sterile protection against parasite challenge, a, Two-dose immunization regimen. Created with Biorender.com. b, CSPj5c-specific IgG titers after the first (day 21-bleed) and the second (day 35-bleed) immunization in C57BL / 6 mice. Bars indicate median values and circles represent individual animals, p-values determined by Kruskal -Wallis test with Dunn’s multiple comparisons test c, PvCelTOS-specific antibody titers after the first (day 21-bleed) and the second (day 35-bleed) immunizations in C57BL / 6 mice. Bars indicate median values and circles represent individual animals, p-values determined by Kruskal -Wallis test with Dunn’s multiple comparisons test, d, Protection level of mice after two immunizations and challenge with 500 PfCSP transgenic P. berghei parasites. The presence of infected RBCs was determined via thin blood smears prepared daily for 4-12 days post challenge. The statistical significance of differences in survival analysis d, was determined via Kaplan-Meier survival analysis. The values for the naive group shown in b, and d, are shared with the data from Fig. A-3b,c.

[0016] Figure A-6. Structure of the CSP-P1-CSP and CSP-P2-CSP complex reveals the structured Pl and P2 core with disordered CSP segments, a, Envelop generated with single particle cryogenic electron microscopy analysis with local resolution of the complex in two orientations, b, The fitted atomic model in two orientations. The Pl portion in the central core is colored dark grey, while the P2 portion surrounding the Pl core is colored light gray.Leydig 774102 NIH E-182-2024-0-PC-015

[0017] Figure A-7. Sterile protection by vaccination with a structure-based design, a, Three-dose vaccination regimen b, CSPj5c-specific IgG titers after the first (day 21 -bleed), the second (day 35-bleed) and the third (day 56-bleed) immunization for C57BL / 6 mice using CSP- P1(S293C)-CSP+CSP-P2-CSP as a vaccine candidate. Bars indicate median values and circles represent individual animals, p-values determined by Kruskal-Wallis test with Dunn’s multiple comparisons test c, Protection level of mice after three immunizations and challenge with 500 PfCSP transgenic P. berghei parasites. The presence of infected RBCs was determined via thin blood smears prepared daily for 4-12 days post challenge. The statistical significance of differences in survival analysis was determined via Kaplan-Meier survival analysis with Bonferroni correction for multiple comparisons.

[0018] Figure A-8 relates to Figure A-l and presents data concerning the engineering of P. falciparum synthase to design a stable, multivalent nanoparticle platform in the form of sizeexclusion chromatograms of protein purification for distinct PfPdxl and PfPdx2 engineered mutants. Pl is PfPdxl K83R K174C S178C and P2 is PfPdx2 H196N N65Q N128Q. Arrows indicates the protein peaks. Dashed line at -13 ml on a superose 6 (10 / 300) or -10.5 mL on a superdex 200 (10 / 300) is the expected elution volume for a double hexamer ring (12-mer) of PfPdxl or the full complex. Dashed line at 15-16 ml on a superose 6 (10 / 300) or -12.5 mL on a superdex 200 (10 / 300) is the expected elution volume for a single hexamer ring (6-mer) of PfPdxl or the full complex. Dashed line at 8- 10ml expected elution volume for aggregated protein. Dashed line at -17 ml is the expected elution volume for PfPdx2. a, Pl on a superdex 200 (10 / 300) column, b, P2 on a superdex 200 (10 / 300) column, c, CSP-P1 on a superose 6 (10 / 300) column, d, Pl-CSP on a superose 6 (10 / 300) column revealed two overlapping peaks consistent with the coexistence of both the double hexamer rings and single hexamer ring suggesting double ring formation was destabilized, e, CSP-P1-CSP on a superose 6 (10 / 300) column revealed a single peak corresponding to double hexamer rings, f, CSP-P2-CSP on a superose 6 (10 / 300) column, g, CSP-P1 in complex with CSP-P2-CSP on a superose 6 (10 / 300) column revealed most of the complex elutes at the aggregation elution volume consistent with the formation of fibrils and higher order structures, h, Pl-CSP in complex with CSP-P2-CSP on a superose 6 (10 / 300) column showing single peak with the elution volume corresponding to double hexamer ring complex. Please also see Fig. A-1B for a similar analysis of the finalLeydig 774102 NIH E-182-2024-0-PC-016 nanoparticles comprised of CSP-P1-CSP in complex with CSP-P2-CSP, PvCelTOS-P2-CSP or CSP-P2-PvCelTOS.

[0019] Figure A-9. Improved production process retains complete sterile protection, a, Reducing SDS-PAGE and b, Superose 6 10 / 300 size-exclusion chromatogram of final coexpressed tag-free CSP-P1-CSP+CSP-P2-CSP. c, Three-dose vaccination regimen, d, CSPj5c- specific IgG titers after the first (day 21), second (day 42) and the third (day 56) immunization of C57BL / 6 mice. Bars indicate median values and circles represent individual animals, e, Protection level of mice after three immunizations with 2.5 pg protein and challenge with 500 PfCSP transgenic P. berghei parasites. The presence of infected RBCs was determined via thin blood smears prepared daily for 4-12 days post challenge. The statistical significance of differences in survival analysis was determined via Kaplan-Meier survival analysis with Bonferroni correction for multiple comparisons.

[0020] Figure A-10. Evaluation of purified CSPj5c and PvCelTOS after freeze / thaw. a, Superdex 75 10 / 300 gel filtration chromatogram and b, Reducing SDS-PAGE gel of CSPj5c purified from Expi293 cell culture, c, Superdex 75 10 / 300 gel filtration chromatogram and d, Reducing SDS-PAGE gel of CSPj5c purified from E. coll, e, Superdex 200 10 / 300 gel filtration chromatogram and f, Reducing SDS-PAGE gel of PvCelTOS purified from E. coli.

[0021] Figure A-ll. CSPj5c fusion to C-terminal of Pl blocks the long fiber formation along six-fold symmetry, a, Structural model for CSPj5c fusion to N-terminal of Pl, which is located planar to the ring and the attached CSPj5c is not located near the proposed fiber interface, b, Structural model for CSPj5c fusion to C-terminal of Pl, which is located at the fiber interface and the CSPj5c is attached at this site precludes any interaction for forming fibrous structure.

[0022] Figure A-12 relates to Fig. A-6 and presents data processing and structure determination of the CSP-P1-CSP+CSP-P2-CSP complex. Shown are 2D classification of the particles that were autopicked from each micrograph with the blob picker from cryoSPARC. The twenty highest populated classes with clear features are shown.

[0023] Figure A-13 relates to Fig. A-6 and presents electron-density (drawn at 1.0 sigma) surrounding region of C293 from cryogenic electron microscopy. Density indicated that thereLeydig 774102 NIH E-182-2024-0-PC-017 was an additional density between the symmetrically related C293s in chain A and M. Cys289 was not involved in disulfide bond formation. Chain A and Chain M are shown in sticks.

[0024] Figure A-14. Top panel, Dynamic light scattering histogram demonstrating that CSP-P1-CSP+CSP-P2-CSP assembles into a uniform complex consistent with the size of a 18- 20 nm diameter Pdxl / 2 complex fused to CSP. Bottom panel, PvCelTOS-specific antibody titers after immunization (left) PvCelTOS-specific antibody titers two weeks after the first immunization (day 21-bleed) of equimolar (0.5 pg dose) PvCelTOS dimer, CSP-P1- CSP+PvCelTOS-P2-CSP nanoparticles, or CSP-Pl-CSP+CSP-P2-PvCelTOS nanoparticles in C57BL / 6 mice, (right) PvCelTOS-specific antibody titers two weeks after the second immunization (day 35-bleed) of equimolar (0.5 pg dose) PvCelTOS dimer, CSP-P1- CSP+PvCelTOS-P2-CSP nanoparticles, or CSP-Pl-CSP+CSP-P2-PvCelTOS nanoparticles in C57BL / 6 mice. All bars indicate median values and squares represent individual animals, p- values determined by Kruskal-Wallis test with Dunn’s multiple comparisons test.

[0025] Figure A-15. PfCSPj5c-specific antibody titers after immunization (left) PfCSPj5c specific titers in C57BL / 6 mice immunized with either PfCSPj5c, CSP-Pl-CSP+PvCelTOS-P2- CSP nanoparticles, or CSP-Pl-CSP+CSP-P2-PvCelTOS nanoparticles on day 21. (right) PfCSPj5c specific titers in C57BL / 6 mice immunized with either PfCSPj5c, CSP-P1- CSP+PvCelTOS-P2-CSP nanoparticles, or CSP-Pl-CSP+CSP-P2-PvCelTOS nanoparticles on day 35. All bars indicate median values and circles represent individual animals, p-values determined by Kruskal -Wallis test with Dunn’s multiple comparisons test.

[0026] Figure A-16. Data processing and structure determination of the CSP-P1- CSP+CSP-P2-CSP complex. After 2D classification, 405,784 particles were used to generate two ab initio models. The class that has a clear map of two components complex and with higher number of particles were selected for non-uniform refinement to generate a 2.95 A map.

[0027] F igure A- 17. a, C ompari son b etween the structure of C SP-P 1 -C SP+C SP-P2-C SP complex and PbPdxl-PfPdx2 complex (PDB ID: 4ADS). Top and side views of the double ring structures as well as individual P1P2 complex structures were represented in ribbons. Structures were superimposed on the right panel, b, Electron-density surrounding region of C293 from cryogenic electron microscopy. Density indicated that there was an additional density between the symmetrically related C293s in chain A and M.Leydig 774102 NIH E-182-2024-0-PC-018

[0028] Figure A-18. Top panel, Optimized production scheme for co-expressed tag-free CSP-P1-CSP+CSP-P2-CSP nanoparticle. Bottom panel, Data demonstrating that CSPJ5C display on the engineered nanoparticle achieves 100% protection from PbPfCSP.

[0029] Figure B-l. Structural overview of PfCpn60 structure with new potential N- and C- termini after circular permutations. (A) Tetradecamer structure of Cpn60. The original N- and C- termini, which orients towards to inner cavity. (B) Monomer structure of Cpn60. The original N- and C- termini were linked together by a short GS linker. The new N- and C- termini were opened at other positions to orient new N- and C- termini outwards to allow fusion of antigens. (C) Monomer structure of Cpn60CP after circular mutation through opening the new N- and C- termini at the position N388.

[0030] Figure B-2. Characterization of Cpn60CP-N388. (A) Elution profile of Cpn60CP- N388 showing majority of protein elutes at 13.81 ml, close to the position of tetradecamer assembly. (B) SDS Page to indicate it is relatively pure protein with subunit molecular weight about 60 kDa. (C) Negative stain electron microscopy to demonstrate that the protein assembles as a ring-shaped nanoparticle close to dimension of 100 A.

[0031] Figure B-3. Characterization of CSP-Cpn60CP-CSP. (A) Elution profile of CSP- Cpn60CP-CSP showing the protein elutes at 12.0 ml, close to the position of tetradecamer assembly. (B) SDS Page to indicate it is relatively pure protein with subunit molecular weight about 86 kDa. (C) BLI binding curves for CSP monoclonal antibody CIS43 to CSP-Cpn60CP- CSP (up panel) and mAb317 to CSP-Cpn60CP-CSP (lower panel), showing strong binding of these monoclonal antibodies to CSP-Cpn60CP-CSP nanoparticle.

[0032] Figure B-4. Data processing for the cryo-EM movies of CSP-Cpn60CP-CSP circular permutated nanoparticle. (Top Panel) Local resolution for CSP-Cpn60CP-CSP showing equatorial domain in highest resolution, intermediate in middle and apical domain in lowest resolution due to the different flexibility. (Bottom Panel) Local resolution for CSP-Cpn60HA- CSP showing equatorial domain in highest resolution, intermediate in middle and apical domain in lowest resolution due to the different flexibility.

[0033] Figure B-5. Electron-density from cryogenic electron microscopy. (A) Electron density for the original N- and C- terminal region. Density clearly indicated Gly359-Ser360 linked to terminal together. (B) Electron density for bound ATP molecule. (C) Electron densityLeydig 774102 NIH E-182-2024-0-PC-019 for Arg465Cys mutation region. (D) Electron density for Asp296Cys mutation region. Figures were draw using Pymol. The electron densities were shown in mesh contoured at 5.0 sigma level. The amino-acid residues and ATP molecule were drawn in sticks while the metal atoms were drawn as balls.

[0034] Figure B-6 graphically illustrates the disulfide bond cross-linked structure. Shown is the electron density for Arg465Cys mutation region between subunit A and K. The electron densities are shown in mesh contoured at 8.0 sigma level.

[0035] Figure B-7. CSP-Cpn60HA-CSP vaccine protected mice from malaria challenge. (A) P. berghei transgenic PbPfCSP sporozoite challenge experiment. C57BL / 6 mice (n = 10) were immunized subcutaneously with 2.5 ug of CSP-PfCpn60HA-CSP diluted in 100 ul PBS mixed with 100 ul AddaS03 three times at 3 -week intervals. (B) Antibody titers were measured on day 14 (2 weeks after the first immunization) and day 35 (2 weeks after the second immunization) and 56 (2 weeks after the third immunization) by ELISA for C57BL / 6. Data are shown for individual mice, average from triplicate. Statistical comparisons were made using a Kruskal- Wallis ANOVA followed by Dunn’s comparison with the control group, corrected for multiple comparisons, n = 10 animals per group. (C) Probability of protection after 500 sporozoite challenge. CSP-Cpn60HA-CSP (top trace) showed 100% protection. The middle trace represents CSP; the lower trace represents naive mice treated with PBS. Statistical analysis showed P value as 0.0023.

[0036] Figure B-8. Characterization of CSP-Cpn60CP-N388. (A) Elution profile of CSP- Cpn60CP-N388 showing majority of protein elutes at 12.3 ml, close to the position of tetradecamer assembly. (B) SDS Page to indicate it is relatively pure protein with subunit molecular weight about 74.2 kDa. (C) Cryo electron microscopy screen shot to demonstrate that the protein assembles as a ring-shaped nanoparticle.

[0037] Figure B-9. Negative stain electron microscopy to demonstrate that the protein assembles as a ring-shaped nanoparticle close to dimension of 100 A. The scale was shown as 50 nm. Left panel for CSP-Cpn60CP-CSP without the presence of ATP. Right panel for CSP- Cpn60CP-CSP in the presence of 1 mM ATP.

[0038] Figure B-10. Superimposition of using subunit A between the current structure of CSP-Cpn60CP-CSP and the structure of 7K3Z. (A) Superimposition of intra-ring subunits ofLeydig 774102 NIH E-182-2024-0-PC-0110 subunit A between the current structure and 7K3Z. (B) Superimposition of subunits in the opposite ring of subunit A between the current structure and 7K3Z indicating more than 10° rotation difference.

[0039] Figure C-l. Purification of PfClpP, PfClpPss, PfClpPss-CSP and CSP-PfClpPss- CSP. Engineering of disulfide bond mutations shifted the elution volume from 16.98 ml to 16.02 ml, implying the formation of tetradecamer form heptamer form. Fusing of CSP at N- or C- terminals further shift elution volume to higher molecular weights. Size-exclusion chromatography was run using superose 6 (10 / 300) column.

[0040] Fig. C-2. Data processing of the cryo electron microscopy movies of CSP-PfClpP- CSP. (Top Panel ) 2D classification for the particles which were autopicked from each micrography with the blob picker from cryoSPARC. The twenty highest populated classes with clear features are shown. (Bottom Panel) Local resolution of complex in two orientations.

[0041] Fig. C-3 Electron-density from cryogenic electron microscopy. (A) Electron density for the surrounding region of Cys306. Density clearly indicated the disulfide bond formation of the side chains of two symmetrically related Cys306 in chains A and M, which are in the opposite heptamer ring, respectively. Similar electron density can be observed in other six pairs of disulfide bonds between chains B and L, chains C and K, chains D and J, chains E and I, chains F and H, and chains G and N, respectively. (B) Electron density for the surrounding region of Cys337. No disulfide bond formation was observed between their symmetrically related Cys337. The electron densities were shown in mesh contoured at 4.0 sigma level. The amino-acid residues were drawn in sticks.

[0042] Figure C-4. Binding of Fab of neutralizing CSP monoclonal antibodies, CIS43 and mAb317 to CSP-PfClpPss-CSP. (A) FPLC chromatograms showing incubation of CIS43 or mAb317 with CSP-PfClpPss-CSP increases the molecular weight. (B) Negative stain electron microscopy analysis for CSP-PfClpPss-CSP+CIS43 (upper panel) and CSP-PfClpPss- CSP+mAb317 (lower panel). The small pictures are 2D classification.

[0043] Figure C-5. Vaccine induced protection against the challenge of PfCSP transgenic P. berghei parasites. (A) P. berghei transgenic PbPfCSP sporozoite challenge experiment. C57BL / 6 mice (n = 10) were immunized subcutaneously with 2.5 ug of CSP-PfClpPss-CSP diluted in 100 ul PBS mixed with 100 ul AddaS03 twice at 3-week intervals. (B) Antibody titers after the firstLeydig 774102 NIH E-182-2024-0-PC-0111 immunization (day 21) and the second immunization (day 35) measured using ELISA for C57BL / 6 and CD-lmice. Statistical comparisons were made using a Kruskal-Wallis ANOVA followed by Dunn’s comparison with the control group, corrected for multiple comparisons, n = 10 animals per group. (C) Protection level of mice challenged by 500 PfCSP transgenic P. berghei parasites for C57BL / 6 mice after two doses of immunization. The parasitemia was verified by eye for the thin blood smear prepared daily for 4-14 days after challenge. (D) P. berghei transgenic PbPfCSP sporozoite challenge experiment. C57BL / 6 mice (n = 10) were immunized subcutaneously with 2.5 ug of CSP-PfClpP(T306C)-CSP diluted in 100 ul PBS mixed with 100 ul AddaS03 twice at 3-week intervals. (E) Antibody titers after the first immunization (day 21), the second immunization (day 35) and the third immunization (day 56) measured using ELISA for C57BL / 6. (F) Protection level of mice challenged by 500 PfCSP transgenic P. berghei parasites for C57BL / 6 mice after three doses of immunization. The parasitemia was verified by eye for the thin blood smear prepared daily for 4-12 days after challenge. The top trace represents CSP-ClpP(T306C)-CSP; the middle trace represents CSP; the lower trace represents naive mice (treated with PBS).

[0044] Figure D-l presents data concerning purification and characterization of proteins nanoparticle ClpR from P. berghei. (A) FPLC chromatogram of proteins with superdex S200 (10 / 300) size column. The elution volumes were consistently with their forming tetradecamer nanoparticles. (B) Negative stain electron microscopy showing the size of nanoparticle, the rule is 50 nm long. Clearly, PbClpR assembles as double ring structure in solution corresponding to tetradecamer nanoparticle.

[0045] Figure D-2 presents data demonstrating that a PbClpR nanoparticle induced some degree of protection against the challenge of P. berghei parasites. (A) P. berghei sporozoite parasite challenging experiment, CD-I mice (n = 5) were immunized subcutaneously with 20 ug of immunogens diluted in 100 ul PBS mixed with 500 ul CFA / IFA three times at 3-week intervals. (B) Antibody titers were measured on day 14 after the first immunization, day 35 after the second immunization and day 56 after the third immunization by ELISA for CD-I mice. Data are shown for individual mice, average from triplicate. Statistical comparisons were made using a Kruskal -Wallis ANOVA followed by Dunn’s comparison with control. (C) Parasitemia verified with eye by analyzing Giemsa-stained thin blood smears after 1500 sporozoiteLeydig 774102 NIH E-182-2024-0-PC-0112 challenge. Dashed trace represents naive mice treated with PBS; solid trace represents mice treated with PbClpR

[0046] Figure D-3 presents data procession for the Cryo-movies of CSP-PfClpRss. ( Top Panel) 2D classification for the particles which were autopicked from each micrography with the blob picker from cryoSPARC. The twenty highest populated classes with clear features are shown. (Bottom Panel) Local resolution of complex in two orientations.

[0047] Figure D-4 graphically presents structural comparison of tetradecameric form of CSP-PfClpRss with CSP-PfClpPss-CSP and heptameric form of PfClpR. (A) Tetradecameric form of CSP-PfClpP-CSP showing heptameric rings interact each other using concave faces. N- terminal is oriented towards convex face (B) Tetradecameric form of CSP-PfClpRss showing heptameric rings interact each other using convex faces. N-terminal is oriented towards concave face. (C) Heptameric form of PfClpR (PDB 4HNK) in two different orientations. Conformational changes were indicated in comparison with the heptameric ring of the tetradecameric form of CSP-PfClpRss. (D) Heptameric ring structure of the tetradecameric form of CSP-PfClpRss.

[0048] Figure D-5 graphically illustrates a mechanism model for ClpR regulation. (A) Current ClpR regulation model by forming hetero-tetradecamer structure with ClpP using concave face. (B) The proposed new ClpR regulation model by forming complex with the intact tetradecamer oligomer of ClpP using convex face.

[0049] Figure D-6 presents data concerning vaccine induced protection against the challenge of PfCSP transgenic P. berghei parasites. (A) P. berghei transgenic PbPfCSP sporozoite challenge experiment. C57BL / 6 mice (n = 10) were immunized subcutaneously with 2.5 ug of CSP-PfCpn60CP-CSP diluted in 100 ul PBS mixed with 100 ul AddaS03 twice at 3 -week intervals. (B) Antibody titers after the first immunization (day 21) and the second immunization (day 35) measured using ELISA for C57BL / 6 and CD-lmice. (C) Protection level of mice challenged by 500 PfCSP transgenic P. berghei parasites for C57BL / 6 mice after two doses of immunization. The parasitemia was verified by eye for the thin blood smear prepared daily for 4- 14 days after challenge. The top trace represents mice treated with CSP-PfClpRss-CSP; the lower trace represents naive mice treated with PBS.

[0050] Figure D-7 presents data concerning the expression and purification of the designed PfCSP (A) FPLC chromatogram showing the purification of CSP using SUPERDEX 200Leydig 774102 NIH E-182-2024-0-PC-0113(10 / 300) column. The designed CSP was expressed using Expi293 mammalian cell expression system. (B) The reduced SDS-PAGE gel for purified CSP.

[0051] Figure D-8 presents size-exclusion chromatograms of protein purification. Arrows indicates the protein peaks. Black dotted line: the expected elution volume for double ring. (A) PfClpP with SUPERDEX 200 (10 / 300) column. (B) PfClpR SUPERDEX 200 (10 / 300) column. (C) CSP-PfClpRss with SUPEROSE 6 (10 / 300) column. (D) CSP-PfClpRss-CSP SUPEROSE 6 (10 / 300) column.DETAILED DESCRIPTION OF THE INVENTION

[0052] The invention provides a composition comprising a Plasmodium species multimeric protein capable of forming a nanoparticle and at least one antigen of interest, wherein the multimeric protein comprises a monomer chosen from Plasmodium species pyridoxal 5’- phosphate synthase (PLP), chaperone 60 protein (Cpn60), and caseinolytic protease (Clp).

[0053] While the monomer can be or comprise any suitable Plasmodium species PLP, Cpn60, or Clp, the monomeric unit can also be derived from such polypeptide. In this context, the monomeric unit can be “derived from” the Plasmodium species polypeptide by containing mutations that facilitate formation of the inventive nanoparticle, and / or which ablate functions unrelated to particle assembly, such as enzymatic activity. For example, one type of mutation rendering a monomeric unit “derived from” a source Plasmodium species polypeptide includes an amino acid mutation (stabilizing mutation) that stabilizes the multimeric nanoparticle of the composition, when the monomers are assembled. For example, mutations can introduce one or more cysteine residues to facilitate disulfide bonds within the multimeric structure.

[0054] In an embodiment, the monomeric unit within the inventive composition is, comprises, or is derived from a Plasmodium species PLP polypeptide, which can be, comprise, or be derived from Pdxl, Pdx2, or a combination thereof. In addition to the multivalency, ease of production (e.g., using E. coll), and improved antigenic features shared by the nanoparticles of the present invention, when such includes monomeric unit within the inventive composition is, comprises, or is derived from a Plasmodium species PLP polypeptide, such additionally presents of an advantage of limiting the propensity for undesirable autoimmunity, as the Plasmodium species PLP polypeptides have no known human orthologs.Leydig 774102 NIH E-182-2024-0-PC-0114

[0055] In the context of PLP, a stabilizing mutation can comprise one or more cysteine substitutions, which can facilitate disulfide bonds within the multimeric structure. For example, as demonstrated in the Examples herein, a stabilizing mutation suitable for Pdxl comprises K174C, S178C, or both, relative to a wild-type (WT) sequence, such as accession XP_966196 (SEQ ID NO: 1). In the context of Pdx2, a stabilizing mutation can comprise, for example, a substitution at H196N, relative to a WT sequence, such as accession XP_001347840 (SEQ ID NO:2). Such mutation can abolishes the PLP glutaminase activity to stabilize the complex formation between Pdxl and Pdx2 within the multimeric protein.

[0056] While, in embodiments in which the monomeric unit within the inventive composition is, comprises, or is derived from a Plasmodium species PLP, in addition to a stabilizing mutation, the Pdxl and Pdx2 can comprise other mutations, relative to the WT sequences, as desired. For example, when the PLP is Pdxl, such can comprise a K83R substitution mutation relative to accession XP 966196 (SEQ ID NO: 1). Such mutation can abolish enzymatic activity, which otherwise could pose a risk of potential side-effects to a subject receiving such nanoparticle as a vaccine. Similarly, when the PLP is Pdx2, such can comprise a N65Q and / or N128Q substitution mutation relative to accession XP_001347840 (SEQ ID NO:2). Such mutations can eliminate potential glycosylation sites for expression in systems that introduce glycosylation. Additionally, when the PLP comprises both Pdxl and Pdx2, such can be generated to engineer a disulfide bond between the two moi eties, such as described in the Example 1 (see also Figure A- 17).

[0057] In another embodiment, the monomeric unit within the inventive composition is, comprises, or is derived from a Cpn60 polypeptide. Chaperone protein Cpn60 is a highly immunogenic protein, which has been identified as an antigen recognized by the immune response in a wide range of bacterial infections. Plasmodium Cpn60 is similarly highly immunogenic and expression is maintained during hepatic stage development of sporozoites. Two Cpn60 genes were identified in the Plasmodium genomes. One is located on chromosome 12 with a 4-kb transcript, and its encoded protein is believed to function inside the mitochondria. The second Cpn60 gene is located on chromosome 10 and its protein functions specifically inside the apicoplast. Cpn60 orthologs have been shown to induce both CD4+ and CD8+ T-cell responses.Leydig 774102 NIH E-182-2024-0-PC-0115

[0058] Desirably, such Cpn60 polypeptide for inclusion in the inventive composition differs from the WT protein to facilitate its inclusion into the inventive nanoparticle compositions. For example, because Cpn60 transitions through multiple oligomeric states during function, this native flexibility and activity can hinder its usability as a stable nanoparticle presentation platform. Second, both N- and C- termini of WT native Cpn60 orient inward into the central cavity of the protein structure, thus preventing attachment of antigens through genetic fusion for surface display. Accordingly, for inclusion in the inventive composition, a Cpn60 polypeptide can be mutated to engineer a circular permutation through linking the original (native) N- and C- terminals together, using other positions as new N- and C-termini, which are exposed (as opposed to projecting internally) to allow the attachment of antigens. Thus, as discussed more fully in Example 2 below, the Cpn60 for inclusion within the inventive composition can comprises a short (two amino acid) GS linker between the N- and C- termini relative to the WT PfCpn60 sequence (SEQ ID NO: 11). Using this configuration, a designed exposed N-terminus can be engineered, such as, but not limited to, at residue K435 or N388. Similarly, a designed exposed C-terminus can be engineered such as, but not limited to, at residue S434 or N376. In certain embodiments, this designed C-terminus can comprise an element such as a his-tag (e.g., HHHHHH).

[0059] Additionally, in embodiments in which the monomeric unit within the inventive composition is, comprises, or is derived from a Cpn60 polypeptide, such can be mutated to lack the loop insert L378 to N387 of WT PfCpn60 sequence (SEQ ID NO: 11), as discussed in Example 2.

[0060] Stabilizing mutations suitable for Cpn60 polypeptide, to facilitate stability of the nanoparticle structure, can include, for example, either of substitutions mutation R175C, D567C, or both of them, relative to the WT PfCpn60 sequence (SEQ ID NO: 11). These cysteine substitutions can facilitate the formation of one or more disulfide bonds within the multimeric protein. Additionally or alternatively, a stabilizing mutation suitable for Cpn60 polypeptide can comprise comprises either or both of D474A, DI 18A substitutions mutations, relative to the WT PfCpn60 sequence (SEQ ID NO: 11). As discussed in Example 2, such mutations can Stabilize the ATP bound conformation of the multimeric protein comprising a Cpn60 polypeptide.Leydig 774102 NIH E-182-2024-0-PC-0116

[0061] In yet another embodiment, the monomeric unit within the inventive composition is, comprises, or is derived from a Plasmodium species ATP-dependent caseinolytic Clp protease. Protease is a large family of proteins playing vital roles in protein quality control by removing misfolded, damaged, and regulatory proteins. In order to prevent proteases from digesting the normal proteins, these proteins often form a compartment structure by assembling many subunits together into nanoparticle-size of proteolytic component with the active sites inside the internal chamber protected from the cellular environment. Since malaria parasite infects both mammalian and mosquito hosts and involved in several different parasite forms during its complex life cycle, an efficient system to maintain protein quality is essential for parasite to survive. The ClpP protease is a highly conserved serine protease found in bacteria and most eukaryotes. In Plasmodium, two ClpP homolog genes have been identified, one active version (termed ClpP) and one inactive version (termed ClpR). Both ClpP and ClpR form homo-heptameric rings and two heptameric rings may interact each other further to form homo-tetradecam er ClpP-ClpP or ClpR-ClpR, or hetero-tetradecameric, ClpP-ClpR, nanoparticles. The crystal structure ofPfClpP has demonstrated that it self-assembles into a l00 A * 100 A >< 100 A nanoparticle with 14 subunits

[0062] As discussed in Example 3, the full PfClpP gene encodes 370 amino acid residues, however, the mature form ofPfClpP has molecular weight of approximately 25 kDa. Since the C-terminal amino acid residues are in intact, the mature PfClpP must be N-terminal truncated form. Thus, as noted in Example 3, the Clp polypeptide can be truncated, for example, but not limited to, the 179 amino acid version (ClpR) disclosed in Example 3, or such can be the longer, 370 amino acid form (ClpP).

[0063] In embodiments in which the Clp is ClpP, the ClpP can comprise a mutation that eliminates its protease activity, so as to avoid potentially adverse side-effects when used within a vaccine. For example, one such mutation includes a S264G substitution mutation relative to the amino acid sequence of accession XP_001351149.1 (SEQ ID NO:21). Also, when the monomeric unit within the inventive composition is, comprises, or is derived from a Plasmodium species Clp, a stabilizing mutation can, as with other mutations of other monomers discussed herein, facilitate the formation of one or more disulfide bonds within the multimeric protein. ForLeydig 774102 NIH E-182-2024-0-PC-0117 example, ClpP can suitably be mutated to comprise a cysteine substitution at T306 (T306C) relative to the WT Clp sequence XP_001351149.1 (SEQ ID NO:21)

[0064] Within the inventive composition, the PLP, Cpn60, or Clp is derived from a species of Plasmodium. The non-limiting exemplary sequences discussed in the Examples herein have been derived from P. falciparum (Pf), however, the PLP, Cpn60, or Clp proteins of other species of Plasmodium can similarly be adapted for use in a composition according to the present invention. Thus, the monomeric unit within the inventive composition can be, comprise, or be derived from common malaria pathogens, such as Pf, P. vivax, or indeed other species of Plasmodium, which are known to those of ordinary skill in the art.

[0065] The inventive composition also comprises one or more (z.e., at least one) antigen of interest. As discussed below, the “antigen of interest” can be any polypeptide moiety able to elicit an immunogenic response within a patient. Such antigen of interest can be all or a portion of a Plasmodium sp. protein or an antigenic polypeptide from a non- lasmodium organism, such as a bacterium, virus, parasite, or other organism.

[0066] To facilitate such, the multimeric protein comprises a mutation to facilitate display of multiple copies of the at least one antigen of interest. For instance, as noted above, when the monomeric unit within the composition is, comprises, or is derived from Cpn60, such can be designed with engineered N- and / or or C-termini to be exposed, as opposed to orientated inward (“buried”), which facilitates N- and / or C-terminal fusion with or attachment of desired antigens.

[0067] Desirably, the inventive nanoparticle (e.g., within the composition) displays multiple copies of the at least one antigen of interest. For example, the PLP synthase-based nanoparticles discussed herein can display up to 4 distinct (i.e., unique) antigens, for a total of 48 copies.Similarly, the Clp-based nanoparticles discussed herein can display up to 2 distinct (i.e., unique) antigens, for a total of 28 copies, as can the Cpn60-based nanoparticles discussed herein.Similarly, the ClpR-based nanoparticles discussed herein can display up to 2 distinct (i.e., unique) antigens, for a total of 14 copies. In embodiments, the inventive nanoparticles can achieve protection against antigens (such as both CSP and CelTOS) by displaying copies of each on the same nanoparticle. For example, two (or more) copies of CSPJ5C displayed on Pdxl / 2, CPN60, or ClpP nanoparticles have achieved protection against CSP. Data have demonstrated that CelTOS can replace one copy of CSP on such nanoparticle, especially Pdxl / 2. Accordingly,Leydig 774102 NIH E-182-2024-0-PC-0118 the invention includes an embodiment in which Pdxl / 2 nanoparticles display 1, 2, or 3 copies of CelTOS.

[0068] The antigens for use in the inventive composition can be, comprise, or be derived from any suitable pathogen or other organism against which it is desired to immunize a patient. In the context of malaria, the antigen can, thus, be, comprise, or be derived from a species of Plasmodium (e.g., P. falciparum, P. vivax, etc.). However, antigens to non-Plasmodium organisms also can be employed and are within the scope of the present invention. Also, it will be understood that the inventive nanoparticle, having up to 48 valencies for antigens (14, 28, or 48 valencies) can therefore display multiple (up to 48) antigens. Within such nanoparticles, the antigens can be the same or different. Thus, in certain embodiments, the nanoparticles within the inventive composition can comprise up to 4 separate antigen species (e.g., a first, second, third, fourth antigen), with each being the same or different (unique). Indeed, in certain embodiments, the inventive reagent can include antigens from different organisms. Thus, for example, a first antigen of interest can be derived from a Plasmodium species and a second antigen of interest within the same nanoparticle can be derived from a non- Plasmodium pathogen.

[0069] While any suitable antigen can be employed in the context of the invention, in the Examples below, the P. falciparum circumsporozoite protein (CSP) (SEQ ID NO:3) and the P. vivax cell-traversal protein for ookinetes and sporozoites (CelTOS) are demonstrated to result in sterile immunity from Plasmodium . For example, as noted in Example 1, CSPj5c the sequence consists of the junction region, NPDPNANPNVDPNA (SEQ ID NO:33), five NPNA repeats and the C-terminal region that includes the TSR domains (residues 310-383, XP 001351122).CSPj5c was fused to both ends of N- or C-terminus of Pl and P2, respectively, to create CSP-P1- CSP and CSP-P2-CSP constructs. The sequence of PvCelTOS consists of residues 35-195 (XP_001617263) was fused to either A- or C-terminus of P2 with CSPj5c at the other terminus to create PvCelTOS-P2-CSP and CSP-P2-PvCelTOS. While such antigens can be employed in the context of the present invention, variations are possible. For example, with respect to CSPj5c, an antigenic polypeptide segment (such as comprising between 14 and 50, or between 20 and 40, or between 25 and 35 amino acids) within residues 80-130 (which encompasses the junction epitope) can be employed in some contexts. Moreover, while the particular antigen discussed in the Examples comprises five NPNA repeats, between three and nine NPNA repeats can suitablyLeydig 774102 NIH E-182-2024-0-PC-01 be employed in embodiments of the invention. Also, embodiments wherein the antigen comprises a polypeptide segment (such as comprising between 14 and 50, or between 20 and 40, or between 25 and 35 amino acids) within residues 270-397 of XP_001351122, which will include C-domains, similarly are contemplated.

[0070] Other suitable antigens include, but are not limited to, Plasmodium sp. (e.g., P. falciparum or P. vivax) CelTOS, reticulocyte-binding protein homolog 5 (RH5), cysteine-rich protective antigen (CyRPA), apical membrane antigen-1 (AMA1), merozoite surface protein 1 (MSP1), Gametocyte surface protein P230 (Pfs230), Pfs48 / 45, 25 kDa ookinete surface antigen (Pfs25), or polypeptides derived from such antigens. For example, an antigen of interest can have or comprise at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least 99% identity to a wild-type Plasmodium sp. Additionally, the nanoparticle platforms described herein can be used to present any desired antigen, including non- Plasm odium antigens, such as those known or suspected of eliciting an immune response to other parasites, bacteria, viruses (e.g., HIV, SARS-CoV-1, SARS-CoV-2, influenza, RSV, poliovirus, and others), cancers, and the like.

[0071] It will be observed that the monomeric units assemble to form a multimeric nanoparticle. For example, such nanoparticle can take the form of a two-component dodecamer (e.g., P1 / P2), which can comprise from 1-4 unique antigens for a total of 48 individual antigen copies (i.e., from 1-48 antigens). Alternatively, such nanoparticle can take the form of a single component tetradecamer (e.g., when based on ClpP or Cpn60), having 1 or 2 unique antigens, for a total of 28 individual antigen copies (i.e., from 1-28 antigens). In yet another configuration (e.g., when based on ClpR), such nanoparticle can take the form of a single component heptamer, having 1 or 2 unique antigens, for a total of 14 individual antigen copies (i.e., from 1-48 antigens).

[0072] While not intending to be bound to specific type specimens, some exemplary multimeric proteins according to the present invention are disclosed herein as CSP-P1-CSP (SEQ ID NO:4), CSP-P1(S293C)-CSP (SEQ ID NO:5), CSP-P2-CSP (SEQ ID NO:6), PvCelTOS-P2- CSP (SEQ ID NO:7), CSP-P2-PvCelTOS (SEQ ID NO:8), Tag-free CSP-P1(S S293C)-CSP (SEQ ID NOV), Tag-free CSP-P2-CSP (SEQ ID NO: 10), PfCpn60CP-K435 (SEQ ID NO: 13), PfCpn60CP-N388 (SEQ ID NO: 14), CSP-PfCpn60CP-N388 (SEQ ID NO: 15), CSP-Leydig 774102 NIH E-182-2024-0-PC-0120PfCpn60CP-CSP (SEQ ID NO: 16), CSP-PfCpn60HA -CSP (SEQ ID NO: 17), PfCpn60HA (SEQ ID NO: 18), CSP-PfClpPss-CSP (SEQ ID NO:22), and CSP-PfClpP(T244C)-CSP (SEQ ID NO:23), PfClpP(T244C) (SEQ ID NO:24), PbClpR (SEQ ID NO:28), CSP-PfClpRss (SEQ ID NO:29), PfClpRss-CSP (SEQ ID NO:30), CSP-PfClpRss-CSP (SEQ ID NO:31), and CSP- PfClpR-CSP (SEQ ID NO: 32).

[0073] Also, it will be observed that in the tables of sequences below, certain sequences comprise an N-terminal segment from cloning (the start Met and cloning scars), and a C-terminal his tag with linker and cloning scars. These portions need not be present in a polypeptide when such is employed in the context of the present invention.

[0074] The inventive composition can be formulated using standard formulation technology for administration to subjects. Thus, the composition can comprise, in addition to the multimeric protein capable of forming a nanoparticle and at least one antigen of interest, the composition can include a pharmaceutically acceptable carrier. Such carriers are known to those of ordinary skill, and, depending on formulation, the composition can be suitable for administration by any desired route ( .g, orally, by intraperitoneal injection, etc.).

[0075] In certain embodiments, it can be advantageous to formulate the nanoparticle with one or more adjuvants. Any adjuvant known to persons of ordinary skill in the art to enhance immune response of vaccines or promote stability can be suitably employed. Non-limiting examples of such adjuvants, which can be included in the inventive composition, include AddaS03™, MatrixM, oligomerization domain of C4-binding protein (C4bp), aluminum hydroxide, aluminum phosphate, aluminum sulfate, monophosphoryl lipid A (MPL), QS-21, TQL1O55, QS-18, QS-17, QS-7, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IF A), oil in water emulsions, CpG, polyglutamic acid, polylysine, AddaVax™, MF59®, and / or combinations thereof, among others known to persons of ordinary skill in the art.

[0076] In another aspect, the invention provides a nucleic acid encoding the multimeric protein and the at least one antigen of interest as described herein. The exact sequence of such nucleic acid can vary, based on the degeneracy of the genetic code. However, given the sequences of proteins and polypeptides set forth herein, persons of ordinary skill in the art can deduce a DNA or RNA sequence encoding the inventive multimeric protein capable of forming a nanoparticle and at least one antigen of interest.Leydig 774102 NIH E-182-2024-0-PC-0121

[0077] Such nucleic acid can be engineered into a suitable expression vector, as desired (e.g., a plasmid or viral vector, such as are known to persons of ordinary skill in the art. Such vectors can then introduce the nucleic acid into cells to produce the inventive multimeric protein capable of forming a nanoparticle and at least one antigen of interest. While any suitable producing cell line can be employed (e.g., HEK, Chinese Hamster Ovary cells, and the like), one advantage attendant to the inventive nanoparticles is that they can be readily manufactured using prokaryotic expression systems. Thus, in an embodiment, the invention provides a prokaryotic organism (e.g., E. coll), which comprises the inventive nucleic acid, and which desirably expresses the nucleic acid to produce the multimeric protein and the at least one antigen of interest.

[0078] Indeed, using the inventive nucleic acid and prokaryotic organism (e.g., E. coll), the invention provides, as a further aspect, a method for producing the inventive multimeric protein and the at least one antigen of interest. In accordance with the method, the inventive nucleic acid is introduced into a prokaryotic organism (e.g., E. coll) cell or population thereof. Thereafter, the cell or population thereof is cultured under conditions suitable to express the nucleic acid therein, so as to produce the inventive multimeric protein and the at least one antigen of interest encoded by the nucleic acid. Such culturing can be achieved using standard methods known to those of ordinary skill in the art to be suitable for protein production.

[0079] Further in accordance with the inventive method, once the cells (e.g., E. coll) have produced the inventive multimeric protein and the at least one antigen of interest, such can thereafter be isolated from the culture and purified, as desired. Methods of isolating proteins from eukaryotic and prokaryotic cells are known to those of ordinary skill in the art and can be employed in the context of the inventive method to produce the inventive multimeric protein and the at least one antigen of interest. Indeed, Example 1 herein demonstrates the production and purification of Pdxl / 2 using a system in which the proteins are expressed with a tag (histidine), which facilitates purification. Such method involves separately expressing the tagged Pdxl and Pdx2 components, mixing them, and then repurifying the complexes using metal -affinity chromatography. The Example also describes an improved production and purification protocol, eliminating the need for the tag and reducing the steps involved. In accordance with this embodiment, untagged Pdxl and Pdx2 (which are included within the scope of the presentLeydig 774102 NIH E-182-2024-0-PC-0122 invention) are co-expressed using a plasmid (or other vector) engineered to express both untagged Pdxl and Pdx2.

[0080] As noted, the inventive composition comprising the inventive multimeric protein and the at least one antigen of interest is suitable for use as a vaccine. Accordingly, the invention provides the use of the inventive to immunize a subject against Plasmodium species infection, / .e., a method of immunizing a subject in need thereof against Plasmodium species infection. The use / method comprises administering at least one dose of the inventive composition as described herein to a subject. In accordance with the inventive method, the composition can be administered to the subject using any mode of delivery suitable for administering vaccines, and can, therefore, be via oral, injection, or other suitable routes of administration.

[0081] While, in performing the inventive method of immunizing a subject, the composition can be administered once, in some applications, it can be desirable to administer the composition multiple times (e.g., 2, 3, 4, 5, 6, 7, etc.) times to the patient, such as to prime the immune system and boost response. Such doses can be administered, for example, at regular intervals, such as weekly, monthly, quarterly, etc., as is common in administering vaccines.

[0082] To elicit an immune response against the antigen(s) present in the inventive composition, the composition is administered at a suitable dosage. The exact dosage will be determined by a treating physician or veterinarian, or via laboratory personnel. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration. In the Examples below, mice were dosed with 2.5 pg of vaccine. Those of ordinary skill in the art can extrapolate from this to determine a suitable dose for a human patient, taking into consideration factors such as mentioned above.

[0083] The inventive method for immunizing a subject can be employed in any desired animal capable of mounting an immune response against antigens (e.g., antigens derived from Plasmodium sp. or other antigens of interest) and typically is a mammal. Such can be, for example, livestock (e.g., cattle, swine, goats, sheep, horses, and the like), pets (e.g., cats and dogs), laboratory subjects (e.g., mice, rats, etc.), non-human primates (e.g., monkeys, babboons, and apes), and of course, human beings (Homo sapiens).Leydig 774102 NIH E-182-2024-0-PC-0123

[0084] In another aspect, the inventive composition can be used to detect the presence of pathogen-specific antibodies (e. , Plasmodium species) in a sample. The method entails labeling the multimeric nanoparticle of the present invention with a detectable label, mixing the sample with the labeled nanoparticle, and detecting the label. As is known to those of ordinary skill in the art from other immunoassays, the presence of the label indicates the presence of pathogen (e.g., Plasmodium') species-specific antibodies in the sample.

[0085] In the context of the inventive method for detecting the presence of pathogen- e.g., Plasmodium)-specific antibodies, the label can be any type of label typically employed in diagnostic assays, such as a fluorescent moiety (e.g., Green Fluorescent Peptide (GFP), a radiolabeled moiety, an enzyme (e.g., luciferase, alkaline phosphatase, etc.). Such label can be fused, conjugated, or affixed to the inventive nanoparticle using conventional technology.

[0086] The sample to be assayed in accordance with the inventive method for detecting the presence of pathogen- (e.g., Plasmodium)-specd c antibodies can be any substance in which it is desired to discover the presence of antibodies. Thus, the sample can be a tissue sample or a fluid sample drawn from a subject (e.g., a human or other animal subject, as noted above), such as blood, plasma, saliva, urine, tears, etc.EXAMPLES

[0087] The following examples further illustrates the invention but, of course, should not be construed as in any way limiting its scope. In brief, the experiments discussed in the Examples demonstrates the engineering and successful production and use of compositions comprising nanoparticles comprising Plasmodium species pyridoxal 5 ’-phosphate synthase (PLP), chaperone 60 protein (Cpn60), and caseinolytic protease (Clp) and exemplary, proof-of-principal antigens (P. falciparum circumsporozoite protein (CSP) and P. vivax cell -traversal protein for ookinetes and sporozoites (CelTOS)). These nanoparticle platforms each effected sterile protection of test animals from malarial challenge. The nanoparticle platforms can be conveniently produced in E. coli, and they can present multiple antigens to generate a robust immune response against pathogens. The data reveal that such platforms can achieve superior protection against malaria than current vaccines, such as R21 and RTS,S / AS01.Leydig 774102 NIH E-182-2024-0-PC-0124EXAMPLE 1

[0088] This example demonstrates the successful engineering of a potent multivalent malaria vaccine using an engineered P. falciparum pyridoxal 5 ’-phosphate (PLP) synthase as a nanoparticle that presents a designed P. falciparum circumsporozoite protein (CSP) and the P. vivax cell-traversal protein for ookinetes and sporozoites (CelTOS).Materials and MethodsProtein design, plasmid construction, protein expression, and purification

[0089] The protein sequences used are summarized in Table Al . The full sequence of P. falciparum (isolate 3d7) Pdxl (PfPdxl: XP 966196 (SEQ ID NO: 1)) and Pdx2 (PfPdx2: XP 001347840 (SEQ ID NO:2)) genes of PLP were retrieved from NCBI protein database. K174C and S178C mutations were introduced into PfPdxl to create potential disulfide bonds to cross-link the inter-hexamer rings to increase the stability of the double-ring structure. K83R mutant was introduced into the active site of PfPdxl to abolish enzymatic activity. For PfPdx2 protein design, H196N was made to abolish the glutaminase activity to stabilize the complex formation between Pdxl and Pdx2. In addition, N65Q and N128Q mutations were introduced to eliminate the potential glycosylation sites for expression in systems that introduce glycosylation. For simplicity we refer to the engineered PfPdxl component as Pl (PfPdxl K83R K174C S178C), and the engineered PfPdx2 component as P2 (PfPdx2 H196N N65Q N128Q). Note, K83R, K174C and S178C of PfPdxl correspond to K198R, K289C and S293C of the CSP-P1- CSP sequence (SEQ ID NO:4) respectively, and H196N, N65Q, and N128Q of the PfPdx2 sequence correspond to H310N, N179Q, N242Q of the CSP-P2-CSP sequence (SEQ ID NO:6) respectively.

[0090] CSPj5c sequence consists of the junction region, NPDPNANPNVDPNA (SEQ ID NO:33), five NPNA repeats and C-terminal TSR domain (residues 310-383, XP_001351122). CSPj5c was fused to both ends of N- or C-terminus of Pl and P2, respectively, to create CSP-P1- CSP and CSP-P2-CSP constructs. PvCelTOS sequence consists of residues 35-195 (XP_001617263) was fused to either N- or C-terminus of P2 with CSPj5c at the other terminus to create PvCelTOS-P2-CSP and CSP-P2-PvCelTOS. The DNA sequences of these constructs were synthesized by GenScript and cloned into a pET28a vector with kanamycin antibiotic resistanceLeydig 774102 NIH E-182-2024-0-PC-0125 gene. All proteins were expressed in Rosetta (DE3) with six histidine residues at C-terminal end to facilitate purification. The plasmid was transformed into E. coli Rosetta (DE3) cells and expressed in IL of LB media. After centrifugation at 5,000 g for 10 min, the cell pellets were resuspended in 50 ml of lysis buffer containing 100 mM (hfflThSCL, 10 mM MgCh, 10 mM KC1, 20 mM Tris pH 8.0 and 10% (w / v) glycerol with a tablet of protease inhibitor (Roche) and 35 ml of 0.1 mM PMSF. After incubating with 250 ml of 25 mg / ml lysozyme on the ice for 5 min, cells were lysed by sonication. The lysate was centrifuged at 50,000 x g for 10 min, and the supernatant was applied to a 3 ml of Ni resin and washed with 20 ml of binding buffer containing 20 mM NaPCL pH 7.4 and 500 mM NaCl and 20 ml of washing buffer containing 20 mM imidazole, 20 mM NaPCL pH 7.4 and 500 mM NaCl. Proteins were eluted by 15 ml of eluting buffer containing 500 mM imidazole, 20 mM NaPCL pH 7.4 and 500 mM NaCl. The eluted proteins were further purified by a Superdex 200 or a Superose 6 gel filtration columns in the PBS buffer. The proteins were aliquoted and stored in -80 °C freezer.Optimized CSP-P1-CSP+CSP-P2-CSP co-expression and tag-free purification

[0091] The N65Q and N128Q mutations in P2 were reverted to the native Asn because aberrant N-linked glycosylation is not a concern during E. coli expression. Genes encoding CSP-P1(S178C)-CSP and CSP-P2-CSP were codon optimized for expression in E. coli, synthesized, and cloned into the pRSFDuet-1 plasmid by Genscript. Plasmid was transformed into BLR(DE3) E. coli and a cell bank was generated and stored at -80 °C. For expression, the cell bank was used to inoculate a 10 mL culture of LB + 50 pg / mL kanamycin and grown overnight at 37 °C. The overnight culture was diluted into 1 L LB + 50 pg / mL kanamycin and grown to an ODeoo of 0.6. Temperature was decreased to 20 °C and expression was induced with 1 mM IPTG overnight. Cells were harvested by centrifugation and resuspended in 3 mL lysis buffer (50 mM Na-Tris pH 8.0, 100 mM NaCl) per gram of wet cell paste. A protease inhibitor tablet (Roche) and 0.25 mg / mL lysozyme were added before sonication. Lysate was centrifuged 30 minutes at 21,000 xg to remove cell debris and supernatant was isolated. Ammonium sulfate was added to the supernatant to reach 20% saturation and incubated at 4 °C for 1 hour. Insoluble material was removed by centrifugation for 15 minutes at 12,000 xg and ammonium sulfate was added to the supernatant to reach 40% saturation. After incubation at 4 °C for 1 hour, insoluble material containing the P1 / P2 nanoparticle was isolated by centrifugation for 15 minutes atLeydig 774102 NIH E-182-2024-0-PC-012612,000 xg. The pellet was resuspended in 10 mL 50 mM Na-Tris pH 8.0, 50 mM NaCl and loaded onto a capto HiRes Q column (Cytiva). Flow through was collected and loaded onto a Superose 6 column (Cytiva) equilibrated in lx PBS. Fractions corresponding to assembled nanoparticle were pooled and endotoxin was removed using a Proteus MIDI Endotoxin removal spin column (Bio-Rad). Protein was sterile filtered, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C before analysis or immunization. The final product had very little nucleic acid contamination (Abs26o / 28o < 0.7) and very low endotoxin levels (< 1.5 EU / kg in C57BL / 6 mice immunized with 2.5 pg protein).CSPj5c production

[0092] The CSPj5c gene sequence, encoding P. falciparum 3d7 isolate CSP junctional region (NPDPNANPNVDPNA), 5 NPNA repeats and the C-terminal TSR domain (amino acids 310- 383) (Table Al), was optimized to human codon, synthesized, and cloned into pHLsec plasmids (GenScript). The plasmids were transformed into XL- 10 A. coli cells, harvested, and purified using Qiagen maxipreparation kit following the manufacturer’s instructions. The plasmids were transfected into Expi293F cells for expression according to manufacturer instructions. Cell-free supernatant was harvested by centrifugation at 3,000 x g. The protein was purified by Ni-affinity column, followed by Superdex 200 gel filtration column as other proteins with his-tag. The designed minimal CSPj5c can be easily expressed in Expi293 cells at high yields (93 mg / L culture), confirming its potential for use as a stable antigen (Figure A-10 a and b).

[0093] For A. coli production, the CSPj5c gene was codon optimized for expression in E. coli and cloned into a modified pET28a vector (GenScript). BLR(DE3) cells were transformed, grown to an ODeoo of 0.6 and induced with 1 mM IPTG. Expression continued overnight at 20 °C before cells were harvested by centrifugation and resuspended in 3 mL lysis buffer (50 mM Na-Tris pH 8.0, 100 mM NaCl) per gram of wet cell paste. Protease inhibitor tablet (Roche) and 0.25 mg / mL lysozyme were added before sonication on ice. Supernatant was clarified by centrifugation at 50,000 xg for 10 minutes and purified by Ni-affinity chromatography. Eluant was concentrated and further purified on a Superdex 75 column (Cytiva) equilibrated in lx PBS. Fractions were pooled and endotoxin was removed using a Proteus MIDI Endotoxin removal spin column (Bio-Rad). Protein was sterile filtered, aliquoted, snap frozen in liquid nitrogen, andLeydig 774102 NIH E-182-2024-0-PC-0127 stored at -80 °C before analysis or immunization. The final product had endotoxin levels < 1.5 EU / kg in C57BL / 6 mice immunized with 2.5 pg protein).PvCelTOS production

[0094] PvCelTOS was expressed in E. coli and purified as previously described. Briefly, PvCelTOS-6xHis in the pET28a vector was transformed into BL21(DE3) E. coli and grown at 37°C until an O.D.600 of 0.6 at which point expression was induced by IPTG and cultures were harvested 3 hours post induction and frozen at -80°C. Thawed cell pellets were lysed using sonication and the lysate centrifuged. The PvCelTOS-6xHis was then purified from the supernatant using Nickel HTC Agarose resin (Goldbio, St. Louis, MO). The resin was equlibrated with 50 mM Tris, pH 8, 0.1 M NaCl and 10 mM imidazole. The PvCelTOS-6xHis then incubated with the resin. The resin was then washed with 50 mM Tris, pH 8, 0.1 M NaCl and 10 mM imidazole. Finally, the PvCelTOS-6xHis was eluted with 30 mM Tris, pH 8, 0.1 M NaCl supplemented with 500 mM imidazole. The eluted protein was then concentrated and further purified by size exclusion chromatography using an Superdex 200 increase column (Cytiva, Marlborough, MA) and PBS buffer.Antibody production

[0095] The fragment sequences of monoclonal antibodies, mAb317 and CIS43 were obtained from literatures and synthesized with a human IgG Fc sequence and cloned into pHLsec plasmids (GenScript). Plasmids with human codon optimized full-length IgG gene were transformed into XL-10 E. coli cells. Plasmids were harvested and purified using Qiagen maxipreparation kit following the manufacturer’s instructions. Equal mole amount of heavy chain and light chain plasmids were transfected into Expi 293F cells according to manufacturer instructions. Cell-free supernatant was harvested by centrifugation and batched incubated with protein A agarose resin (GoldBio) for 1 hour at room temperature. After washing the collected resin with 10 column volumes (CV) protein A IgG binding buffer, antibodies were eluted with 10 CV IgG elution buffer and neutralized with 1 CV 1 M Tris buffer pH 9.0. Antibodies were concentrated using an Amicon centrifugal filter and further purified by a Superdex 200 increase 10 / 300 size exclusion column using PBS pH 7.4 buffer. The Fab fragments of mAb317 andLeydig 774102 NIH E-182-2024-0-PC-0128CIS43 were expressed using Expi293F cells like IgG antibodies but purified using His-tag affinity column follow by Superdex 200 size-exclusion column.Dynamic Light Scattering

[0096] Purified CSP-P1-CSP+CSP-P2-CSP complex stored at -80 °C was thawed, passed through a 0.2 pm filter, and diluted to 2 mg / mL with lx PBS. Triplicate readings were collected at 25 °C on a Zetasizer Nano-S (Malvern Panalytical) and average values are reported with standard deviation.Negative stain electron microscopy

[0097] The sample quality and structural features were assessed by negative stain transmission electron microscopy in a FEI TT20 microscopy equipped with a TVIPS 16 megapixel charge-coupled device (CCE) camera. The protein samples (100 ng / ml) were applied to freshly plasma cleaned carbon-coated grids (Quantifoil), followed by negative staining with 2% uranyl acetate. Datasets of more than 100,000 particles were collected. The 2D class average were generated in RELION.Cryo-EM grid preparation and data collection

[0098] Before grid preparation for cryo-EM, the protein sample was centrifuged at 13,000 g for 2 min to remove any protein aggregates. A 3.5 ml of protein sample at the concentration of 0.5 mg / ml, which was measured with a Nanodrop spectrometer (Thermo Fisher Scientific), was applied to a glow charged Quantifoil 300 mesh 1.2 / 1.3 carbon grid that had been glow discharged for 90s at 10 mA with PELCO easiGlow Discharge Set. The samples were blotted for 3s with a blot force of 3 using 55 / 20 mm filtered paper (Ted Pella) before being plunged into liquid ethane with a Vitrobot Mark VI (FEI) set at 16°C and 100% humidity. The data were collected on the 300-keV Titan Krios with Gatan BioQuantum Image Filter in the National Institutes of Health (NIH) National Cancer Institute (NCI) / NIH IRP Cryo-EM Facility (NICE) facility. The images were recorded with a 20-eV slit post-GIF K2 Summit camera in superresolution counting mode at a nominal magnification of 81,000 and a defocus range from -0.7 to -2.0 pm. Exposures of 4 s were dose fractionated into 50 frames, with an exposure rate of 15Leydig 774102 NIH E-182-2024-0-PC-0129 electrons pixel1s ', resulting in a total exposure of 53.8 electrons A2. The data collection was automated using the SerialEM software package.Image processing

[0099] We collected 3,242 dose-fractionated videos of CSP-P1-CSP and CSP-P2-CSP complex. The processing was done within cryoSPARC (v4.1.1). Motion correction was done by cryoSP ARC’S Patch motion correction with an output F-crop factor of one-half. CTF estimation for each micrograph was calculated with Patch CTF estimation. Particles were autopicked from each micrograph with the blob picker from cryoSPARC and then sorted by two-dimensional (2D) classification to exclude bad particles; 516,166 particles were selected. These 2D templates were then used for further template particle picking resulting in 2,242,732 starting particles. Subsequent rounds of 2D classification resulted in 405,784 particles. The particles were used to generate two an ab initio classes in cryoSPARC. One superior class had a total of 251,050 particles and a clear map of the two components complex and these particles were selected to conduct NU-refinement to generate a 2.95 A map.Model building and refinement

[0100] To build the complex, two models PDB:4ADT and PDB:2ABW were used to docked into the EM map using UCSF chimera for CSP-P1-CSP and CSP-P2-CSP, respectively. De novo model building was carried out manually in COOT and real-space refinement was performed using PHENIX. The statistics of model refinement are shown in Table A3.Bio-layer interferometry

[0101] The binding of IgG of mAb317 and CIS43 to CSPj5c-fused nanoparticles was measured using bio-layer interferometry (Octet Red; Pall ForteBio). CSPj5c-fused nanoparticles were loaded onto IgG biosensors (Pall ForteBio, cat No 18-5019) at 10 pg / ml in kinetics buffer (TBS + 0.002% Tween20 and 0.01% BSA). The loaded sensors were dipped into solutions containing mAb317 or CIS43 IgG in kinetics buffer at a concentration of 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0015625 and 0.00078125nM. The binding experiments were performed in the following steps: 1) baseline for 60 s; 2) loading of CSPj5c-fused nanoparticle for 60 s; 3)baseline for 60 s; 4) association of antibody for 60 s; and 5) disassociation of antibody into kinetics buffer for 300 s. A reference well with no CSPj5c-fused nanoparticle loaded onto the sensor was run in all experiments and subtracted from sample wells, The assay were performedLeydig 774102 NIH E-182-2024-0-PC-0130 at 22 °C and data was analyzed using Octet Red Data Analysis software version fitting with a 2:1 binding model.Animal immunization and challenge studies

[0102] All mouse studies were reviewed and approved under protocol LMIV IE by the Institutional Animal Care and Use Committee (IACUC) at the National Institutes of Health (approval code: LMIV IE and approval date: 10 / 1 / 2024) and performed in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited facility (AAALAC file #000777, last accredited in 2021). The PHS Animal Welfare Assurance (File Number # DI 6-00602) was last approved 05 / 30 / 2023. Studies were designed to reduce animal numbers where possible, and no more than momentary pain or distress was anticipated. All housing, husbandry practices and pain management were in accordance with AAALAC guidelines, standards, and regulations.Mouse Immunizations

[0103] Sterile filtered antigens and nanoparticle samples were formulated with equal volume of the sterile AddS03 mixed by pipetting up and down 10 times prior to immunization. C57BL / 6 mice were immunized with two doses of 0.5 pg PvCelTOS-6xHis or two doses of 2.5 pg of the vaccines in lOOpL administered 3 weeks apart via subcutaneously injection and the sera was collected from each mouse on day 35 of the study. C57BL / 6 mice were immunized with two or three 2.5 pg doses in lOOpL of the tested vaccines with each dose administered 3 weeks apart via subcutaneously injection. Sera was collected from each mouse 21 days after the first immunization and 14 days after the second vaccination for the two-dose regimen (Figure A-3a). For three-dose regimen (Figure A-7a), C57BL / 6 mice were immunized three doses with 2.5 pg of the nanoparticles or proteins formulated in AddaS03 with each dose administered 3 weeks apart via subcutaneously injection. Sera were collected 21 days after the first immunization, 14 days after the second immunization and the third immunization (day 21, day 35 and day 56, respectively) to quantify CSPj5c-specific antibody titers via quantitative ELISA. All groups immunized contained 10 mice each.Challenge studies

[0104] Transgenic Pb ANKA line 2257, PfCS(r)PbCS GFP::LucPbeefla sporozoites expressing P. falciparum (PI) CSP were prepared through cyclic transmission in BALB / c miceLeydig 774102 NIH E-182-2024-0-PC-0131 and Anopheles stephensi mosquitoes at the Laboratory of Malaria Immunology and Vaccinology (LMIV) Insectary, National Institutes of Health (NIH) in Bethesda, MD, USA. The growth cycle began by inoculating mice with blood-stage Pb PfCSP 2257 parasites, which were then used to feed female Anopheles stephensi mosquitoes. Mosquito salivary gland sporozoites (SPZ) were isolated and harvested 18-27 days post blood meal, as previously described — . Briefly, infected mosquitoes were dissected to remove their salivary glands, which were then placed in 0.3 ml of 0.22pm filter (Merck Millipore, #SLGPM33RS) -sterilized medium E199 (Quality Biologicals, #112-022-101) containing 0.2% bovine serum albumin (MP Biomedicals, #160069) in 1.5 ml Protein LoBind® Tubes (Eppendorf, #022431081). The salivary glands were triturated 20 times using a 1.0 ml syringe (BD, #309701) and a 26G needle (BD, #305110) to release the SPZ. The SPZ were counted using a disposable hemocytometer (INCYTO, #DHCN015), and their count per ml was determined. All study and control mice were intradermally (ID) challenged at the base of the tail at two sites with live SPZ using a 29G 1.0 ml insulin syringe (Exelint, #26028), administering 500 SPZs in lOOpL of E199 containing 0.2% BSA (50 pL per site) to ensure reliable infection of all mice. Control mice, immunized with lx PBS (pH 7.4) / adjuvant only and challenged with live Pb PfCSP 2257 SPZ, were used as comparisons.

[0105] Breakthrough to blood-stage infection was assessed by Giemsa-stained thin blood smears from day 4 to day 12 or 14 post-challenge. A negative smear by the end of this period was considered indicative of sterile protection. Mice were considered positive if infected red blood cells (RBCs) were detected in more than one out of twenty random fields of view per blood smear. Mice with three consecutive days of positive parasitemia were euthanized, and a final blood smear was collected. Mice that remained negative for blood-stage infection were euthanized at the end of the study, either on day 12 or 14 post-challenge. Kaplan-Meier curvesLeydig 774102 NIH E-182-2024-0-PC-0132 were used to illustrate the time to developing parasitemia during days 4-12 or 14 post-challenge with live Pb PfCSP 2257 SPZ.Determination of antibody titers in response to vaccination

[0106] Bleeds were performed after 3 weeks of the first dose and two weeks after the second dose. The serum was used to determine the CSPj5c-specific antibody titers. Immune responses to antigens and nanoparticles were assessed by ELISA.

[0107] For samples from C57 / BL6 mice: Nunc MaxiSorp plates (ThermoFisher Scientific) were coated with 100 pl 0.01 mg / ml purified CSPj5c and PvCelTOS diluted in 50 mM Na- carbonate buffer pH 9.5. Plates were stored at 4 °C overnight. The plates were washed three times with PBST, then were blocked with 2% BSA in PBST for 1 hour at room temperature. After washing three times, each well of plates was added with 100 pl of diluted serum in 2% BSA in PBST and incubated at room temperature for 1 hour. Plates were washed three times with PBST, 200 pl of 1 : 10,000 diluted peroxidase-conjugated anti-mouse IgG with 2% BSA in PBST was added (Jackson ImmunoResearch Laboratories, Inc ). Plates were incubated at room temperature on a shaker for 30 minutes. After washing three times with PBST, 70 pl of tetramethylbenzidine (TMB) was added and plates with aluminum foil covered were incubated at room temperature for 20 minutes. Finally, 70 pl of 0.16 M H2SO4 was added to each well to stop reaction.

[0108] For the comparison of PvCelTOS equimolar immunizations: Nunc MaxiSorp plates (ThermoFisher Scientific) were coated with 100 pl 0.02 mg / ml purified PvCelTOS diluted in Na-carbonate buffer pH 9.5. Plates were stored at 4 °C overnight. The plates were washed three times with PBST, then were blocked with 2% BSA in PBST for 1 hour at room temperature. After washing three times, each well of plates was added with 100 pl of diluted serum in 2% BSA in PBST and incubated at room temperature for 1 hour. Plates were washed three times with PBST, 100 pl of 1 :5,000 diluted peroxidase-conjugated anti-mouse IgG with 2% BSA in PBST was added (Jackson ImmunoResearch Laboratories, Inc.). Plates were incubated at room temperature for 30 minutes. After washing three times with PBST, 100 pl of tetramethylbenzidine (TMB) was added and plates with aluminum foil covered were incubated atLeydig 774102 NIH E-182-2024-0-PC-0133 room temperature for 20 minutes. Finally, 100 pl of 2 M H2SO4 was added to each well to stop reaction.

[0109] The Absorbance at 450 nm was measured for all ELISA plates using a Biotek Synergy Hl plate reader. Absorbance values for each individual animal were measured in triplicate experimental replicates and the average is reported.Quantification and statistical analysis

[0110] All statistical tests are indicated in Figure legends. These include Kruskal-Wallis ANOVA followed by Dunn’s comparison, corrected for multiple comparisons or a two-tailed Mann-Whitney U test when comparing two groups. P-values <0.05 were considered significant. Statistically significant differences in survival analysis were calculated by the Kaplan-Meier method. All statistical tests were performed in Prism 9 (GraphPad Software, La Jolla, CA, USA).Experimental model and subject detailsMammalian cell lines

[0111] Expi293F cells were used for protein expression (Thermo Fisher Scientific RRID:CVCL_D615). This is a transformed cell line derived from human female kidney cells. Authentication was not performed after purchase. Cells were cultured at 37 °C, >80% humidity, 8% CO2, suspended in Expi293 Expression Medium by shaking at 125 rpm (Thermo Fisher Scientific cat#A1435103).Monse studies

[0112] Mouse studies were performed in an AAALAC-accredited facility under the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) at the National Institutes of Health. Five or Ten naive 5-6 week old female C57BL / 6 (Charles River Laboratories) per group were immunized with 2.5 pg antigen each. Mice were randomly assigned to each group. Experimental units comprised single animals and groups of 5 or ten mice receiving the same immunization were co-housed. Mice were maintained in a specific pathogen-Leydig 774102 NIH E-182-2024-0-PC-0134 free vivarium, housed in a climate-controlled room on a 12-hour day / night cycle, and had access to food and water ad libitum.Table AlBiological Sequences relevant to Example 1Leydig 774102 NIH E-182-2024-0-PC-01Leydig 774102 NIH E-182-2024-0-PC-01Table A2CSPj5c median titers and PvCelTOS median titers (related to Figs. A-3, A- 5 and A-7)PvCelTOS medianCSPj5c median titers titersLeydig 774102 NIH E-182-2024-0-PC-01Table A3Cryo-EM data collection and refinementData collection and processingMagnification 81,000Voltage(kV) 300Electron exposure(e- / A2) 53.8Defocus range (pm) -0.7 to -2.0Pixel size (A) 0.528 (1.056 binned)Symmetry imposed D6Initial particle images (no.) 2,242,732Final particle images (no.) 251,050Map resolution (A) at FSC threshold of 0.143 2.95RefinementInitial model used (PDB code) 4ADT, 2ABWAverage B factor (A2) 61.01Model compositionNonhydrogen atoms 40,677Protein residues 5,287R.M.S. deviationBond length (A) 0.004Bond angles (°) 0.496ValidationMolProbity score 1.61Clashscore 5.69Poor rotamers (%) 0.89Ramachandran plotFavored (%) 95.66Allowed (%) 4.34Disallowed (%) 0ResultsIdentification of native nanoparticles from Plasmodium falciparum

[0113] We identified potential self-assembling nanoparticles that lacked orthologs in humans by searching the malaria parasite genomes and protein data bank (PDB) for high-order oligomers. This search identified pyridoxal 5 ’-phosphate (PLP) synthase (PDB ID: 4ADS) as a suitable candidate for further development as an antigen presentation platform because PLP synthase is essential for parasite survival but is absent in mammalian and human genomes. PLP consists of two components, Pdxl and Pdx2. The Pdxl-Pdx2 complex structures wereLeydig 774102 NIH E-182-2024-0-PC-0138 determined from representative bacteria, Geobacillus kaustophilus, Thermotoga maritima and Bacillus subtilis. The mixed species complex structure of P. berghei (Pb) Pdxl and P. falciparum (Pf) Pdx2 has also been reported (PDB ID: 4ADS).Molecular engineering of PLP synthase

[0114] An effective nanoparticle display platform for vaccine development should have high antigen valency and optimal particle size and stability. Three issues exist with using PLP synthase as an antigen display platform. First, the complex between Pdxl and Pdx2 is unstable as the attachment of wild-type PfPdx2 is random and not cooperative. Similar phenomena have been reported for PLP synthases from other organisms. Second, PfPdxl forms a stable dodecamer core but the addition of wild-type PfPdx2 to PfPdxl can result in long fibers that subsequently aggregate and precipitate. This precludes stable homogenous nanoparticle development and has hindered the determination of a PfPdxl-PfPdx2 complex structure. Third, PfPdxl and PfPdx2 can potentially dissociate during catalytic and mechanistic cycles resulting in complex heterogeneity.

[0115] We performed molecular engineering of P. falciparum PLP synthase to resolve these issues (Figure A-l). First, we mutated the active sites of both PfPdxl and PfPdx2 as catalytic inactivity will stabilize the complex. The active site residue Lys83 of PfPdxl was mutated to Arg and this residue is consistent with ArglOl at the equivalent position of the orthologous Arabidopsis thaliana pseudoenzyme Pdxl.2. Separately, the H199N mutant of P. berghei Pdx2 forms a fully occupied and stable PLP synthase complex with PbPdxl, and the equivalent mutation, H196N, was introduced into PfPdx2. Second, we found that antigen attachment to the C-terminus of PfPdxl prevents fiber formation upon complex formation (Figure A-8h). Third, the structure (PDB ID: 4ADS) of the PbPdxl -PfPdx2 complex included residues at the ring-ring interface that could be used to stabilize the complex. We designed a pair of potential disulfide bonds by introducing K174C and S178C (K289C and S293C in the CSP-P1-CSP numbering) to cross-link the two opposite hexamer rings and stabilize the double-ring structure. Finally, we eliminated potential glycosylation sites by introducing the N65Q and N128Q mutations into PfPdx2 to promote potential eukaryotic cell expression. These mutations were included to allow for future development of expression systems that introduce aberrant glycosylation and that may be needed for complex disulfide-bridged antigens even though all the designed constructsLeydig 774102 NIH E-182-2024-0-PC-0139 evaluated here could be readily produced in the cost-effective Escherichia coh expression system. This structure-based engineering process resulted in very stable homogenous particles with the ability to display up to 48 antigens with four distinct antigen attachment sites (Figure A- 8, Figure A-la). For simplicity, we refer to the engineered PfPdxl component as Pl and the engineered PfPdx2 component as P2.CSP antigen design

[0116] We sought to evaluate the effectiveness of the engineered PLP synthase nanoparticles by displaying two high-priority malaria vaccine antigens. The first antigen evaluated was circumsporozoite protein (CSP). P. falciparum CSP is a 397-amino acid sporozoite surface protein that plays a critical role in parasite migration from the skin to the liver and during hepatocyte infection. The CSP protein consists of a conserved A-terminal region with a charged protease-cleavage site known as region I, a central diverse repeat region of 25-49 NANP repeats, and a conserved C-terminal region comprising a short, conserved region III and a thrombospondin-like type 1 repeat (TSR) domain (Figure A-la). The central NANP repeats are a target for protective antibodies and the structurally conserved C-terminal TSR domain contains CD4+and CD8+T-cell epitopes with proposed roles in protection from disease. The most advanced malaria vaccines, RTS,S / AS01 and R21 / MM, contain 19 NANP repeats and the C- terminal region. The junctional region between the A-terminal domain and the NANP repeats has also been identified as a target for potent infection-blocking antibodies. An examination of the CSP NANP repeats indicated that five repeats of NANP is the optimal number of repeats for determining the antibody titer and potency.

[0117] We designed a minimal CSP antigen based on the available protection data, which included the junction region (NPDPNANPNVDPNA), five NPNA repeats and the C-terminal domain from residue 310 to residue 383 (CSPj5c) (Figure A-la, Figure A-lOa-d). CSPj5c contains B-cell epitopes for the three most potent mAbs: CIS43, which preferentially recognizes the junctional epitope; mAb317, which recognizes the three consecutive NANP type I turns in an extensive interaction; and L9, which preferentially binds NVDP minor repeats. CSPj5c also retains the T-cell epitopes located in the C-terminal domains.Leydig 774102 NIH E-182-2024-0-PC-0140Engineered nanoparticles displaying CSPj5c are stable and highly expressed

[0118] The A-termini and C-termini of Pl and P2 are all surface accessible resulting in four distinct attachment sites for target antigens. A bacterial expression system was evaluated because a high yield from a cost-effective bacterial expression system has the potential to enable low-cost vaccine development. The designed CSPj5c attached to the N- and / or C-termini of either Pl or P2 were successfully expressed and purified to homogeneity by expression in Escherichia coli (Figure A-lb and c, Figure A-8a-f). The A-terminal CSPj5c fusion protein of Pl (CSP-P1) aggregated when complexed with P2 (Figure A-8g) , as observed in other studies due to the formation of long tubular fiber structures of various lengths. This is likely because the N- terminus of Pl is located planar to the ring and because the attached CSPj5c is not located near the proposed fiber interface (Figure A-l la). In contrast and consistent with the structural analysis, CSPj5c attached to the C-terminus of Pl (Pl-CSP) created stable P1-P2 complexes, which could be generated as long as Pl contained a C-terminal minimal CSPj5c (Figure A-lb, Figure A-8h, Figure A-l lb).

[0119] We focused on nanoparticles of a complex of CSP-P1-CSP and CSP-P2-CSP for further analysis as these two proteins form a stable complex with high CSP valency. Size exclusion chromatography of the complex showed that the elution volume shifted, indicating an increase in molecular weight, upon incubation of both components (Figure A-lb). SDS-PAGE analysis indicated the presence of both CSP-P1-CSP and CSP-P2-CSP in the complex (Figure A- 1c). Dynamic light scattering (DLS) indicated a highly monodisperse sample (PDI = 0.055 ± 0.015) with a diameter of 23.76 ± 0.52 nm (Figure A-14 (top panel)). These data suggest the uniform formation of an 18-20 nm P1 / P2 24mer complex with CSP displayed on the exterior. Finally, we examined the nanoparticles by negative stain electron microscopy (EM) which revealed a stable dodecamer core composed of CSP-P1-CSP surrounded by CSP-P2-CSP consistent with 24 heteromers (yida infra) of the P1-P2 complex (Figure A-ld).Neutralizing epitopes on the engineered nanoparticle vaccine are accessible

[0120] Having established the stable engineering of multivalent nanoparticles, we assessed whether the epitopes in the nanoparticles are accessible to neutralizing antibodies. The Fab fragments of the CSP junctional region monoclonal antibody CIS43 and the CSP major repeat monoclonal antibody mAb317 were incubated with the CSP-P1-CSP+CSP-P2-CSP. SubsequentLeydig 774102 NIH E-182-2024-0-PC-0141 size exclusion chromatography showed a shift in elution volume (13.1 to 12.9 ml for CIS43 fab and 13.1 to 12.5 ml for 317 fab) suggesting complex formation that was further evaluated by direct biophysical interaction studies (Figure A-lb and Figure A-2a). Biolayer interferometry (BLI) binding experiments were performed to measure the kinetics of binding of CIS43 and mAb317 IgG to the particles. Both the CIS43 and mAb317 IgGs strongly bound to the CSP-P1- CSP+CSP-P2-CSP nanoparticle with extremely high apparent binding rates (CIS43 ka = 7.19 ± 0.01 x 108M^s’1; mAb317 ka= 6.35 ± 0.01 x 108M'1s'1) and negligible apparent dissociation rates (CIS43 kdis ~2 x 10'7s'1; mAb317 kdiS=~3 x 10'7s'1;) that were so slow they could not be reliably measured (Figure A-2b). The apparent association rates are consistent with the theoretical maximum of ~106if they are corrected for the number of epitopes per CSP molecule (~5-7) and CSP molecules per nanoparticle. The extremely low dissociation rates are approximately two orders of magnitude slower than previously reported for monomeric CSP suggesting that the high local concentration of epitopes on the nanoparticle prevented mAb dissociation upon binding. These results indicate that the epitopes were correctly exposed and presented and that the antibody bound the nanoparticles with extremely high apparent affinity.Vaccination with engineered nanoparticles elicits high titers of specific antibodies and provides sterile protection against malaria parasite challenge

[0121] C57BL / 6 mice were immunized twice with 2.5 pg of the designed nanoparticles formulated in AddaS03 to evaluate immunogenicity (Figure A-3a). Serum CSPj5c-specific IgG titers were measured 21 days after the first immunization and 14 days after the second immunization (at 21 and 35 days, respectively) by quantitative ELISA. The antigen group elicited higher CSPj5c-specific IgG titers after the second immunization than in the naive control groups (Figure A-3b and Table A2). We further evaluated whether vaccination with the designed nanoparticles could confer sterile protection against malaria upon challenge of the C57BL / 6 mice with PfCSP transgenic P. berghei sporozoites. C57BL / 6 mice immunized with two doses of the nanoparticles formulated in AddaS03 exhibited significant sterile protection (80% sterile protection, p value = 0.0022) compared to control mice upon intradermal challenge with 500 PfCSP transgenic P. berghei sporozoites (Figure A-3c).Leydig 774102 NIH E-182-2024-0-PC-0142Engineered multivalent nanoparticles displaying CSP and CelTOS

[0122] We evaluated whether PLP could display multiple antigens. Recent studies have shown that antibodies to either PfCelTOS or PvCelTOS can provide cross-species sterile protection upon passive transfer in a mouse challenge model to both antigens, suggesting inclusion of either PfCelTOS or PvCelTOS would improve vaccine efficacy and allow for crossspecies protection. Cross-species protection could be especially valuable in regions were both P. falciparum and P. vivax are coendemic. Fusion of an oligomeric antigen to an oligomeric nanoparticle carrier can lead to aberrant higher order structures and aggregation due to incompatible symmetries. The dimeric nature of CelTOS can hamper its addition to existing multimeric nanoparticles. Consistent with this, fusion of P. vivax (Pv) CelTOS to Pl affected the correct assembly of Pl resulting in aggregation. A major advantage of using PLP as a display platform is that P2 is a monomer allowing for the attachment of multimeric antigens. The attachment of CelTOS to either the A-terminus (PvCelTOS-P2-CSP) or the C-terminus of P2 (CSP-P2-PvCelTOS) resulted in the stable expression and formation of 24mer complexes with CSP-P1-CSP (Figure A-4a,b). Negative stain EM revealed correct nanoparticle assemblies (Figure A-4c).Vaccination with multiantigen nanoparticles elicits high titers of CSP- and PvCelTOS- specific antibodies and confers sterile protection against malaria parasite challenge

[0123] C57BL / 6 mice were vaccinated with two 2.5 pg doses of CSP-Pl-CSP+PvCelTOS-P2-CSP or CSP-Pl-CSP+CSP-P2-PvCelTOS nanoparticles containing two antigens (Figure A- 5a). The CSP- and PvCelTOS-specific IgG titers were quantified using ELISA for sera collected 21 days after the first immunization and 14 days after the second immunization (day 21 and day 35, respectively). Both nanoparticle types resulted in significantly greater IgG titers against CSPj5c and PvCelTOS in C57BL / 6 mice, demonstrating the correct folding, presentation, and efficient display of both antigens (Figure A-5b,c). The CSPj5c- and PvCelTOS-specific IgG titers in the CSP-Pl-CSP+CSP-P2-PvCelTOS group were 1.7 and 2.8 fold greater for CSPj5c and 3 and 4 fold greater for PvCelTOS than those in the CSP-Pl-CSP+PvCelTOS-P2-CSP group on days 21 and 35 respectively, although the differences were not statistically significant (Figure A-5b,c and Table A2).Leydig 774102 NIH E-182-2024-0-PC-0143

[0124] C57BL / 6 mice were subsequently challenged intradermally with 500 PfCSP transgenic P. berghei sporozoites to evaluate sterile protection in vivo. Consistent with the higher titers, the CSP-Pl-CSP+CSP-P2-PvCelTOS (90% sterile protection p value =0.0005) and CSP- Pl-CSP+PvCelTOS-P2-CSP (70% sterile protection p value = 0.0253) groups provided better protection against infection than the naive control group. The naive control group for this study is shared with the CSP-P1-CSP + CSP-P2-CSP study (Figure A-3c). These results demonstrated that the addition of CelTOS did not hamper the protective effect of the CSPj5c presented by the nanoparticles and that potent protective responses were elicited by the nanoparticles containing both antigens. The greater protection afforded by CSP-Pl-CSP+CSP-P2-PvCelTOS (90%) than by CSP-Pl-CSP+PvCelTOS-P2-CSP (70%) suggested that the orientation of antigens attached to nanoparticles may affect the immune response.

[0125] Nanoparticle display has the potential to improve the immune response to the displayed antigen. The designed nanoparticles each contain one copy of PvCelTOS per repetitive unit for a total of 12 PvCelTOS antigens per particle. In order to evaluate if this ordered display improved the immune response we compared CelTOS IgG titers elicited by the nanoparticles to the titers elicited by an equimolar dose of PvCelTOS (Figure A-14 (bottom panel)).Approximately 20% of the mass of the CSP-Pl-CSP+CSP-P2-PvCelTOS and CSP-P1- CSP+PvCelTOS-P2-CSP nanoparticles is comprised of PvCelTOS leading to an equimolar dose of 0.5 pg for PvCelTOS alone. Immunization with 0.5 pg of PvCelTOS had median PvCelTOS- specific IgG titers of 3.8 for day 21 and 4,350 for day 35. In contrast, an equimolar dose of the CSP-Pl-CSP+PvCelTOS-P2-CSP or CSP-Pl-CSP+CSP-P2-PvCelTOS nanoparticles had median PvCelTOS specific IgG titers of 5,842 and 6,583 antibody units, respectively, for day 21 and 38,750 and 118,183, respectively, for day 35 and the IgG titers for both nanoparticles were significantly better on both day 21 (p value 0.0015 and <0.0001) and day 35 (p value 0.0275 and <0.001) when compared to equimolar PvCelTOS (Figure A-14 (bottom panel)).

[0126] CSPj 5c-specific IgG titers were also determined and compared between CSPj 5c (yida infra), CSP-Pl-CSP+PvCelTOS-P2-CSP, and CSP-Pl-CSP+CSP-P2-PvCelTOS (Figure A-15). CSP-Pl-CSP+PvCelTOS-P2-CSP had median CSPj5c titers of 107,507 on day 21 and 343,000 on day 35 which was 59,726-fold and 52,908-fold higher titers than CSPj 5c immunized mice on days 21 and 35 respectively. CSP-Pl-CSP+CSP-P2-PvCelTOS had median CSPj5c IgG titers ofLeydig 774102 NIH E-182-2024-0-PC-0144183,707 on day 21 and 1,636,833 on day 35 which was 101,105-fold and 252,481 -fold higher IgG titers than CSPj5c immunized mice on days 21 and 35 respectively. All of these increases in CSPj5c IgG titers were statistically significant (Figure A-15). These results indicate the P1-P2 nanoparticle system improves the immune response to both CSP and PvCelTOS.Cryogenic EM structures of the PLP synthase complex

[0127] The structure of the P. falciparum PLP synthase complex has not been characterized, as fiber formation prevents the formation of homogeneous PfPdxl-PfPdx2 complexes. A complete structure of the PLP synthase complex would aid in vaccine design as described here and also provide the complete structure of a druggable complex from the deadliest malaria parasite. The high homogeneity and particle stability of the nanoparticle vaccines CSP-P1-CSP and CSP-P2-CSP prompted their high-resolution structural determination by cryogenic EM (Table A3, Figure A-6, Figure A-16).

[0128] The CSP-P1-CSP and CSP-P2-CSP complex was determined to 2.95 A resolution, with the core CSP-P1-CSP at 2.5 A and the external CSP-P2-CSP at 4.0 A resolution (Figure A- 6a and Figure A-16). No additional density was observed for the CSPj5c antigens consistent with the flexibility provided by the linkers between the antigen and the nanoparticle. Pl assembles into a 12-mer cylindrical structure of two hexamer rings, and twelve P2 subunits associate around the Pl core (Figure A-6b). The overall architecture is highly similar to thePbPdxl :PfPdx2 complex (PDB ID: 4ADS) with respect to the individual Pl and P2 domains as well as the overall 24-mer (Figure A- 17a). The structure demonstrated the successful engineering of a 24-mer nanoparticle consisting of two components with D6 symmetry. The extent of assembly is difficult to quantify but appears to be high based on the size and monodispersity measured by DLS as well as the size shift observed by size-exclusion chromatography (SEC) upon addition of P2 to Pl (Figure A-lb and Figure A-14 (top panel)). One of the two ab initio models generated during cryo-EM data processing may indicate the presence of a single hexameric CSP-P1-CSP ring, but the majority of particles are classified as the two-ring 12-mer model, and the extremely low poly dispersity seen by DLS suggests a largely uniform 12-mer in solution (Figure A-14 (top panel) and Figure A-16). In the cryo-EM structure, CSP-P2-CSP is lower resolution than CSP-P1-CSP, which could be due incomplete association with the CSP-P1- CSP ring or flexibility when attached. The difference in SEC elution volume between CSP-P1-Leydig 774102 NIH E-182-2024-0-PC-0145CSP with and without CSP-P2-CSP suggests that numerous CSP-P2-CSP molecules associate with the Pl core, but the precise number is uncertain (Figure A-lb). Ultimately, we have shown that large monodisperse nanoparticles are present in solution displaying up to 48 copies of CSP. Fusion of CSP to neither the N nor the C termini of the two components affected the assembly of the 24-mer nanoparticles, establishing a platform capable of displaying multiple antigens with high valency.

[0129] The structure revealed additional density between symmetrically related residues near the C293 residue (Figure A- 17b), which implies potential disulfide bond formation across two opposite hexamer rings. In contrast, disulfide bonds are unlikely to exist between symmetrically related C289 residues or between C293 and a symmetrically related C289 in the opposite hexamer ring, as the structure indicates that these residue pairings are too distant and that no density was observed. Therefore, it is unlikely that the K289C mutation contributed to the stabilization of the two hexamers into dodecamers.Structure-based redesign and optimized vaccination regimen resulting in complete sterile protection

[0130] Ciyo-EM structure determination of the CSP-P1-CSP+CSP-P2-CSP nanoparticle established that only C293 likely formed a disulfide bond across hexamer rings and that the second introduced cysteine C289 plays no role in stabilization. We therefore created a single disulfide bond mutant nanoparticle that contained only C293 termed CSP-P1(S293C)-CSP. Given the strong (80%) but incomplete sterile protection observed after treatment with two doses of nanoparticles (Figure A-3) we opted to evaluate a three-dose regimen with the updated nanoparticle design (Figure A-7a). Monomeric CSPj5c was expressed and purified from mammalian cells and included as a positive control to evaluate whether nanoparticle display enhances sterile protection. C57BL / 6 mice were immunized three times with 2.5 pg of the nanoparticles or proteins formulated in AddaS03 (Figure A-7a). Sera were obtained to evaluate immunogenicity after each dose, and the mice were challenged intradermally with 500 PfCSP transgenic P. berghei sporozoites to evaluate protection. Sera were collected 21 days after the first immunization, 14 days after the second immunization and 14 days after the third immunization (day 21, day 35 and day 56, respectively) to quantify CSPj5c-specific IgG titers via quantitative ELISA (Figure A- 7b). On day 56, the CSP nanoparticle group exhibitedLeydig 774102 NIH E-182-2024-0-PC-0146 significantly greater CSPj5c-specific IgG titers than did the CSPj5c monomer or naive groups (Figure A-7b). The median CSPj5c titers for the nanoparticle group were 4,830-fold greater than the CSPj5c monomer which is consistent with the enhanced immunogenicity conferred by nanoparticle display (Figure A- 7b and Table A2). In addition, we analyzed the IgG titers longitudinally for both the CSPj5c and the CSP-P1-CSP+CSP-P2-CSP nanoparticle on day 35 and 56. The titers for CSPj5c and the CSP-P1-CSP+CSP-P2-CSP nanoparticle were significantly increased on day 56 compared to day 35 with p values of 0.0115 and 0.0001, respectively, as determined by a Mann-Whitney U-test. Consistent with the CSPj5c-specific IgG titers, the sterile protection of the mice immunized with the CSP nanoparticles was significantly greater (100% protection, p value=0.0004) than that of the no treatment group and was significantly greater than that of the CSPj5c monomer group (50% sterile protection, p value=0.0126) (Figure A-7c).Complete sterile protection is retained with an improved production process

[0131] The CSP-P1-CSP+CSP-P2-CSP nanoparticle achieves 100% protection with 3 vaccinations, but there are several elements of the production process that required optimization for future manufacturing and clinical use. First, we greatly simplified the purification process by co-expressing CSP-P1-CSP and CSP-P2-CSP in BLR(DE3) E. coli from a single pRSF-Duet plasmid. CSP-P1-CSP+CSP-P2-CSP assembles during expression / purification, enabling a single purification scheme for the complete P1 / P2 complex rather than individually purifying each component, assembling the complex, and repurifying the assembled complex (Figure A- 18 (top panel)). We also removed the histidine tags initially used to purify CSP-P1-CSP and CSP-P2- CSP because of safety concerns for clinical use, and we developed a purification scheme to isolate highly pure tag-free CSP-P1-CSP+CSP-P2-CSP nanoparticle (see Figures A-9a (SDS- PAGE analysis) and A-9b (SEC analysis of purity).

[0132] Mice immunized three times with 2.5 pg doses of tag-free co-expressed CSP-P1- CSP+CSP-P2-CSP had median IgG titers almost identical to the original His-tagged CSP-P1- CSP+CSP-P2-CSP product (921,167 vs. 870,500 antibody units). Furthermore, all animals were completely protected from challenge, demonstrating that the improved product retains potent protection (Figure A-9e). We also immunized mice with CSPj5c monomer expressed in E. coli to serve as a control for the nanoparticles expressed in E. coli (Figure A9e and A- 10c). The miceLeydig 774102 NIH E-182-2024-0-PC-0147 immunized with monomeric CSPj5c were not protected at all, demonstrating a significant (p < 0.0001) and dramatic enhancement conferred by P1 / P2 nanoparticle display of CSPj5c. Additional data from the mouse challenge study (Figure A- 18 (bottom panel)) leads to the following conclusions: Firstly, that the Pdxl / 2 nanoparticle produced using the improved production process achieves 100% protection. Secondly, that reverting the engineered S178C mutation to the native serine amino acid (S178S) decreases protection, providing evidence that the engineered changes indeed have a biological effect. Thirdly, that the ferritin nanoparticle is inferior to Pdxl / 2 whether it is produced in bacterial or mammalian expression systems.Fourthly, that monomeric CSP5JC does not confer protection.

[0133] In conclusion, the engineered vaccines disclosed in this Example elicited high titers of anti-CSP and anti-CelTOS antibodies and provided sterile protection against malaria. PLP synthase has no identifiable human ortholog limiting its propensity for autoimmunity or preexisting immunity, and the engineered nanoparticles possess desirable manufacturing characteristics. These studies established an effective nanoparticle platform for malaria and infectious disease vaccines.EXAMPLE 2

[0134] This example demonstrates the successful engineering of potent multivalent malaria vaccines using engineered P. falciparum Cpn60 (PfCpn60).

[0135] Consistent with structures of Cpn60 in other organisms, PfCpn60 is a barrel -shaped tetradecamer consisting of two stacked heptameric rings with a total molecular weight of 882 KDa. PfCpn60 forms a complex with P. falciparum CpnlO (PfCpnlO), an 81kDa heptameric ring shaped protein that caps PfCpn60 in vivo. Together, these proteins work to assist in protein folding. Although Cpn60 has potential as a nanoparticle display platform, it suffers from several hurdles that must be overcome. First, Cpn60 transitions through multiple oligomeric states during function and this protein flexibility and activity hinders its usability as a stable nanoparticle presentation platform. Second, because both N- and C- termini of Cpn60 orient inward into the central cavity and prevent attachment of antigens through genetic fusion for surface display (Fig. B-l). To overcome these hurdles, stability enhancing mutations in PfCpn60 were identified and PfCpn60 with was engineered with a circular permutation to link the original N- and C- terminals together and to use other positions as new Af- and C-terminals that areLeydig 774102 NIH E-182-2024-0-PC-0148 exposed to allow the attachment of antigens. This circular permutated PfCpn60 (PfCpn60CP) nanoparticle platform can display as many as 28 subunits of a designed version of CSP in two different orientations. The fusion antigen CSP was shown to be accessible and strongly bound to the CSP monoclonal antibodies CIS43 and mAb317. The CSP fusion nanoparticle, in the final designed disulfide-bond linked stable tetradecamer form, CSP-PfCpn60HA-CSP, when formulated with AddaS03 adjuvant, induced a strong CSP-specific antibody response as measured by an enzyme-linked immunosorbent assay (ELISA). The adjuvanted CSP- PfCpn60HA-CSP conferred sterile protection in mice when challenged with transgenic P. berghei sporozoites demonstrating its viability as a potent malaria vaccine.Materials and MethodsPreparation of Cpn60CP and other proteins with his-tag.

[0136] The Cpn60CP-K435 and Cpn60CP-N388 were constructed using K435 and N388 as new A-terminus, respectively, while S434 and N376 with HHHHHH his-tag as new C-terminus by linking original N- and C- termini together with a two-amino acid peptide, GS, linker (Fig.B- 1). In Cpn60CP-N388 construct, the extra loop insert (L378 to N387) of PfCpn60 in comparison to the sequences of homolog Cpn60 in other organisms was removed. The residue R465 (equivalent to R175 in the wild-type PfCpn60) and the residue D296 (equivalent to D567 in the wild-type PfCpn60) were mutated to cysteines for potential disulfide bond formation between two opposite heptameric rings. In addition, two ATP binding site residues, D203 and D408 (the equivalent residues D474 and DI 18 in the wild-type PfCpn60), were mutated to Ala to stabilize the ATP bound conformation.

[0137] The DNA sequence was codon optimized for E. coli, synthesized, and cloned into pET28AKS expression vector (GenScript). The vector was transformed into E. coli Rosetta (DE3) cells and expressed in IL of LB media. After centrifugation at 5,000g for 10 min, cell pellets were resuspended in 35 ml of PBS buffer with a table of protease inhibitor (Roche) and 35 ul of 0.1 mM PMSF. After incubating with 250 ul of 25 mg / ml lysozyme for 5 min, cells were lysed by sonication. The lysate was centrifuged at 50,000 g for 10 min, and the supernatant was applied to a 3 ml of Ni resin and washed with 20 ml of binding buffer containing 20 mM NaPO4 pH 7.4 and 500 mM NaCl and 20 ml of washing buffer containing 20 mM imidazole, 20 mMLeydig 774102 NIH E-182-2024-0-PC-0149NaP04 pH 7.4 and 500 mM NaCl. Cpn60CP-N388 was eluted by 15 ml of eluting buffer containing 500 mM imidazole, 20 mM NaPCh pH 7.4 and 500 mM NaCl. The eluted Cpn60CP- K435 and Cpn60CP-N388 was further purified by a Superose 6 gel filtration column using the PBS buffer. All other proteins with CSP fusion were expressed and purified using a similar method.

[0138] Antibody production. The production of CSP monoclonal antibodies, mAbs311 and CIS43 have been described previously. In brief, the Fv sequences of mABS311 and CIS43 were obtained from literature and their corresponding DNA sequences were synthesized with a human IgG Fc sequence and cloned into pHLsec plasmids (GenScript). Plasmids with human codon optimized full-length IgG gene were transformed into XL- 10 E. coll cells. Plasmids were harvested and purified using Qiagen maxipreparation kit following the manufacturer’s instructions. Equal mole amount of heavy chain and light chain plasmids were transfected into Expi 293F cells according to manufacturer instructions. Cell -free supernatant was harvested by centrifugation and batched incubated with protein A agarose resin (GoldBio) for 1 hour at room temperature. After washing the collected resin with 10 column volumes (CV) protein A IgG binding buffer, antibodies were eluted with 10 CV IgG elution buffer and neutralized with 1 CV 1 M Tris buffer pH 9.0. Antibodies were concentrated using an Amicon centrifugal filter and further purified by a superdex 200 increase 10 / 300 size exclusion column using PBS pH 7.4 buffer.

[0139] CSP production. The preparation and purification of a designed version of CSP was as is described in Example 1.

[0140] Negative stain electron microscopy. The sample quality and structural features were assessed by negative stain transmission electron microscopy in a FEI TT20 microscopy equipped with a TVIPS 16 mega-pixel charge-coupled device (CCE) camera. The protein samples (0.01 mg / ml) were applied to freshly plasma cleaned carbon-coated grids (Quantifoil), followed by negative staining with 2% uranyl acetate. Datasets of more than 100,000 particles were collected. The 2D class average were generated in RELION.

[0141] Bio-layer interferometry. The binding of IgG of mAb317 and CIS43 to CSP-fused nanoparticles was measured using bio-layer interferometry (Octet Red; Pall ForteBio). CSP- PfCpn60CP-CSP nanoparticles were loaded onto human IgG biosensors (Pall ForteBio, cat NoLeydig 774102 NIH E-182-2024-0-PC-015018-5019) at lOpg / ml in kinetics buffer (TBS + 0.002% Tween20 and 0.01% BSA). The loaded sensors were dipped into solutions containing m Ab317 or CIS43 IgG in kinetics buffer at a concentration of 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0015625 and 0.00078125 nM. The binding experiments were performed in the following steps: 1) baseline for 60 s; 2) loading of CSP -fused nanoparticle for 60 s; 3) baseline for 60 s; 4) association of antibody for 60 s; and 5) disassociation of antibody into kinetics buffer for 120 s. A reference well with no CSP-fused nanoparticle loaded onto the sensor was run in all experiments and subtracted from sample wells, The assay was performed at 22 °C and data were analyzed using Octet Red Data Analysis software version fitting with a 2: 1 binding model.

[0142] Cryo electron microscopy. Before grid preparation for cryo-EM, the protein sample was centrifuged at 13,000 g for 2 min to remove any protein aggregates. A 3.5 pl of protein sample at the concentration of 1.0 mg / ml, which was measured with a Nanodrop spectrometer (Thermo Fisher Scientific), was applied to a glow charged Quantifoil 300 mesh 1.2 / 1.3 carbon grid that had been glow discharged for 90s at 10 mA with PELCO easiGlow Discharge Set. The samples were blotted for 3s with a blot force of 3 using 55 / 20 mm filtered paper (Ted Pella) before being plunged into liquid ethane with a Vitrobot Mark VI (FEI) set at 16°C and 100% humidity. The data were collected on the 300-keV Titan Krios with Gatan BioQuantum Image Filter in the National Institutes of Health (NIH) National Cancer Institute (NCI) / NIH IRP Cryo- EM Facility (NICE) facility. The images were recorded with a 20-eV slit post-GIF K2 Summit camera in super-resolution counting mode at a nominal magnification of 130,000A~ and a defocus range from -0.7 to -2.0 pm. Exposures of 2.5 s were dose fractionated into 50 frames, with an exposure rate of 15 electrons pixel1s resulting in a total exposure of 53.8 electrons A2. The data collection was automated using the SerialEM software package

[0051] ,

[0143] Image processing. The structure of CSP-Cpn60-CSP R465C and D296C was determined from 3,007 dose-fractionated videos that were processed in cryoSPARC (v4.1.1). Motion correction was done by cryoSPARC’ s Patch motion correction with an output F-crop factor of one-half. CTF estimation for each micrograph was calculated with Patch CTF estimation. 1,327,652 particles were autopicked from each micrograph with the blob picker from cryoSPARC and then sorted by two-dimensional (2D) classification to exclude bad particles. 155,341 particles were selected and used to generate 4 ab initio classes in cryoSPARC. TheLeydig 774102 NIH E-182-2024-0-PC-0151 major class with 76,647 particles revealed a clear map of the protein and these particles were selected to conduct NU-refmement to generate a 3.16 A map.

[0144] The structure of CSP-Cpn60HA-CSP was determined from 3,074 dose-fractionated videos that were processed in cryoSPARC (v4.1.1). Motion correction was done by cryoSP ARC’S Patch motion correction with an output F-crop factor of one-half. CTF estimation for each micrograph was calculated with Patch CTF estimation. Particles were autopicked from each micrograph with the blob picker from cryoSPARC and then sorted by two-dimensional (2D) classification to exclude bad particles; 5,140 particles were selected. These 2D templates were then used for further template particle picking resulting in 1,220,882 staring particles. Subsequent rounds of 2D classification resulted in 77,068 particles that were selected and used to generate two ab initio classes in cryoSPARC. One of these classes had a total of 67,149 particles and a clear map of the protein and these particles were selected to conduct NU-refmement to generate a 3.17A map.

[0145] Animal immunization and challenge experiment. The samples were formulated with equal volume of the sterile AddS03 (100 pl) and incubated on the shaker for 5 min prior to immunization. Ten C57BL / 6 mice were immunized with two 2.5 pg doses of samples vaccines with each dose administered 3 weeks apart via subcutaneously injection. Three weeks after the second immunization, all mice were challenged with 500 P. berghei sporozoites in 100 pl in the tail via intramuscularly injection. Bleeds were performed after 3 weeks of the first dose and two weeks after the second dose. The serum was used to determine the CSP-specific antibody titers. Four days after challenge, bleeds were performed daily for blood smear to establish if mice develop blood stage parasitemia. Parasitemia was determined by analyzing Giemsa-stained thin blood smears prepared with blood from each individual mouse 4 to 14 days after challenge except day 13. Mice who lived more than 14 days after the challenge are considered protective. Animal immunizations and challenge experiments were conducted at the Laboratory of Malaria Immunology and Vaccinology (LMIV) under the guideline and approval of the Institutional Animal Care and Use Committee (IACUC) at the National Institute of Health.

[0146] Serum antibody titer ELISA. Nunc MaxiSorp plates (ThermoFisher Scientific) were coated with 100 ul 0.01 mg / ml purified CSP diluted in 30 mM Na-carbonate buffer pH 9.5. Plates were stored at 4 °C overnight. The plates were washed three times with PBST, then wereLeydig 774102 NIH E-182-2024-0-PC-0152 blocked with 2% BSA in PBST for 1 hour at room temperature. After washing three times, each well of plates was added with 100 ul of 1 :10,000 or 1 : 100,000 diluted serum in 2% BSA in PBST and incubated at room temperature for 1 hour. Plates were washed three times with PBST, 200 ul of 1 :10,000 diluted peroxidase-conjugated anti-mouse IgG with 2% BSA in PBST was added (Jackson ImmunoResearch Laboratories, Inc ). Plates were incubated at room temperature on a shaker for 30 minutes. After washing three times with PBST, 70 ul of tetramethylbenzidine (TMB) was added and plates with aluminum foil covered were incubated at room temperature for 20 minutes. Finally, 70 ul of 2 M H2SO4 was added to each well to stop reaction and absorbance at 250 nm was measured using a Biotek Synergy Hl plate reader. Absorbance values for each individual animal were measured in triplicate on separate plates and the average is reported.Results

[0147] Circular permutation design of PfCpn60 to expose the N- and C-termini. The crystal structure of PfCpn60 was previously determined to 3.7 A resolution. The overall structure of PfCpn60 is similar to that of its orthologs in other organisms. In all structures of Cpn60, the N- and C-termini orient towards to the internal chamber, precluding availability for large antigen fusion. Therefore, Cpn60 was engineered to redirect the N- and C-termini outwards using circular permutation. Since the native N- and C- termini of Cpn60 are located nearby and can be joined together using an engineered short linker (Fig. B-l). New engineered N- and C- termini can then be created by opening the structure at other appropriate positions that allow antigen attachment and display on the surface of the particle. Careful inspection of the PfCpn60 structure (PDB ID: 7K3Z) revealed that two positions suitable for introduction of the new A- and C- termini (Fig. B-l). The loop region between al 1 and al2 (T433 to K435), which is located on the outermost surface of the Cpn60 tetradecamer cylindrical ring in the ATP binding structure, and the unique insertion loop between L378 and Asn387 in the PfCpn60, both have potential as the engineering new exposed N- and C- terminals.

[0148] Cpn60 was engineered such that the original N- and C- terminus were linked together by a two amino acid GS linker. Two different versions were then evaluated: Cpn60CP-K435 where the new A- and C-termini were introduced at residue K435, and Cpn60CP-N388 whereLeydig 774102 NIH E-182-2024-0-PC-0153 the new N- and C-termini were introduced at residue N388 (Fig. B-l). Cpn60CP-K435 expressed well but the majority of protein eluted from the size exclusion at the elution volume close to the monomer or dimeric position (17.75 ml at 10 / 300 superose 6 size exclusion column, Fig. B-8). This suggested the engineering in Cpn60CP-K435 while enabling Cpn60 expression abolished formation of larger oligomeric state. In contrast, Cpn60CP-N388 is also highly expressed, and the majority of protein eluted from the size exclusion column at the elution volume close to the position of tetradecameric assembly (13.81 ml, Fig. B-2A). SDS-PAGE analysis confirmed that the major peak is relatively pure Cpn60 with a monomeric molecular weight close to 60 kDa corresponding to the Cpn60CP-N388 subunit molecular weight (Fig. B-2B). Negative stain electron microscopy of the sample in solution clearly established that Cpn60CP-N388 (for clarity, Cpn60CP-N388 is abbreviated as Cpn60CP hereafter) assembled as an expected tetradecameric ring (Fig. B-2C).

[0149] Active-site mutants that stabilize Cpn60 conformation. Cpn60 can exist in different conformations and oligomeric states, and the transient nature hinder its use as a stable platform for antigen presentation. A previous negative stain electron microscopy study has shown that different forms of the wild-type PfCpn60 structures coexist in solution, including single-ring, double-ring, half-football, and whole-football depend on the presence of ATP, ADP or protein substrate and / or CpnlO binding state. Therefore, modifications were made to stabilize PfCpn60 in a favorable conformation.

[0150] D398 in the ortholog E. coli GroEL is a key catalytic residue and D398A mutation reduces the ATP hydrolysis to 2% of wild-type GroEL. Furthermore, another active site mutation, D52A, has been shown to reduce ATP hydrolysis activity even more to <0.01% of wild-type GroEL. The equivalent residues for PfCpn60 are D474 and DI 18, respectively. Negative stain electron microcopy showed that PfCpn60 single D474A mutant existed most in a single-ring structure in solution, which resulted in successfully crystallization for structural determination. Therefore, the circular permutation mutant PfCpn60CP was engineered by introducing D203A and D408A double active site mutations (equivalent to D474A and DI 18ALeydig 774102 NIH E-182-2024-0-PC-0154 mutations in the wild-type PfCpn60) to demolish further the ATP hydrolysis activity and thus reduce conformational flexibility to minimum.

[0151] R465C and D296C mutations potentially introduce disulfide bonds to stabilize the tetradecamer. Negative stain electron microscopy analysis demonstrated that wild type PfCpn60 exists in multiple oligomeric states. The single- and double-ring structures are likely in dynamic equilibrium depending on substrate binding and ATP status. Whether cysteine disulfide bonds could be introduced to crosslink the two opposite heptameric rings and stabilize the double-ring structure was examined. Successful disulfide bond formation requires a Coc-Coc distance of less than or equal to 6.5 A and a C0-C0 distance of less than or equal to 4.5 A. The available EcCpn60 tetradecameric structure revealed that residue A109 meets the requirements for disulfide bond formation across two heptameric rings (PDB: 1XCK, Coc-Coc and C0-CP distances of 5.82 and 3.73 A, respectively for A109). In addition, the A109C mutation has been shown to form a disulfide bond that locks EcCpn60 in double ring structure (PDB: 6OPX). The equivalent residue in PfCpn60CP is R465. Additional potential residues were examined, and S463 in EcCpn60 was identified as a potential location with the Coc-Coc and C0-C0 distances of 6.97 and 4.98 A. The equivalent residue in PfCpn60CP is D296. Therefore, R465C and D296C mutations (the equivalent residues in the wild-type PfCpn60 are R175 and D567) were introduced to stabilize the larger tetradecameric double ring barrel shaped structure through the potential disulfide bonds.

[0152] The Cpn60CP nanoparticle effectively presents CSP. Whether the engineered Cpn60CP is suitable for use as antigen presenting platform was evaluated. A minimal version of CSP that contains the A'-t erm in al junction region, five NANP central repeats and the C-terminal thrombospondin (TSR) domain was designed and fused to the A-terminus or both termini of Cpn60CP. This minimal version of CSP was designed to contain epitopes for three most potent mAbs: CIS43, that preferentially recognized the junctional sequence of DPNA NVNV containing two type I 0 turns, mAb317, that recognized the three consecutive NPNA type I turns in an extensive interaction and L9, that preferentially bound NVDP minor repeats

[0057] ,

[0153] CSP fused to either the A'-terminus (CSP-Cpn60CP) or both the N- and C-termini (CSP-Cpn60CP-CSP) were successfully expressed and purified. The proteins were eluted at 12.7 and 12.0 ml on a 10 / 300 superose 6 size-exclusion column, respectively and these elutionLeydig 774102 NIH E-182-2024-0-PC-0155 volumes are consistent with the formation of a tetradecamer (Fig. B-3A and Fig. B-8). SDS- PAGE analysis demonstrated highly pure protein with a monomer molecular weight close to 86 kDa, corresponding the subunit molecular weight of CSP-Cpn60CP-CSP (Fig. B-3B).

[0154] Whether the nanoparticle fusion retained binding to CSP monoclonal antibodies was examined, thereby identifying whether the critical epitopes in CSP are retained in the nanoparticle. Biolayer interferometry (BLI) binding experiments demonstrated that CSP junction region monoclonal antibody, CIS43, and repeat region monoclonal antibody, mAb317 strongly binds to CSP-Cpn60CP-CSP nanoparticle (Fig. B-3C). Extremely high apparent association rates and negligible apparent dissociation rates were observed indicating the epitopes are exposed and presented correctly, and that the antibodies bound the nanoparticles with extremely high apparent affinity.

[0155] Negative stain electron microscopy for CSP-Cpn60CP-CSP in absence (Fig, B-9, left panel) and presence of 1 mM ATP (Fig. B-9, right panel) confirmed that the CSP fusion did not affect the formation of nanoparticle, demonstrating Cpn60CP is suitable to be an antigen display platform.

[0156] Cryogenic EM structure of CSP-Cpn60CP-CSP. To further confirm the molecular architecture as designed, the engineered nanoparticle vaccine CSP-Cpn60CP-CSP was submitted to high-resolution structure analysis by cryogenic EM (Table Bl). The structure was determined to 3.2 A resolution in general with equatorial domain at 3.2 A resolution, intermediate domain at 4.0 A resolution and the apical domain at 4.8 A resolution due to their different flexibility (Fig. B-4). No significant density was observed for CSP due to its extreme flexibility. Since most of engineered mutations are in the equatorial domain, they can be examined reliably. First, the original N- and C- terminal were checked. In the current circular permutation, the two terminals were linked by a short two residue peptide linker, GS (current numbering G359-S360. Electron density map in this region clearly showed the linkage and the linker is in ordered position (Fig. B-5A). Second, the ATP binding site was checked. Even though two active site mutations D408A and D203A were introduced, the mutations will allow ATP to bind in the active site. Electron density clearly indicated the presence of intact ATP in the conserved binding site in the same manner as wild-type Cpn60 (Figure B-5B), suggesting that active site mutations successfully disrupted ATP hydrolytic activity. Finally, whether the R465C and D296CLeydig 774102 NIH E-182-2024-0-PC-0156 mutations allowed to form the disulfide bonds to cross-link the two opposite heptamer rings was checked. Even though the Ca atom of C465 is 6.75 A from the Ca atom of R465 in opposite ring, electron density did not indicate they are forming a disulfide bond. Instead, a bulky extra electron-density extended across the interface between two opposite rings (Fig. B-5C). They are likely involved in chelating metal-bridging structure linking two opposite heptamer rings with H467. The distance between the Ca atoms of C296 is 9.0 A, too far to form disulfide bond. However, the extra electron-density linked two C296 across the interface. Cys296 is likely also involved in metal bridging linkage between two opposite rings with K300 and possible waters (Fig. B-5D). In comparison to the wild-type structure (PDB 7K3Z), the subunit structures are very close each other with RMSD of 0.77 A by superimposition of subunit A together even though circular permutation was performed in the engineered structure. The intra-ring subunit organization is also close to each other since other inter-ring subunits superimpose well too (Fig. B-10A). However, the opposite rings need to rotate more than 10° to form the current metalbridging double ring structure (Fig. B-10B). Therefore, Arg465 and Asp296 mutations to cysteines did not build the disulfide bridges to cross-link the opposite heptamer rings but assisted the tetradecamer formation likely through the metal-bridging linkages.

[0157] Creation of true disulfide bond linked Cpn60CP tetradecamer. To create a true disulfide bond linked Cpn60CP tetradecamer, residue 296 was mutated back to Asp to remove the metal bridging structure (Fig. B-5D), and His467 was mutated to Ala to destroying potential chelating structures (Figure B-5C). As expected, the CSP fusion protein with the new mutant, named CSP-Cpn60HA-CSP, eluted at 12.35 ml on a superose 6 size exclusion column, similar to CSP-Cpn60CP-CSP, indicating the formation of a tetradecameric structure. To confirm this, samples were submitted for cryo-electron microscopy analysis. The electron density clearly shows that Cys465 truly forms a disulfide bond with Cys465 in the opposite heptameric ring (Fig. B-6A), with ideal disulfide bond geometry (%3 = 99° for the disulfide bond and the Ca-Ca and CP-CP distances of 6.87 and 4.16 A between chain A and K). To confirm that the electron density was due to the formation of a disulfide bond between two symmetries related Cys465s and not the artifact by D7 symmetry averaging, the electron density between these two residues in all chains was examined using the Cl symmetry electron density map. Apparently, disulfide bonds were formed between all seven pairs of symmetrically related C465 residues. InLeydig 774102 NIH E-182-2024-0-PC-01 comparison to the metal bridging structure of CSP-Cpn60CP-CSP, the rotation for opposite ring between CSP-Cpn60HA-CSP and the wild-type structure (7K3Z) is much less degree (about 2- 3°) (Fig. B-6B). Overall, the core Cpn60 structure of CSP-Cpn60HA-CSP is more ordered with lower temperature factors than that of CSP-Cpn60CP-CSP (Table Bl). Same as the structure of CSP-Cpn60CP-CSP, ATPs were found in the active sites in all 14 subunits.

[0158] CSP-Cpn60HA-CSP elicited high CSP-specific antibodies and conferred sterile protection. C57BL / 6 mice are an established mouse model for malaria that has been used to evaluate P. berghei transgenic parasites that have the PbCSP gene replaced with PfCSP. This model allows for evaluation of sterile protection for CSP-based vaccines. Ten C57BL / 6 mice were immunized with CSP-Cpn60HA-CSP and CSP monomer. Three dose vaccination strategies were used, as shown in Fig. B-7A. As shown in Fig. B-7B, CSP-Cpn60HA-CSP elicited significantly higher titers of CSP-specific antibody than CSP monomer, demonstrating the nanoparticle platform certainly enhanced the antigen-specific antibody response. The vaccination also demonstrated that CSP-Cpn60HA-CSP provided better protection (10 / 10) than CSP monomer (5 / 10) for 500 bp challenge of PfCSP generic P. berghei parasites (Fig. B-7C).

[0159] In conclusion, the engineered vaccines disclosed in this Example can display an immunodominant antigen, CSP, to elicit high enough neutralizing antibody titer to provide highly efficacious sterile protection in a mouse model of malaria. The synergistic action from all components derived entirely from Plasmodium is expected to provide better immune protection against malaria than current vaccines. These studies established an effective nanoparticle platform for malaria and infectious disease vaccines.Table BlBiological Sequences relevant to Example 2Leydig 774102 NIH E-182-2024-0-PC-01Leydig 774102 NIH E-182-2024-0-PC-01Table B2Group CSP-Cpn60-CSP ParisitemiaLeydig 774102 NIH E-182-2024-0-PC-0160Table B3Cryo-EM data collection and refinementLeydig 774102 NIH E-182-2024-0-PC-0161EXAMPLE 3

[0160] This Example demonstrates the successful engineering of a potent malaria vaccine using engineered P. falciparum ATP-dependent caseinolytic Clp proteases as nanoparticles that display P. falciparum circumsporozoite protein (CSP).

[0161] P. falciparum ClpP proteases were engineered to overcome several limitations hampering its use as an effective nanoparticle presentation platform. The final designed nanoparticle vaccine platform described here can display up to 28 copies of antigens and contains two distinct antigen attachment sites allowing for multiple distinct antigens to be displayed resulted in a multivalent vaccine. This Example demonstrates the effectiveness of this nanoparticle by simultaneously presenting a designed version of P. falciparum circumsporozoite protein (CSP) at both N- and C- terminals resulting in sterile protection from malaria.Molecular engineering of Clp proteases

[0162] PfClpP was shown to be a heptamer in solution but to be a tetradecamer in crystal structure (2F6I), demonstrating that PfClpP is in the dynamic equilibrium between heptamer and tetradecamer in solution. It was believed that the protease dynamics resulted in the temporary unstructuring of interacting domains (termed handle domain), opening transient equatorial side pores that for product peptide egress.

[0163] An effective nanoparticle display platform for vaccine development should have high antigen valency, particle size and optimal stability. The full PfClpP gene (Accession No XP_001351149.1 - SEQ ID NO:21) encodes 370 amino acid residues, however, the mature form of PfClpP has molecular weight of approximately 25 kDa determined by Western blot analysis of lysates of infected red blood cells. Since the C-terminal amino acid residues are in intact, the mature PfClpP must be TV-terminal truncated form. Like the structural studies (PDB ID: 2F6I), a truncated version comprising residues 179 to 370 was used in the current study, referred to here as “ClpP” for simplicity. To prevent the ClpP from causing a harmful effect to the host when it is used as nanoparticle platform, the active site residue S264 was mutated to G264 as the corresponding position in the inactive form of ClpR to eliminate the protease activity.Furthermore, to stabilize the double-ring tetradecamer structure the T306 was mutated to C306 for di-sulfide bond formation. The corresponding residue Al 53 (equivalent to T306) in E. coli ClpP has been used for formation of inter-ring disulfide bond.Leydig 774102 NIH E-182-2024-0-PC-0162CSP antigen design

[0164] The effectiveness of the engineered Clp protease nanoparticle was investigated by displaying designed circumsporozoite protein (CSP) in both N- and C-terminals. The designed CSP consists of a conserved A-terminal region with a charged protease-cleavage site known as region I, a central diverse repeat region of 25-49 NANP repeats, and a conserved C-terminal region comprised of a short-conserved region III and thrombospondin-like type 1 repeat domain. The central NANP repeats are a target for protective antibodies and the structurally conserved C- terminal thrombospondin type I domain contains CD4+and CD8+T-cell epitopes with proposed roles in protection from disease. The most advanced malaria vaccines, RTS,S / AS01 and R21 / MM, contains 19 NANP repeats and the C-terminal region. Recently, the junctional region between the A-terminal domain and the NANP repeats have also been identified as a target for potent infection-blocking antibodies. However, vaccination using the single epitope of this region displaying on QP virus-like particle did not confer sterilizing immunity against malaria in mouse challenging model. Examination of the CSP NANP repeats indicated five repeats of NANP is the optimal number of repeats for antibody titer and potency.

[0165] A minimal CSP antigen based on the available protection data was designed that includes the junction region (NPDPNANPNVDPNA (SEQ ID NO:33)) followed by five NPNA repeats and the C-terminal domain from residue 310 to residue 383 (Table Cl). This minimal version of CSP contains B-cell epitopes for three most potent mAbs: CIS43, that preferentially recognized the junctional sequence of DPNANVNV (SEQ ID NO:34); mAb317, that recognized the three consecutive NANP type I turns in an extensive interaction; and L9, that preferentially bound NVDP minor repeats. This minimal CSP also retains the T-cell epitopes located in the C- terminal domains.

[0166] Production of CSP has proven challenging due to its inherent complexity. Successful production of CSP has been reported for full length of CSP in E. coli, a short version of CSP in Pichia pastor is, full length of CSP in Pseudomonas fluor escens, and CSP fusions to the hepatitis B virus surface antigen in RTS,S and R21 produced in Saccharomyces . The designed minimal CSP described in this Example can be easily expressed in mammalian Expi293 cells in high yields with 93 mg in IL culture confirming its potential for use as a stable antigen.Leydig 774102 NIH E-182-2024-0-PC-0163Engineered nanoparticles displaying CSP are stable and highly expressed

[0167] Without T244 and R275 mutation to cysteine, the PfClpP eluted around 16.98 ml in superose 6 10 / 30 mm size exclusion column, corresponding to the single heptamer molecular weight (Fig. C-1A, Table C3). After these residues were mutated to cysteine, the PfClpPss eluted around 16.02 ml (Fig. C-3B), which corresponds to the double heptamer molecular weight. CSP fusion to N or C or both ends seem not to affect the formation of nanoparticle. The elution volumes from superose 6 (10 / 300) size column for PfClpPss-CSP and CSP-PfClpPss-CSP were 13.38 and 13.10 ml, respectively (Fig. C-1C-D).

[0168] To further confirm the CSP fusion did not affect the assemble of nanoparticles, all samples were submitted to the negative stain electron microscopy analysis. Although no clear double ring structure was observed probably due to orientation preference, all PfClpP fusing proteins were clearly shown to assemble as a nanoparticle ring structure.Cryogenic EM structures of the ClpP protease

[0169] The crystal structure of the P. falciparum ClpP has been reported. To confirm the engineered structure of the core display platform, the structures of the nanoparticle vaccines CSP-PfClpPss-CSP was determined at high-resolution using cryogenics EM (Table C2, Fig. C- 2).

[0170] The CSP-PfClpPss-CSP was determined to 2.72 A resolution (Fig. C-4). No significant density was observed for antigens consistent with the flexibility provided by the linkers between antigen and nanoparticle. The core nanoparticle assembles into a 14-mer cylindrical structure of two seven-fold rings (Fig. C-2D). The structures demonstrated the successful engineering of stable 14mer nanoparticles consisting of two heptamer rings in D7 symmetry. CSP fusion to both A and C termini did not affect the assemble of tetradecamer nanoparticle, potentially providing us a platform to be able be used as multivalent vaccines. Since the structure was determined at high resolution, the most of sidechains were clearly defined. Electron density clearly demonstrated that the residue C244 formed disulfide bonds with C244 in symmetrically related chain in the opposite heptamer ring (Fig. C-3A). To confirm that the electron density is due to the formation of the disulfide bond between two symmetrically related Cys244 rather than it is caused by the D7 symmetry averaging, the electron density between these two residues in all chains were checked using the Cl symmetry map. Clearly, theLeydig 774102 NIH E-182-2024-0-PC-0164 disulfide bonds were formed between all seven pairs of symmetrically related C306 residues. The subunit structure is very similar to the structure deposited in PDB (2F6I) with RMSD of 0.6 A by superimposition of subunit A together. Furthermore, both 2F6I and the current disulfide cross-linked model are in a compact conformation, which are catalytically inactive state. Superimposition of all chains between 2F6I with the current model results in RMSD of 1.2 A, mainly by a rigid rotation of each subunit. As a result of the disulfide bond linkages between two opposite heptamer rings, the general shaper of the entire particle becomes 1.0-1.5 A shorter in the axial direction, but 1.0-1.5 A wider in ring diameter in equatorial direction in comparison to the deposited PfClpP structure (2F6I). The structure also clearly indicated that the residue C275 does not form disulfide bond with C275 in symmetrically related chain in the opposite heptamer ring (Fig. C-3B) even though they are located close each other, indicating this mutation did not make any contribution for tetradecamer formation. In the final PfClpP construct (named as PfClpP (T306C), this residue was mutated back to original R275. The expression and sizeexclusion chromatogram of CSP-PfClpP(T244C)-CSP is similar to the double mutation construct.Neutralizing epitopes on the engineered nanoparticle vaccine are accessible

[0171] To confirm whether the CSP neutralizing epitopes are accessible for neutralizing antibodies, the Fab fragments of the CSP junctional region monoclonal antibody, CIS43, and the CSP major repeat monoclonal antibody, mAb317, were incubated with CSP-PfClpPss-CSP, and superose 6 size exclusion chromatography showed a significant change in elution volume (13.57 to 12.85 ml for CIS43 and 13.85 to 12.52 ml for mAb317, respectively) (Fig. C-4A). The slightly larger offset of mAb317 means that mAb317 binds more than CIS43 to CSP-PfClpPss-CSP, which is consistent with only one junction region in one CSP, but five repeats in our designed CSP. To further confirm the binding of their Fab fragments to CSP-PfClpPss-CSP, the Fab fragment-binding fractions eluted from the column were subjected to negative staining electron microscopy analysis. Negative-stained photomicrographs clearly demonstrated the appearance of additional density around the central core nanoparticles (Fig. C-4B). Furthermore, in consistent with more mAb317 binding than CIS43, the density for mAb317 bound sample is stronger than that for CIS43 bound sample.Leydig 774102 NIH E-182-2024-0-PC-0165Vaccination with engineered nanoparticles elicit high titers of CSP specific antibodies and resulted in sterile protection against malaria

[0172] To determine the immunogenicity of CSP-PfClpPss-CSP, inbred C57BL / 6 and outbred CD-I mice were immunized twice with 2.5 pg of proteins supplemented with AddaS03 (Fig. C-5A). Serum collected 21 days after the first and the second immunization (day 21 and day 35, respectively) was used to quantify CSP-specific antibody titers using quantitative ELISA (Fig. C-5B). Compared with the PBS control group, CSP-PfClpPss-CSP elicited high CSP- specific antibody titers. The CSP-specific antibody titer levels are very similar for CD-I mice (Fig. C-5B).

[0173] Whether vaccination of CSP-PfClpPss-CSP could induce sterile protection against malaria upon challenge with PfCSP transgenic P. berghei sporozoites was examined. The C57BL / 6 mice immunized with two doses of the nanoparticles formulated in AddaS03 were challenged by injecting 500 PfCSP transgenic P. berghei sporozoites. Mice immunized with the nanoparticles induced statistically significant sterile protection compared to adjuvant control mice inducing 90% protection against parasitemia (Fig. C-5C). In CSP-PfClpPss-CSP group, the infected mouse (No. 4) has the lowest CSP-specific antibody titer among the tested 10 mice. Whether it's coincidence or correlation, more data and further research are needed.

[0174] After the cryo-EM structure of CSP-PfClpPss-CSP was determined, it was found that only C244 formed disulfide bond across opposite heptamer rings, but not C275. In the final construct, C275 was mutated back to original R275. This final construct was named as CSP- PfClpP(T244C)-CSP. To confirm the immunogenicity of CSP-PfClpP(T244C)-CSP, inbred C57BL / 6 were immunized three times with 2.5 pg of proteins supplemented with AddaS03 (Fig. C-5D). In this animal experiment, CSP alone was included to compare the difference between CSP alone and CSP fusion with nanoparticle. Serum collected 14 days after the first, the second and the third immunization (day 21, day 35 and day 56, respectively) was used to quantify CSP- specific antibody titers using quantitative ELISA (Fig. C-5E). Compared with the CSP monomer group and PBS control group, CSP nanoparticle group elicited significantly higher CSP-specific antibody titers, but no statistically significant difference between CSP monomer group and PBS control group even though the mean value of CSP monomer group was two order higher than the PBS control group (Fig. C-5E). In consistent with the CSP specific antibody titers, miceLeydig 774102 NIH E-182-2024-0-PC-0166 immunized with the CSP nanoparticles induced statistically significant sterile protection (100 % protection against parasitemia) compared to the CSP monomer group (50%) and adjuvant control group (20%) (Fig. C-5F).

[0175] In conclusion, the vaccines disclosed in this Example elicited high titers of anti-CSP antibodies and demonstrated sterile protection against malaria. The CSP-PfClpP-CSP elicited sufficiently high CSP-specific antibody titers and induced 90-100% immune protection in mice challenged with lethal doses of the parasite.

[0176] The ATP-dependent caseinolytic Clp protease plays an important role in the proteostasis of blood-stage parasites, and the engineered nanoparticles possess desirable manufacturing characteristics. The current study highlights the potential of the ClpP system from Plasmodium as an antigen display platform in next-generation malaria vaccine design. The synergetic effects from both nanoparticle itself and fused antigen may provide more durable and effective anti-malaria vaccines since the whole system is entirely prepared from the Plasmodium proteins that may induce stronger immune response from both humoral and cellular arms. This new strategy has broad implication for the future development of vaccine for malaria and other infectious diseases. The results of this study establish an effective nanoparticle platform for malaria and infectious diseases vaccines.Table ClBiological Sequences relevant to Example 3Leydig 774102 NIH E-182-2024-0-PC-01EXAMPLE 4

[0177] This example demonstrates the successful engineering of a potent malaria vaccine using engineered P. falciparum ATP-dependent caseinolytic Clp proteases as nanoparticles that display P. falciparum circumsporozoite protein (CSP).Materials and MethodsPlasmid construction, protein expression, and purification of PfClpR, PbClpR and their CSP fusions[00178J The full sequence of P. falciparum (isolate 3D7) PfClpR (XP_001348522) and PbClpR (XP_034420620) were retrieved from NCBI database. For PfClpR, the DNA sequence encoding protein residues between Ser49 and Glu244 were synthetized (GenScript) and cloned into expression vector. The corresponding residues, N191 and R222 were mutated to C191 and C222, respectively, to create PfClpR. For PbClpR, the DNA sequence encoding protein residues between Ser42 and Glu240 were synthetized (GenScript) and cloned into the expression vector. A short version of CSP that consists of the junction region, NPDPNANPNVDPNA (SEQ ID NO:33), five NPNA repeats and C-terminal PSR domain (residues 310-383, XP_001351122) was designed. The corresponding designed CSP gene was fused to either end or both ends of N-Leydig 774102 NIH E-182-2024-0-PC-0168 or C-terminus of PfClpR to create CSP-PfClpR CSP-PfClpR-CSP fusion protein. The protein sequences used in the current study are presented in Table DI.

[0179] All proteins were expressed in Rosetta (DE3) with 6 histidine residues at C-terminal end to facilitate purification and prepared in the similar way as described as following. The vector was transformed into E. coli Rosetta (DE3) cells and expressed in 2L of LB media. Cell pellets were resuspended in 50 ml of lysis buffer containing 100 mM (NH^SCU, 10 mM MgCh, 10 mM KC1, 20 mM Tris pH 8.0 and 10% (w / v) glycerol with a table of protease inhibitor (Roche), 35 ul of 0.1 mM PMSF. After incubating with 250 ul of 25 mg / ml lysozyme for 5 min., cells were lysed by sonication. The lysate was centrifuged at 50,000 g for 15 min, and the supernatant was applied to a 2 ml of Ni resin and washed with 20 ml of binding buffer containing 20 mM NaPCL pH 7.4 and 500 mM NaCl and 20 ml of washing buffer containing 20 mM imidazole, 20 mMNaPCL pH 7.4 and 500 mM NaCl. Proteins were eluted by 15 ml of eluting buffer containing 500 mM imidazole, 20 mM NaPCL pH 7.4 and 500 mM NaCl. The eluted proteins were further purified by a superdex 200 or a superose 6 gel filtration columns in the PBS buffer. The proteins were aliquoted and stored in -80 °C freezer for animal experiments.CSP production

[0180] The short version of CSP gene sequence, encoding P. falciparum 3D7 isolate CSP junctional region (amino acids 101-114), 5 NPNA repeats and the C-terminal TSR domain (amino acids 310-383) (Table DI), was optimized to human codon, synthesized, and cloned into pHLsec plasmids (GenScript). The plasmids were transformed into XL-10 E. coli cells, harvested, and purified using Qiagen maxipreparation kit following the manufacturer’s instructions. The plasmids were transfected into Expi 293F cells for expression according to manufacturer instructions. Cell-free supernatant was harvested by centrifugation at 3,000 g. The protein was purified by Ni-affinity column, followed by Superdex 200 gel filtration column as other proteins with his-tag.Antibody production

[0181] The fragment sequences of monoclonal antibodies, mAb317 and CIS43 were obtained from literatures and synthesized with a human IgG Fc sequence and cloned into pHLsec plasmids (GenScript). Plasmids with human codon optimized full-length IgG gene were transformed into XL-10 E. coli cells. Plasmids were harvested and purified using QiagenLeydig 774102 NIH E-182-2024-0-PC-0169 maxipreparation kit following the manufacturer’s instructions. Equal mole amount of heavy chain and light chain plasmids were transfected into Expi 293F cells according to manufacturer instructions. Cell-free supernatant was harvested by centrifugation and batched incubated with protein A agarose resin (GoldBio) for 1 hour at room temperature. After washing the collected resin with 10 column volumes (CV) protein A IgG binding buffer, antibodies were eluted with 10 CV IgG elution buffer and neutralized with 1 CV 1 M Tris buffer pH 9.0. Antibodies were concentrated using an Amicon centrifugal filter and further purified by a superdex 200 increase 10 / 300 size exclusion column using PBS pH 7.4 buffer. The Fab fragments of mAb317 and CIS43 were expressed using Expi293F cells like IgG antibodies but purified using His-tag affinity column follow by Superdex s200 size-exclusion column.Negative stain electron macroscopy

[0182] The sample quality and structural features were assessed by negative stain transmission electron microscopy in a FEI TT20 microscopy equipped with a TVIPS 16 megapixel charge-coupled device (CCE) camera. The protein samples (100 ng / ml) were applied to freshly plasma cleaned carbon-coated grids (Quantifoil), followed by negative staining with 2% uranyl acetate. Datasets of more than 100,000 particles were collected. The 2D class average were generated in RELION.Cryo electron microscopy

[0183] Before grid preparation for cryo-EM, the protein sample was centrifuged at 13,000 g for 2 min to remove any protein aggregates. A 3.5 pl of protein sample at the concentration of ~1.0 mg / ml, which was measured with a Nanodrop spectrometer (Thermo Fisher Scientific), was applied to a glow charged Quantifoil 300 mesh 1.2 / 1.3 carbon grid that had been glow discharged for 90s at 10 mA with PELCO easiGlow Discharge Set. The samples were blotted for 3s with a blot force of 3 using 55 / 20 mm filtered paper (Ted Pella) before being plunged into liquid ethane with a Vitrobot Mark VI (FEI) set at 16°C and 100% humidity. The data were collected on the 300-keV Titan Krios with Gatan BioQuantum Image Filter in the National Institutes of Health (NIH) National Cancer Institute (NCI) / NIH IRP Cryo-EM Facility (NICE) facility. The images were recorded with a 20-eV slit post-GIF K2 Summit camera in superresolution counting mode at a nominal magnification of 130,000A~ and a defocus range from -0.7 to -2.0 pm. Exposures of 8 s were dose fractionated into 40 frames (200 ms per frame),Leydig 774102 NIH E-182-2024-0-PC-0170 with an exposure rate of 8 electrons pixel1s ’, resulting in a total exposure of 57 electrons A2. The data collection was automated using the SerialEM software package.Animal immunization and challenge experiment

[0184] The tested CSP-PfClpR-CSP fusion samples was formulated with equal volume of the sterile AddS03 (100 l) and incubates on the shaker for 5 min prior to immunization. Ten C57BL / 6 mice and ten CD-I mice were immunized with two 2.5 pg doses of the tested vaccines with each dose administered 3 weeks apart via subcutaneously injection. Three weeks after the second immunization, the C57BL / 6 mice were challenged with 500 P. berghei sporozoites in 100 pl in the tail via intramuscularly injection.Determination of antibody titers in response to PbClpR and CSP-PfClpR-CSP / adjuvant vaccinations

[0185] Bleeds were performed after 3 weeks of the first dose and two weeks after the second dose. The serum was used to determine the antigen specific antibody titers for PbClpR and CSP- specific antibody titers for CSP-PfClpR-CSP. Immune responses against PbClpR and CSP- PfClpR-CSP fusion were assessed by ELISA. Nunc MaxiSorp plates (ThermoFisher Scientific) were coated with 100 ul 0.01 mg / ml purified CSP diluted in 30 mM Na-carbonate buffer pH 9.5. Plates were stored at 4 °C overnight. The plates were washed three times with PBST, then were blocked with 2% BSA in PBST for 1 hour at room temperature. After washing three times, each well of plates was added with 100 ul of 1 :10,000 or 1: 100,000 or even 1 : 1,000,000 diluted serum in 2% BSA in PBST and incubated at room temperature for 1 hour. Plates were washed three times with PBST, 200 ul of 1 : 10,000 diluted peroxidase-conjugated anti-mouse IgG with 2% BSA in PBST was added (Jackson ImmunoResearch Laboratories, Inc.). Plates were incubated at room temperature on a shaker for 30 minutes. After washing three times with PBST, 70 ul of tetramethylbenzidine (TMB) was added and plates with aluminum foil covered were incubated at room temperature for 20 minutes. Finally, 70 ul of 2 M H2SO4 was added to each well to stop reaction and absorbance at 250 nm was measured using a Biotek Synergy Hl plate reader. Absorbance values for each individual animal were measured in triplicate on separate plates and the average is reported.Leydig 774102 NIH E-182-2024-0-PC-0171Determination of the infection following the parasite challenge and assessment of in vivo protection

[0186] Four days after challenge, bleeds were performed daily for blood smear to establish if mice develop blood stage parasitemia. Parasitemia was determined by analyzing Giemsa-stained thin blood smears prepared daily with blood from each individual mouse 4 to 14 days after challenge except day 13. Mice that showed no evidence of parasitemia 14 days after challenge were considered protected.ResultsNanoparticle protein PbClpR itself provide some degree of protection against malaria

[0187] Potential self-assembling high-order oligomers nanoparticle proteins were identified by searching the malaria parasite genomes and protein data bank (PDB). Plasmodium Clp proteases were identified to assemble as nanoparticle size of oligomers. Size-exclusion chromatography and negative stain electron microscopy analysis confirmed ClpR from P. berghei (PbClpR) forms multimer nanoparticles (Fig. D-l A-B). To evaluate whether this nanoparticle protein itself can induce some degree of protection against malaria, PbClpR were submitted to animal experimentation to set up a baseline before fusing any antigen. After three doses of immunization (Fig. D-2A), the PbClpR induced high titer of immunogen-specific antibody (Fig. D-2B). Even though all animals developed parasitemia eventually, the PbClpR group had 3 animals among 5 who had 2-3-day delay in parasitemia (Fig. D-2C), implying that PbClpR itself may provide some degree protection against malaria.Molecular engineering of PfClpR

[0188] PfClpR has high sequence similarity to PfClpP (47.3%) but is non-catalytic because its equivalent active site residue S264 in PfClpP becomes G148 in PfClpR. PfClpR was shown to be a heptamer or tetradecamer in solution, but to be a heptamer in crystal structure (4HNK and 4GM2), demonstrating that PfClpR are in the dynamic equilibrium between heptamer and tetradecamer in solution. It was believed that ClpR interacted with ClpP to form heterotetradecamer to regulate the ClpP activity. Therefore, PfClpR plays an essential role in apicoplast biogenesis as a potential regulator of PfClpP protease activity in vivo.Leydig 774102 NIH E-182-2024-0-PC-0172

[0189] To potentially stabilize the double-ring tetradecamer structure, two potential disulfide bonds were introduced based on prediction of the similar structure to. The residues N191 and R222 in PfClpR (PfClpRss) were mutated to cysteine for potential disulfide bond formation to crosslink opposite heptamer rings. However, when double ring structure of CSP-PfClpRss was determined using cryo-EM (see section below). It was found that PfClpR formed tetradecamer nanoparticle using convex face rather than PfClpP using concave face and these mutations are not in close proximity to each other.CSP antigen design

[0190] The effectiveness of the engineered Clp protease nanoparticle was evaluated by displaying circumsporozoite protein (CSP) in both N- and C-terminals. CSP consists of a conserved -terminal region with a charged protease-cleavage site known as region I, a central diverse repeat region of 25-49 NANP repeats, and a conserved C-terminal region comprised of a short-conserved region III and thrombospondin-like type 1 repeat domain. The central NANP repeats are a target for protective antibodies and the structurally conserved C-terminal thrombospondin type I domain contains CD4+and CD8+T-cell epitopes with proposed roles in protection from disease. The most advanced malaria vaccines, RTS,S / AS01 and R21 / MM, contains 19 NANP repeats and the C-terminal region. Recently, the junctional region between the A-terminal domain and the NANP repeats have also been identified as a target for potent infection-blocking antibodies. However, vaccination using the single epitope of this region displaying on Q0 virus-like particle did not confer sterilizing immunity against malaria in mouse challenging model. Examination of the CSP NANP repeats indicated five repeats of NANP is the optimal number of repeats for antibody titer and potency.

[0191] A minimal CSP antigen was designed based on the available protection data that includes the junction region (NPDPNANPNVDP (SEQ ID NO:33)) followed by five NANP repeats and the C-terminal domain from residue 311 to residue 377 (Table DI). This minimal version of CSP contains B-cell epitopes for three most potent mAbs: CIS43, that preferentially recognized the junctional sequence of DPNANVNV (SEQ ID NO:34); mAb317, that recognized the three consecutive NANP type I turns in an extensive interaction; and L9, that preferentially bound NVDP minor repeats. This minimal CSP also retains the T-cell epitopes located in the C- terminal domains.Leydig 774102 NIH E-182-2024-0-PC-011

[0192] Production of CSP has proven challenging due to its inherent complexity. Successful production of CSP has been reported for full length of CSP in E. coll, a short version of CSP in Pichia pastoris, full length of CSP in Pseudomonas fluorescens, and CSP fusions to the hepatitis B virus surface antigen in RTS,S and R21 produced in Saccharomyces . The designed minimal CSP described here can be easily expressed in mammalian Expi293 cells in high yields with 93 mg in IL culture confirming its potential for use as a stable antigen (Fig. D-7).CSP fusion to N-terminal and both N- or C- terminals of PfClpR are stable and highly expressed

[0193] In comparison to PfClpR alone, the expression level of the CSP fusion to the N- terminal of PfClpRss was much higher. It was also interesting to observed that the oligomer state of CSP-PfClpRss was different when the running buffer changed from PBS to Heps buffer (20 mM Heps and 100 mM NaCl, pH = 7.5). The elution volume of CSP-PfClpRss in PBS buffer was 15.55 ml corresponding to heptamer in solution while the elution volume changed to 14.54 ml in Heps buffer corresponding to tetradecamer in solution. The phenomenon also confirmed that no disulfide bond formation for N191C mutation. When CSP fusion to both N- and C- terminals of PfClpRss, both heptamer and tetradecamer oligomer states co-existed in the solution, but majority in heptamer state. To further confirm the double-ring structure of CSP- PfClpRss, the samples were submitted to ciyo-EM analysis.Cryogenic EM structures of CSP-PfClpR

[0194] Collecting high quality cryogenic EM data for CSP-PfClpRss-CSP was unsuccessful due to strong orientation preference, and probably the majority of CSP-PfClpRss-CSP is in heptamer oligomer state, as observed in crystal structure of PfClpR. Instead, cryogenic EM data for CSP-PfClpRss were collected in hopes it can represent the double ring structure of CSP- PfClpRss-CSP in tetradecamer oligomer state. Even though the strong orientation preference still existed, the data allowed construction of a 3.49 A resolution electron density map for CSP- PfClpRss (Fig. D-4). Interestingly, no docking solution was identified using the whole structure (heptamer) of PfClpR which was deposited in PDB (4HNK) as a search model. However, the docking solution was easily identified using the individual subunit as a search model plus the D7 symmetric file identified from the electron density map. Surprisingly, the heptamer arrangement to tetradecamer for CSP-PfClpRss was totally different from that of CSP-PfClpPss-CSPLeydig 774102 NIH E-182-2024-0-PC-0174 previously determined. In CSP-PfClpPss-CSP, the concave face of two heptamers oriented towards each other and using the handle-domains to interlock two heptamers together forming tetradecamer as that in the structure of PfClpP without fusion, but in CSP-PfClpRss, the convex face rather than concave face of two heptamers oriented towards each other forming tetradecamer. The handle domains played no role in the formation of such tetradecamer. Therefore, the equivalent disulfide bond mutations of N191C have no contribution for tetradecamer assemble at all. The interaction between two heptamers of CSP-PfClpRss was weak because of very weak electron density in their interaction interface. The careful inspection of size exclusion chromatograms of CSP-PfClpRss and CSP-PfClpRss-CSP indicated that the tetradecamer and heptamer are convertible dependent on the buffer composition and CSP fusion on their terminal of PfClpR.Tetradecameric structure of PfClpR suggests a possible regulation mechanism

[0195] In comparison to the heptamer structure deposited in PDB (4HNK), the heptameric ring of PfClpR in tetradecameric form becomes slightly larger due to the about 5° clockwise rotation of each subunit around vertical axis and 5° anti-clockwise rotation of each subunit around horizontal axis (Fig. D-4). As a result, the radii of the central hole of PfClpR becomes significantly larger than that in single heptamer ring. The present structure represented a novel way of interaction between PfClpR-PfClpR or even PfClpR-PfClpP, even though the intense investigation about PfClpP and PfClpR, the mechanism for PfClpR how to interact PfClpP and how to regulate the catalytic activity of PfClpP is not clear. It has been known that all ClpPs form tetradecamer through the interaction of handle domain on the concave face of two heptamers. AAA+ATPase chaperones such as ClpX and ClpA interact with one end of ClpP tetradecamer to regulate the substrate entry. The current working model for ClpR regulation is ClpP and ClpR form a single hetero-tetradecameric core complex using concave face to regulate the release of substrate degraded fragments. The present tetradecameric structure of PfClpR suggested that the ClpR may not disrupt the intact tetradecameric core of ClpP but directly interact with the intact tetradecameric core of ClpP through another end of convex face to regulate the release of degraded protein fragments (Fig. D-5). The whole ClpP and ClpR system behaves like the structure of proteasome with 14 catalytic active 0 subunits form a central tetradecameric core and the heptamer of the catalytic inactive a subunits at the ends of core.Leydig 774102 NIH E-182-2024-0-PC-0175Furthermore, the heptamer of the catalytic inactive a 7 subunits from human can interact each other using the same interface forming the homo-tetradecam er particle.Vaccination with CSP-PfClpRss-CSP elicit high titers of CSP specific antibodies and resulted in sterile protection against malaria

[0196] To determine the immunogenicity of CSP-PfClpRss-CSP, inbred C57BL / 6 and outbred CD-I mice were immunized twice with 2.5 pg of proteins supplemented with AddaS03 (Fig. D-7A). Serum collected 21 days after the first immunization and 14 days after the second immunization (day 21 and day 35, respectively) was used to quantify CSP-specific antibody titers using quantitative ELISA (Fig. D-6B). Compared with the PBS control group, CSP- PfClpRss-CSP elicited high CSP-specific antibody titers in comparison to the control group. The CSP-specific antibody titer levels are very similar for CD-I mice immunized for both antigens (Fig. D-6B).

[0197] Even though CSP-PfClpRss-CSP did not form stable tetradecamer, whether vaccination of CSP-PfClpRss-CSP could induce sterile protection against malaria upon challenge with PfCSP transgenic P. berghei sporozoites as CSP-PfClpPss-CSP was evaluated. C57BL / 6 mice immunized with two doses of the CSP-PfClpRss-CSP nanoparticle formulated in AddaS03 were challenged by injecting 500 PfCSP transgenic P. berghei sporozoites. CSP-PfClpRss-CSP immunized group induced statistically significant sterile protection compared to adjuvant control mice inducing 90% protection against parasitemia (Fig. D-6C). It is worthy to be noted that the infected mouse (No. 4) in the CSP-PfClpRss-CSP group were not the one with lowest CSP- specific antibody titers among 10 mice, implying the CSP-specific antibody may not be the only factor to provide protection, the PfClpR nanoparticle itself may provide the additional T-cell epitopes for protection in consistent with the PbClpR alone provide some delay for parasitemia.Table DIBiological Sequences relevant to Example 4Leydig 774102 NIH E-182-2024-0-PC-01Leydig 774102 NIH E-182-2024-0-PC-0177Table D2Correlations of elution volume with total molecule weightLeydig 774102 NIH E-182-2024-0-PC-01Table D3Parasitemia of individual mice from animal challenge experiment verified by eye using thin blood smearLeydig 774102 NIH E-182-2024-0-PC-0179= Positive blood smear= Negative blood smear= No smear= Bad Smear= EuthanizedTable D4CSP-specific antibody units for individual mouse[00198J The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover the singular, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e. ., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in theLeydig 774102 NIH E-182-2024-0-PC-0180 specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0199] Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 774102 NIH E-182-2024-0-PC-0181CLAIM(S):

1. A composition comprising a Plasmodium species multimeric protein capable of forming a nanoparticle and at least one antigen of interest, wherein the multimeric protein comprises a monomer chosen from Plasmodium species pyridoxal 5’-phosphate synthase (PLP), chaperone 60 protein (Cpn60), and caseinolytic protease (Clp).

2. The composition according to claim 1, wherein a monomer comprises at least one stabilizing mutation that stabilizes the nanoparticle structure of the composition.

3. The composition according to claim 2, wherein the monomer is a PLP polypeptide selected from Pdxl, Pdx2, or a combination thereof.

4. The composition according to claim 3, wherein the PLP is Pdxl and wherein a stabilizing mutation comprises K174C, S178C, or both, relative to accession XP 966196 (SEQ ID NO: 1).

5. The composition according to claim 4, wherein the stabilizing mutation facilitates the formation of one or more disulfide bonds within the multimeric protein.

6. The composition according to claim 3 or 4, wherein the PLP is Pdxl and wherein the Pdxl comprises a K83R mutation relative to accession XP 966196 (SEQ ID NO: 1).

7. The composition according to claim 6, wherein the K83R mutation abolishes enzymatic activity.

8. The composition according to claim 3, wherein the PLP is Pdx2 and wherein a stabilizing mutation comprises H196N, relative to accession XP_001347840 (SEQ ID NO:2).

9. The composition according to claim 8, wherein the stabilizing mutation abolishes the glutaminase activity to stabilize the complex formation between Pdxl and Pdx2 within the multimeric protein.

10. The composition according to claim 3 or 8, wherein the PLP is Pdx2 and wherein the Pdx2 comprises a N65Q and / orN128Q mutation relative to accession XP_001347840 (SEQ ID NO:2).Leydig 774102 NIH E-182-2024-0-PC-018211. The composition according to any one of claims 3-10, wherein the Pdxl and Pdx2 do not comprise an HHHHHH his-tag.

12. The composition according to claim 2, wherein the monomer is a Cpn60 polypeptide.

13. The composition according to claim 12, wherein the Cpn60 comprises a two amino acid GS linker between the N- and C- termini relative to the Wild Type (WT) PfCpn60 sequence UniProtKB accession: Q8I0V3 (SEQ ID NO: 11).

14. The composition according to claim 13, wherein the Cpn60 comprises an A -terminus at residue K435 or N388.

15. The composition according to claim 13 or 14, wherein the Cpn60 comprises a C-terminus at residue S434 or N376.

16. The composition according to claim 15, wherein the C-terminus comprises an HHHHHH his-tag.

17. The composition according to any one of claims 13-16, wherein the Cpn60 lacks the loop insert L378 to N387 ofWT PfCpn60 sequence UniProtKB accession: Q8I0V3 (SEQ ID NO: 11).

18. The composition according to any one of claims 13-17, wherein a stabilizing mutation comprises R175C, D567C, or both, relative to the WT PfCpn60 sequence UniProtKB accession: Q8I0V3 (SEQ ID NO: 11).

19. The composition according to claim 18, wherein the stabilizing mutation facilitates the formation of one or more disulfide bonds within the multimeric protein.

20. The composition according to any one of claims 13-19, wherein a stabilizing mutation comprises D474A, D118A, or both, relative to the WT PfCpn60 sequence UniProtKB accession: Q8I0V3 (SEQ ID NO: 11).

21. The composition according to claim 20, wherein the stabilizing mutation stabilizes the ATP bound conformation of the multimeric protein.Leydig 774102 NIH E-182-2024-0-PC-018322. The composition according to claim 2, wherein the monomer is a Clp polypeptide.

23. The composition according to claim 22, wherein the Clp polypeptide comprises 179 amino acids (ClpR) or 370 amino acids (ClpP).

24. The composition according to claim 23, wherein the Clp is ClpP and wherein the ClpP comprises a S264G mutation relative to accession XP 001351149.1 (SEQ ID NO:21).

25. The composition according to claim 24, wherein the S264G mutation eliminates protease activity.

26. The composition according to claim 24 or 25, wherein the Clp is ClpP and wherein a stabilizing mutation comprises T306C relative to accession XP_001351149.1 (SEQ ID NO:21).

27. The composition according to claim 26, wherein the stabilizing mutation facilitates the formation of one or more disulfide bonds within the multimeric protein28. The composition according to any one of claims 1-27, wherein the Plasmodium species is Plasmodium falciparum.

29. The composition according to any one of claims 1-28, wherein the multimeric protein comprises a mutation to facilitate display of multiple copies of the at least one antigen of interest.

30. The composition according to claim 29, wherein the N-terminus is exposed for attachment of the at least one antigen of interest.

31. The composition according to claim 29, wherein the C-terminus is exposed for attachment of the at least one antigen of interest.

32. The composition according to any one of claims 1-31, wherein the nanoparticle displays multiple copies of the at least one antigen of interest.

33. The composition according to any one of claims 1-32, wherein the at least one antigen of interest comprises a first antigen of interest and a second antigen of interest, wherein the first and second antigens of interest differ.Leydig 774102 NIH E-182-2024-0-PC-018434. The composition according to any one of claims 1-33, wherein the nanoparticle displays more than one antigen of interest.

35. The composition according to any one of claims 1-34, wherein each antigen of interest is derived from a Plasmodium species.

36. The composition according to any one of claims 1-35, wherein the at least one antigen of interest is chosen from circumsporozoite protein (CSP), Plasmodium vivax cell-traversal protein for ookinetes and sporozoites (CelTOS), and Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites.

37. The composition according to claim 36, wherein the CSP comprises an amino acid sequence represented by SEQ ID NO:3.

38. The composition of any one of claims 1-37, wherein the multimeric protein is selected from CSP-P1-CSP (SEQ ID NO:4), CSP-P1(S293C)-CSP (SEQ ID NO:5), CSP-P2-CSP (SEQ ID NO:6), PvCelTOS-P2-CSP (SEQ ID NO:7), CSP-P2-PvCelTOS (SEQ ID NO:8), Tag-free CSP-P1(S S293C)-CSP (SEQ ID NO:9), Tag-free CSP-P2-CSP (SEQ ID NO: 10), PfCpn60CP- K435 (SEQ ID NO: 13), PfCpn60CP-N388 (SEQ ID NO: 14), CSP-PfCpn60CP-N388 (SEQ ID NO: 15), CSP-PfCpn60CP-CSP (SEQ ID NO: 16), CSP-PfCpn60HA -CSP (SEQ ID NO: 17), PfCpn60HA (SEQ ID NO: 18), CSP-PfClpPss-CSP (SEQ ID NO:22), and CSP-PfClpP(T244C)- CSP (SEQ ID NO:23), PfClpP(T244C) (SEQ ID NO:24), PbClpR (SEQ ID NO:28), CSP- PfClpRss (SEQ ID NO:29), PfClpRss-CSP (SEQ ID NO 30), CSP-PfClpRss-CSP (SEQ ID NO:31), and CSP-PfClpR-CSP (SEQ ID NO:32).

39. The composition according to any one of claims 1-38, wherein the monomers assemble to form a multimeric nanoparticle.

40. The composition according to claim 39, wherein the multimeric nanoparticle forms a two-component dodecamer (P1 / P2).

41. The composition according to claim 39 or 40, which comprises from 1-4 unique antigens.

42. The composition according to claim 39 or 40, which comprises from 1-48 antigens.Leydig 774102 NIH E-182-2024-0-PC-018543. The composition according to claim 39, wherein the multimeric nanoparticle forms a single component tetradecamer (Cpn60 or ClpP).

44. A genetic vector comprising nucleic acid sequences encoding Pdxl and Pdx2.

45. The genetic vector according to claim 44, which is a plasmid suitable for expressing both Pdxl and Pdx2 in a host cell.

46. The genetic vector according to claim 44 or 45, wherein the host cell is prokaryotic.

47. A method for immunizing a patient, the method comprising administering the composition according to any one of claims 1-43 to a patient in a location and according to a regimen sufficient to confer immunity to the patient.

48. Use of the composition according to any one of claims 1-43 for vaccinating a patient to confer immunity to the patient.

49. The method according to claim 47 or the use according to claim 48, wherein the immunity is against Plasmodium species.

50. The method or use according to any one of claims 47-49, wherein the patient is human.

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