Norovirus s particle based vaccines and methods of making and using the same

Abstract

To provide vaccine compositions, in particular, polyvalent icosahedral compositions for antigen presentation.SOLUTION: A polyvalent icosahedral composition for antigen presentation comprising an S particle is provided, where the S particle comprises a recombinant fusion protein comprising a) a norovirus (NoV) S domain protein; b) a linker protein domain operatively connected to the norovirus S domain protein; and c) an antigen protein domain operatively connected to the linker.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 477,481, filed March 28, 2017, the contents of which are incorporated in their entirety and for all purposes.

[0002] Statement regarding federally funded research This invention was made with Government support under R21 AI092434-01A1 and R56 AI114831-01A1 to XJ awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0003] RV causes severe acute gastroenteritis mainly in infants and young children, resulting in approximately 200,000 deaths, 2.3 million hospitalizations, and 24 million outpatient visits in children under 5 years of age worldwide each year [25-27]. The two current RV vaccines, RotaTeq (Merck) and Rotarix (GlaxoSmithKline, GSK), are effective in protecting children from severe RV cases in many developed countries [28, 29]. However, these have not shown satisfactory efficacy in most developing countries in Africa and Asia, where most RV infections, morbidity, and mortality occur [30-32], making an RV vaccine most needed. Summary of the Invention

[0004] Disclosed herein is a vaccine composition, particularly a multivalent icosahedral composition for antigen presentation. The disclosed composition may comprise an S particle composed of a recombinant fusion protein. The recombinant fusion protein may comprise a Norovirus (NoV) S domain protein, a linker protein domain operably linked to the Norovirus S domain protein, and an antigen protein domain operably linked to the linker. The disclosed composition may be used to provide. [Brief description of the drawings]

[0005] The application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0006] Those skilled in the art will understand that the drawings, described below, are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0007] [Figure 1] Native norovirus (NoV) S domain proteins assembled into particles or complexes with low efficiency. (A) Schematic of expression constructs of GST-S domain fusion proteins showing the location of the thrombin cleavage site and hinge. (B and C) SDS-PAGE analysis of GST-S fusion protein (GST-S, ~51 kDa) (B) and free S protein (~25 kDa) (C). (D) EM micrograph of S protein (arrow), showing almost no assembled S particles. (E) Elution curve of gel filtration chromatography of S protein through a size-exclusion column (Superdex 200). The gel filtration column was calibrated with the Gel Filtration Calibration Kit and purified recombinant NoV P particles [21, 22], small P particles

[20] , and P dimer

[11] . The elution positions of blue dextran 2000 (~2000 kDa, void), P particles (~830 kDa), small P particles (~420 kDa), P dimer (~69 kDa), and aprotinin (~6.5 kDa) are indicated. (F) SDS-PAGE analysis of proteins from two peaks, peak 1 (fractions #15 and 16) and peak 2 (fractions #28 and 29). In all SDS PAGEs, lane M is a prestained protein marker of the indicated molecular weight. Trace S protein bands at ~42 kDa and ~16 kDa were seen in (B), (C), and (F), respectively. [Diagram 2] Identification of the exposed protease site in the S domain. (A) N-terminal sequencing of the protease-cleaved S protein resulted in a penta-residue sequence, NAPGE. (B) The S domain sequence shows the same protease residue sequence, NAPGE (underlined), and indicates the protease cleavage site (asterisk). The C-terminal hinge (underlined), the four-residue linker (GGGG), and the terminally fused Hisx6 peptide are shown. The calculated molecular weight of this recombinant S domain protein is also shown. (C) Sequence alignment between all GII Norovirus representations showed that the protease site is highly conserved (positions 69 and 70 highlighted). (D) Inspection of the partial GII NoV shell structure in different colored cartoon representations at the three-fold axis shows the exposed proteinase site formed by R69 (red)-N70 (cyan) in a sphere representation. Left panel: top view, right panel: side view. [Diagram 3]Production and characterization of SR69A protein and S60 particles. (A) Schematic of the expression construct of SR69A protein showing the hinge, linker (GGGG), and Hisx6 peptide (orange balls labeled as H). Its complete sequence is shown in Figure 2B. (B) SDS-PAGE analysis of SR69A protein (~25 kDa). Lanes 1-5 were elution fractions from TALON CellThru Resin. Lane M represents pre-stained protein markers of the indicated molecular weights. (C) EM micrographs of SR69A protein showing self-assembled S60 particles of uniform size. (D and E) Analysis of SR69A protein by gel filtration chromatography (D) followed by SDS PAGE analysis of the elution peak (E). (D) Elution curve of gel filtration chromatography of SR69A protein through a size exclusion column (Superdex 200, 10 / 300 GL). The gel filtration column was calibrated as done in Figure 1E. The elution positions of blue dextran 2000 (approximately 2000 kDa), P particles (approximately 830 kDa), P dimer (approximately 69 kDa), and aprotinin (approximately 6.5 kDa) are indicated by (x) as 1, 2, 3, and 4, respectively. (E) SDS-PAGE analysis of SR69A protein from the three major peaks of gel filtration (D), where lane C is control SR69A protein before loading onto the size-exclusion column, lane M is pre-stained protein markers of the indicated molecular weights, lanes 8 and 9 are from fractions #8 and 9 of peak 1, lane 16 is from fraction #16 of peak 2, while lane 19 is from fraction #19 of peak 3. (F) Electrospray ionization mass spectrometry (ESI-MS) analysis of SR69A protein. ESI-MS was acquired in positive ion mode on 80 μM SR69A protein (based on monomer) in aqueous ammonium acetate (200 mM, pH 6.8 and 25 °C). Both the SR69A domain monomer (25.047 kDa) and dimer (50.095 kDa) were detected. A broad feature centered at m / z ≈15,500 was observed.Although the mass resolution was insufficient to establish the charge state, the MW of these ions was estimated to be approximately 1.47 MDa based on the reported m / z of a large protein complex

[61] , which corresponds to 60 charged S60 particles. [Figure 4] Structural modeling of S60 particles based on the known crystal structure of 60-valent feline calicivirus VLP (PDB#: 4PB6). (A) EM micrograph showing S60 particles. (B-D) Structure of SR69A protein monomer (orange) in cartoon representation (B) and S60 particle in surface representation in 5-fold (C) and 2-fold (D) axes, respectively. The exposed C-terminal hinge (surface representation) is shown in green. (E-G) Structure of SR69A protein monomer (orange) in cartoon representation with C-terminal fused linker (magenta) and Hisx6 peptide (light blue) in dot representation (E) and the resulting surface representation of S60 in 5-fold (F) and 2-fold (G) axes, respectively. The exposed C-terminal hinge, linker, and Hisx6 peptide are shown in dot representation. [Diagram 5]Characterization of S60-VP8 chimeric particles. (A) Schematic of the SR69A-VP8 chimeric protein. The VP8 antigen of rotavirus (green) was fused to the hinge via a linker (HHHH). The Hisx6 peptide (orange) was fused to the C-terminus of the VP8 antigen. (B) SDS-PAGE analysis of the SR69A-VP8 protein (~45 kDa). (C) Gel filtration chromatography of the SR69A-VP8 protein through a size-exclusion column (Superdex 200, 10 / 300 GL). The column was calibrated as done in Figure 1E. The elution positions of blue dextran 2000 (~2000 kDa), P particles (~830 kDa), P dimer (~69 kDa), and aprotinin (~6.5 kDa) are indicated by (x), labeled 1, 2, 3, and 4, respectively. (D) EM micrograph of S60-VP8 particles from peak 1 (C) of gel filtration. (F) Electrospray ionization mass spectrometry (ESI-MS) analysis of SR69A-VP8 protein. ESI-MS was acquired in positive ion mode in aqueous ammonium acetate (200 mM, pH 6.8 and 25 °C) of 80 μM SR69A-VP8 protein (based on monomer). Both SR69A-VP8 monomer (44.950 kDa) and degradation products (19.990 kDa) were detected. A broad feature centered at m / z ≈23,700 was observed. Although the mass resolution was insufficient to establish the charge state, the MW of these ions was estimated to be approximately 3.4 MDa based on the reported m / z of a large protein complex

[61] , which corresponds to the MW of a 60-charged SR69A-VP8 particle. [Figure 6]Further stabilization of S60-VP8 particles by introduction of inter-S domain disulfide bonds. (A and B) Structural analysis of the GII.4 shell structure. (A) The partial shell structure of GIII.4 NoV at the three-fold axis (WJ, unpublished data) revealed that V57 and Q58 of the S domain are sterically close to M140' and S136' of the neighboring S domain, respectively. The six S domains are shown in gray cartoon representation, and the four aforementioned amino acids are shown in sphere representations of different colors. (B) Close-up of the steric relationship between V57 (red) / Q58 (cyan) of one S domain and S136' (green) / M140' (orange) of the neighboring S domain with a distance of 5.7-5.9 Å. (C-E) Characterization of the SR69A / V57C / M140C-VP8 protein. (Protein sequence shown in SEQ ID NO:31.) (C) Expression construct of SR69A / V57C / M140C-VP8 protein. (D) SDS PAGE analysis of SR69A / V57C / M140C-VP8 protein. Lanes 1, 2, 3, and 4 are four eluted protein fractions from the affinity column. 15 μl of each fraction was loaded in each lane. M is prestained protein marker. (E) Elution curve of gel filtration chromatography of SR69A / V57C / M140C-VP8 protein through a size exclusion column (Superdex 200, 10 / 300 GL). The gel filtration column was calibrated as done in FIG. 1E. The elution positions of blue dextran 2000 (approximately 2000 kDa), P particles (approximately 830 kDa), P dimer (approximately 69 kDa), and aprotinin (approximately 6.5 kDa) are indicated with (x) labeled 1, 2, 3, and 4, respectively. (F-J) Characterization of SR69A / V57C / Q58C / S136C-VP8 protein. (Protein sequence shown in SEQ ID NO: 32) (F) Expression construct of SR69A / V57C / Q58C / S136C-VP8 protein. (G) SDS PAGE analysis of SR69A / V57C / Q58C / S136C-VP8 protein. Lanes 1, 2, and 3 are the three eluted protein fractions from the affinity column. 10 μl of each fraction was loaded into each lane.(H) Gel filtration analysis of SR69A / V57C / Q58C / S136C-VP8 protein through a size-exclusion column (Superdex 200, 10 / 300 GL). The gel filtration column was calibrated as done in Figure 1E. The elution positions of four proteins with different MW are indicated in (E). (I) EM micrograph of S60-VP8 particles from peak 1 (H) of the gel filtration. (J) SDS PAGE analysis of proteins from peak 1 (fractions #7-10), peak 2 (fraction #21), and peak 3 (fraction #23). Lane C is the control protein before loading onto the column. [Figure 7] Structure of S60-VP8 particle. (A-C) The 3D structure of S60-VP8 particle was reconstructed by cryoEM technique. (A) Surface structure of S60-VP8 particle at the 5-fold axis. (B and C) Slice structure of the central slice (B) and the latter half (C) of S60-VP8 particle showing the external and internal structure. The internal S60 particle (S) and protruding VP8 antigens are shown. The radii based on the color scheme are indicated. "5" indicates the 5-fold axis. (D-F) Fitting of the 60-valent FCV shell structure (red, cartoon representation) to the cryoEM density map of S60-VP8 particle (transparent gray). The fitting result is shown in three transparent slices showing the first half (D), the central slice (E), and the latter half (F) of the S60-VP8 particle in a frontal view. (G and H) Fitting of 60 copies of the VP8 crystal structure of P[8]RV (PDB code: 2DWR) to the protruding region of the S60-VP8 particle cryoEM density map. The fitted FCV shell crystal structure within the S60 particle region of the S60-VP8 particle is shown in cartoon representation (red), while the fitted VP8 crystal structure within the protruding region is shown in blue cartoon representation. (I) S60-VP8 particle model based on the fitting results above. The internal S60 particle is shown in red cartoon representation, and the 60 protruding VP8 antigens are shown as light blue dot representations. [Figure 8]S60-VP8 particles formed a peak after CsCl density gradient centrifugation. S60-VP8 particles were loaded onto a CsCl density gradient. After ultracentrifugation, S60-VP8 particles in the fractionated gradient were detected by antibodies specific for P[8]RV VP8 (A) and GII.4 NoV VLP (B), respectively. In both cases, a defined peak of S60-VP8 particles was detected in the middle of the gradient. [Figure 9] We demonstrated that S60 particle-displaying VP8 retains ligand-binding function. (A) Glycan-binding assays showed that S60-VP8 particles bound synthetic oligosaccharides representing H1 and Leb antigens, but not those representing Ley antigens. S60 particles without VP8 did not bind any of the three antigens. [Figure 10] We show that S60-VP8 particles enhanced immunogenicity against presented RV VP8 antigen. Mice were immunized (N=6) with the same dose / administration of S60-VP8 particles, free VP8 antigen, and S60 particles without VP8, respectively, followed by measurement of VP8-specific IgG responses (A), as well as the 50% blocking titer (BT50) against RV VP8-ligand interaction (B) and neutralizing activity against RV infection (C) of the resulting mouse sera. (A) VP8-specific IgG responses elicited by S60-VP8 particles, free VP8 antigen, and S60 particles, respectively. (B) BT50 against RV VP8-ligand interaction by mouse sera after vaccination with the same three immunogens, respectively. (C) Neutralizing activity against RV infection of cultured cells by mouse sera after immunization with the same three immunogens, respectively. Statistical differences between data groups are indicated by asterisks (*P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] definition Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the relevant art. In case of conflict, this document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used to practice or test the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0009] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0010] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on the limitations of the method of measuring or determining the value, e.g., the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When a particular value is described in this application and claims, the term "about" should be assumed to be within an acceptable error range of the particular value, unless otherwise indicated.

[0011] As used herein, the term "effective amount" refers to an amount of one or more active ingredients sufficient to produce a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0012] The terms "individual," "host," "subject," and "patient" are used interchangeably to refer to an animal that is the object of treatment, observation, and / or experiment. Generally, the terms refer to human patients, although the methods and compositions may be equally applicable to non-human subjects, such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.

[0013] As used herein, the term "antigen" may be used interchangeably with the terms "immunogen" and "immunogenic antigen," as defined below. Technically speaking, an antigen is a substance that can bind to the products of an immune response, but not necessarily induce an immune response (i.e., all immunogens are antigens, but not vice versa), however, the antigens discussed herein as the subject of the present invention are assumed to be immunogenic antigens, even when referred to as antigens.

[0014] The term "fusion protein" refers to a protein created by translation of a fusion gene, resulting in a single polypeptide possessing functional properties derived from each of the original proteins.

[0015] The term "immunity" refers to the state of having sufficient biological defenses to ward off infection, disease, or other biological invasion by disease-causing organisms.

[0016] The term "immunogenic" refers to the ability of an immunogen to elicit a humoral and / or cellular immune response.

[0017] The terms "immunogen" and "immunogenic antigen" refer to a specific type of antigen that is capable of inducing or eliciting an adaptive immune response in the form of the production of one or more antibodies.

[0018] The terms "immunogenic response" and "immune response" refer to a change in the reactivity of an organism's immune system in response to an immunogen. This may involve antibody production, induction of cell-mediated immunity, complement activation, or the development of acquired immunity, or immune tolerance to a particular disease or pathogen.

[0019] The terms "immunization" and "vaccination" refer to the deliberate induction of an immune response, involving the effective manipulation of the immune system's natural specificity, as well as its inducibility. The principle behind immunization is the introduction of an antigen derived from a disease-causing organism, which stimulates the immune system to develop protective immunity against that organism, but does not itself cause the pathogenic effects of that organism.

[0020] The term "infection" refers to the invasion of animal or plant host tissues by a pathogen and the proliferation of the pathogen within the body, and the body's response to the pathogen and any toxins it may produce.

[0021] The term "norovirus", "NoV", "Norwalk-like virus", or "NLV" refers to viruses of the Norovirus genus of the Caliciviridae family, including, but not limited to, Norwalk virus ("NV"), MOH, Mexico, VA207, VA387, 02-1419, C59, VA115, Hawaii, Snow Mountain, Hillington, Toronto, Leeds, Amsterdam, Idaho Falls, Rosedale, Grimsby, Southampton, Desert Shield, Birmingham, and White River Cap. NoVs cause acute gastroenteritis in humans.

[0022] As used herein, the letter "S" refers to the particles described, e.g., S69A. / 58C / 140CWhen used with -VP8, it means "S domain", which refers to the S-VP8 protein having the 69A / 58C / 140C mutation. In other embodiments, the nomenclature used may be, for example, S, with "69A / 58C / 140C" shown as a superscript.

[0023] The term "vaccine" refers to a biological preparation or composition that improves immunity to a particular disease. Vaccines are examples of immunogenic antigens that are purposefully administered to induce an immune response in a recipient.

[0024] The terms "multivalent vaccine" and "polyvalent vaccine" refer to a vaccine designed to immunize against two or more strains of the same microorganism (such as NoV), or against two or more different microorganisms.

[0025] Norovirus (NoV) is a member of the Norovirus genus in the Caliciviridae family, and causes epidemic acute gastroenteritis in humans that exhibits significant morbidity and mortality [4, 5]. Structurally, NoV virions are encapsulated by a protein capsid composed of a single major structural protein, the capsid protein or viral protein 1 (VP1). The crystal structure of the NoV capsid revealed that NoV VP1 contains two major domains, the N-terminal shell (S) and the C-terminal protrusion (P) domain, connected by a short hinge [6]. The S domain builds the internal icosahedral shell that supports the basic scaffold of the NoV virion, while the P domain constitutes the dimeric protrusion [7–10] that stabilizes the NoV capsid and recognizes cell surface glycans as host adhesins or receptors [11–14].

[0026] In vitro expression of full-length NoV VP1 via a eukaryotic system resulted in the auto-formation of 180-valent virus-like particles (VLPs) structurally and antigenically similar to the authentic viral capsid [6, 15], whereas production of the P domain via an E. coli system formed P dimers structurally indistinguishable from those of the NoV capsid [7-11, 16-19]. In addition, modified NoV P domains assembled into different higher order particles or complexes, including 12-valent small P particles

[20] , 24-valent P particles [21, 22], and 36-valent P complexes

[23] .

[0027] Unlike the P domain, the S domain has been less studied, but "thin-laminated" S particles, possibly equivalent to the 180-valent shell of the NoV capsid, have been reported through expression of the S domain in a baculovirus / insect cell system [11, 24]. In this study, applicant developed a novel technology to produce uniform 60-valent S particles, termed S60 particles, via a simple E. coli system and applied them as a multifunctional vaccine platform for antigen presentation for subunit vaccine development against rotavirus (RV) and other pathogens.

[0028] RV causes severe acute gastroenteritis mainly in infants and young children, resulting in approximately 200,000 deaths, 2.3 million hospitalizations, and 24 million outpatient visits in children under 5 years of age worldwide each year [25-27]. The two current RV vaccines, RotaTeq (Merck) and Rotarix (GlaxoSmithKline, GSK), are effective in protecting children from severe RV cases in many developed countries [28, 29]. However, these have not shown satisfactory efficacy in most developing countries in Africa as well as Asia, where most infections, morbidity, and mortality from RV occur [30-32], making RV vaccines of utmost need. The applicant's recent studies suggested that the low efficacy of RV vaccines in developing countries may be due to a mismatch of vaccine P types with the shift in predominant RV P types in low- and middle-income countries [33, 34]. In addition, both current live attenuated vaccines remain expensive, and replication of vaccine RV in the intestine after oral administration may be responsible for an increased risk of intussusception in vaccinated children.[35–41] Therefore, a neo-RV vaccine that can overcome the aforementioned limitations of the two current live RV vaccines is needed.

[0029] The P type of RV is determined by viral protein 4 (VP4), which constitutes the spike protein of RV virions. Structurally, each spike protein contains two major parts: the stalk formed by VP5 and the distal head constructed by VP8

[42] . VP5 and VP8 are cleavage products of VP4 by trypsin. VP8 is involved in the interaction with RV host attachment factors or receptors, a group of cell surface glycans, including histo-blood group antigens (HBGAs) [33, 43-45]. Previous studies have shown that VP8 antigen induces neutralizing antibodies that inhibit RV infection and replication in cultured cells and protect immunized mice from RV infection [46, 47], and thus VP8 antigen is an important vaccine target against RV [46-49].

[0030] However, many defined neutralizing antigens, including RV VP8, face the common problem of low immunogenicity for non-replicating vaccine development due to their small size with low valency. This problem can be solved by fusion or conjugation to large multivalent protein platforms to enhance the immunogenicity of the antigen. In this study, applicants have provided solid evidence supporting the significant enhancement of the immunogenicity of RV VP8 antigen after being presented by NoV S60 particles as an effective vaccine platform. Applicants' data show that S60-VP8 particles are easily generated, stable, and highly immunogenic for the presented RV VP8 antigen, and thus a promising subunit vaccine against RV infection.

[0031] Homotypic interactions of viral capsid proteins are common and facilitate the self-assembly of viral capsids. By taking advantage of such interactions of the norovirus shell (S) domains that naturally build the inner shell of the norovirus capsid, applicants have developed a method for the production of 60-valent icosahedral S60 particles via a simple E. coli system. This can be achieved by several modifications to the S domain, such as the R69A mutation that destroys the exposed proteinase cleavage site and the triple cysteine ​​mutation (V57C / Q58C / S136C) that establishes an S-domain inter-disulfide bond to strengthen the S-domain interaction. Multivalent S60 particles with 60 exposed S-domain C-termini provide an ideal platform for antigen presentation to improve immunogenicity against presented antigens for vaccine development. This was demonstrated by constructing chimeric S60 particles that display 60 rotavirus (RV) VP8 proteins, the major RV neutralizing antigen. These S60-VP8 particles were easily produced and elicited high IgG responses in mice against the presented VP8 antigen. Mouse antisera after immunization with S60-VP8 particles showed high blocking of RV VP8 binding to its glycan ligands and high neutralizing activity against RV infection in cultured cells. The three-dimensional structures of S60 and S60-VP8 particles were studied. Finally, S60 particles can also present other antigens, supporting the notion that S60 particles are a multifunctional vaccine platform.

[0032] Disclosed herein are methods and compositions that can be used to form multivalent vaccine compositions, particularly using modified Norovirus S particles.

[0033] In one embodiment, a multivalent icosahedral composition for antigen presentation is disclosed.The composition can include S particles, and the S particles can include a recombinant fusion protein comprising a Norovirus (NoV) S domain protein, a linker protein domain operably linked to the Norovirus S domain protein, and an antigen protein domain operably linked to the linker.

[0034] The composition generally has an icosahedral symmetric structure, hi one embodiment, the composition comprises 60 sites for antigen presentation.

[0035] In one embodiment, the Norovirus S domain protein is that of a Calicivirus, which may be characterized as having 180 copies of a single capsid protein.

[0036] In one embodiment, the Norovirus S domain protein comprises a mutation at the proteinase cleavage site of the NoV S domain protein, which mutation renders the site resistant to trypsin cleavage. One or more mutations can be made at the site, provided that the mutation effectively destroys the trypsin cleavage site. Modifications to the site that achieve such effects will be readily understood by those skilled in the art. In one embodiment, the mutation can be at position 69 or 70. In one embodiment, the mutation can be at position R69. In certain embodiments, the mutation can be a change to any amino acid other than K (lysine) sufficient to destroy the proteinase cleavage site. In certain embodiments, the mutation is R69A. In other embodiments, the mutation can be at position N70, for example, the mutation can be at any amino acid other than P (proline) sufficient to destroy the proteinase cleavage site.

[0037] In one embodiment, the Norovirus S domain protein may contain sufficient mutations to provide a non-natural disulfide bond binding site. The Norovirus S domain protein may contain at least two amino acid mutations (sterically close to each other) at cysteine ​​residues sufficient to provide at least one non-natural disulfide bond binding site, or in other embodiments, at least two non-natural disulfide bond binding sites, or at least three non-natural disulfide bond binding sites, between adjacent S domain proteins of a multivalent icosahedral S particle. In certain embodiments, the mutations may be selected from V57C, Q58C, S136C, M140C, or a combination thereof.

[0038] In one embodiment, the linker may comprise an amino acid sequence of sufficient length to provide space and certain flexibility between the S domain protein particle and the presented antigen. The linker is usually a short peptide of 1-10 amino acid units, or 3-6 amino acids, connecting the C-terminus of the S domain to the presented antigen. The linker provides space and some flexibility between the S60 particle and the presented antigen, aiding in independent folding of the S domain and the presented antigen. Longer linkers may be used if necessary. The amino acid length of the linker must be sufficient to allow flexibility of the protein domain to form the claimed composition.

[0039] The disclosed compositions are ideally suited for antigen presentation. Suitable antigens can be easily determined by those skilled in the art. Exemplary antigens are disclosed herein. In certain embodiments, antigen protein domains can be selected by size in addition to immunogenicity, and can encode antigens with sizes from 8 amino acids up to about 300 amino acids, or from 8 amino acids up to about 400 amino acids, or from 8 amino acids up to about 500 amino acids. As can be easily understood by those skilled in the art, the size of antigens can vary widely, and the present compositions can be used to present a variety of different antigens to induce immune responses.

[0040] In one embodiment, the polyvalent icosahedral composition may include an antigenic protein domain that is a rotavirus (RV) antigen. In one embodiment, the antigenic protein domain may include an RV spike protein antigen (VP8 antigen). In further embodiments, the antigen may include a TSR antigen of the sporozoite surface protein (CSP) of the malaria parasite Plasmodium falciparum, a receptor binding domain of the HA1 protein and M2e epitope of influenza A virus, a P domain antigen of hepatitis E, a surface spike protein of astrovirus, and combinations thereof. Again, such antigens are merely exemplary, and such listing is not intended to limit the scope of the claims. Exemplary sequences include those of SEQ ID NO:34 and SEQ ID NO:35: human rotavirus VP8 antigen, SEQ ID NO:42 and SEQ ID NO:43: P domain antigen of Hepatitis E virus (HEV), SEQ ID NO:44 and SEQ ID NO:45: surface spike protein antigen of avian AstV (see, e.g., (GenBank AC#:NP987088, residues 423-630)), SEQ ID NO:46 and SEQ ID NO:47: HA1 antigen (H7) of influenza A virus, SEQ ID NO:48 and SEQ ID NO:49: TSR antigen of sporozoite surface protein of Plasmodium falciparum, and SEQ ID NO:50 and SEQ ID NO:51: M2E epitope of influenza A virus. It will be understood that the antigenic sequence used to generate the antigenic peptides may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a reference nucleic acid sequence, provided that the resulting antigen elicits at least a partial immune response in an individual administered a composition bearing the antigen.

[0041] The recombinant fusion protein is a subunit of the disclosed vaccine composition. Further disclosed herein is a recombinant fusion protein that can form the basis of a multivalent icosahedral composition. The fusion protein can include a Norovirus (NoV) S domain protein having the above-mentioned trypsin site and the above-mentioned mutation to the added cysteine ​​site, a linker protein domain operably linked to the Norovirus S domain protein having the above-mentioned mutation, and an antigen protein domain operably linked to the linker. The characteristics of each part of the fusion protein are as described above.

[0042] In addition to the above S particles, the disclosed compositions may further comprise one or more pharma- ceutically acceptable carriers, which may include any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption retardants, and the like. The disclosed S particles may be provided in saline. Optionally, protective agents, such as antibacterial active agents, such as gentamicin, merthiolate, and the like, may be included. The compositions may further include stabilizers, such as sugars, trehalose, mannitol, saccharose, and the like, to extend and / or maintain the shelf life of the product. Those skilled in the art will appreciate that the compositions herein may incorporate known injectable physiologically acceptable sterile solutions. To prepare a ready-to-use solution for parenteral injection or infusion, isotonic aqueous solutions, such as, for example, saline or corresponding plasma protein solutions, are readily available. In addition, the immunogenic and vaccine compositions of the present invention may include diluents, isotonicity agents, stabilizers, or adjuvants. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonicity agents can include, inter alia, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, inter alia, albumin and alkali salts of ethylenediaminetetraacetic acid. Suitable adjuvants will be understood by those skilled in the art.

[0043] In one embodiment, a container is disclosed that contains at least one dose of the immunogenic composition disclosed herein. The container may contain 1-250 doses of the immunogenic composition, or in other embodiments, 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition. In one embodiment, each of the containers may contain two or more doses of the immunogenic composition and may further include an antibacterial active agent. These agents may include, for example, antibiotics such as gentamicin and merthiolate.

[0044] Further aspects relate to kits. The kit may include any of the above containers and instructions containing information regarding delivery of the above immunogenic compositions. For example, instructions regarding intramuscular application of at least one dose may be provided to reduce the severity of clinical symptoms associated with infection with the antigens disclosed herein. The kit and / or composition may further include an immunostimulant, such as keyhole limpet hemocyanin (KLH) or incomplete Freund's adjuvant (KLH / ICFA). Other immunostimulants known to those skilled in the art may also be used.

[0045] In one embodiment, a method for producing the disclosed polyvalent icosahedral structure is disclosed. The method includes the steps of: a) producing a first region comprising a modified NoV S domain protein, the modification including a mutation sufficient to destroy an exposed protease cleavage site (the mutation prevents proteolysis, preferably the R69A mutation) and introduce one or more mutations in the Norovirus (NoV) S domain protein sufficient to form an S domain interprotein disulfide bond, such as a mutation selected from V57C, Q58C, S136C, and M140C, and combinations thereof; and b) recombinantly expressing the first region with the modified NoV S domain protein using a linker and an antigen. In certain embodiments, the composition can be effectively produced in E. coli.

[0046] In one aspect, a method of eliciting an immune response in an individual in need thereof is disclosed. In this aspect, the method may include administering the vaccine composition disclosed above to an individual in need thereof. The disclosed composition may be administered to an individual according to any method known in the art, and it is readily understood that optimal administration (including route and amount) does not require undue experimentation. The vaccine composition may be administered prophylactically to an individual suspected of future exposure to an antigen incorporated in the vaccine composition. In certain aspects, a method is provided that provides an immune response that protects an individual receiving the composition from infection, or reduces or alleviates the severity of clinical symptoms associated with infection. Infections may include, for example, malaria, influenza A, hepatitis E, and astrovirus infections. The dosing regimen may be a single dose schedule or a multiple dose schedule (e.g., including booster doses) in which unit dosage forms of the composition are administered at different times. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of an antigen composition disclosed herein in an amount sufficient to produce the desired effect, the composition being provided in association with a pharma- ceutically acceptable excipient (e.g., a pharma-ceutically acceptable diluent, carrier, or vehicle). Vaccines may be administered together with other immunomodulatory agents. EXAMPLES

[0047] The following non-limiting implementations are provided to further illustrate the embodiments of the present invention disclosed herein.Those skilled in the art should understand that the techniques disclosed in the following examples represent approaches that have been found to work well in the implementation of the present invention, and therefore can be considered to constitute examples of the modes for its implementation.However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed, and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0048] The generation of new biomaterials by bioengineering is a rapidly growing field in modern medicine. Typical examples include various multivalent protein nanoparticles and complexes constructed by utilizing the self-assembly function of viral capsid proteins [1-3]. Viral capsid proteins are involved in many fundamental functions required for the viral life cycle, especially viral attachment and entry, and therefore can elicit neutralizing antibodies against viral infections after immunization in humans and animals. This supports the notion that viral capsid proteins are excellent vaccine targets against the corresponding viral pathogens. Indeed, various capsid protein particles and complexes have been developed and used as non-replicating subunit vaccines to combat various infectious diseases that claim millions of lives every year [1-3]. Unlike conventional live attenuated and inactivated virus vaccines, which require the cultivation of infectious virions and are therefore associated with certain safety concerns, non-replicating subunit vaccines derived from bioengineered viral capsid proteins do not involve infectious agents and are therefore safer and less costly to produce than conventional vaccines. Non-replicating subunit vaccines therefore represent a new generation of innovative vaccine strategies.

[0049] material and method Plasmid constructs. 1) Expression constructs of glutathione-S-transferase (GST)-tagged S domain proteins. The S domain with the hinge coding sequence of GII.4 NoV strain VA387 (GenBank AC#:AY038600.3; residues 1-221) was inserted into the multiple cloning site of pGEX-4T-1 vector (GST Gene Fusion System, GE Healthcare Life Sciences) via the BamH1 / Sal I sites. The resulting S domain proteins had an N-terminal GST with a thrombin cleavage site in between. 2) Hisx6-fused S R69APlasmid constructs for domain expression. The same NoV S domain hinge coding sequence with the R69A mutation was inserted into the multiple cloning site of the pET-24b vector (Novagen) via the BamH1 / Not I sites. The resulting S domain protein had a Hisx6 peptide fused to its C-terminus. 3) S R69A DNA construct for expression of RV VP8 chimeric protein. A DNA fragment containing the RV VP8 coding sequence of P[8] human RV strain BM14113, corresponding to the amino acid sequence from 64 to 231 of VP8 of the WA strain (GenBank AC#: VPXRWA), was inserted into S. R69A The domains were fused to the hinge end with a linker (four histidines) between them. RV strain BM14113 was isolated directly from an RV-positive stool sample

[45] . A Hisx6-peptide was added to the C-terminus of the VP8 antigen for purification purposes. 4)S R69A / V57C / M140C-VP8, S R69A / V57C / Q58C / S136C-VP8, and S R69A / V57C / Q58C / S136C / M140C-VP8 chimeric protein expression construct. This DNA construct is R69A The other two (V57C / M140C), three (V57C / Q58C / S136C), or four (V57C / Q58C / S136C / M140C) mutations were introduced by site-directed mutagenesis into the expression construct of the S-VP8 chimeric protein. R69A Plasmid construct for expression of the / V57C / Q58C / S136C-mVP8 chimeric protein. This construct contains S R69A The DNA sequence was similar to that of the / V57C / Q58C / S136C-VP8 construct, but the VP8 coding sequence was replaced with that of the murine RV EDIM (epidemic diarrhea of ​​infant mice) strain VP8

[50] . In addition, other S ‐presenting antigens of various pathogens were identified, including the surface TSR antigen of the sporozoite surface protein (CSP) of the Plasmodium falciparum parasite strain 3D7 (GenBank AC#: CAB38998, residues 309–375). R69ADNA constructed for the V57C / Q58C / S136C-based chimeric particles

[51] , the M2e epitope of influenza A virus [52, 53], and the P domain antigen of hepatitis E virus [54–56] were inserted into S , in which the RV VP8 coding sequence was replaced with one encoding the corresponding antigen. R69A The V57C / Q58C / S136C-VP8 chimeric protein was constructed using as the starting construct.

[0050] Recombinant protein production and purification. Recombinant GST-fusion and Hstx6-fusion proteins were expressed in E. coli (BL21, DE3) as previously described (11, 47, 53, 56). The resulting recombinant proteins were purified using Sepharose 4 Fast Flow purification resin (GE Healthcare Life Sciences) for GST-tagged and TALON CellThru Resin (ClonTech) for Hisx6-peptide fusion proteins according to the manufacturer's instructions. GST can be removed from the target protein by cleavage with thrombin (GE Healthcare Life Sciences), while the GST-fusion protein remains bound to the purification resin.

[0051] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein quantification. Purified proteins were analyzed by SDS-PAGE using a 10% resolving gel. Proteins were quantified by SDS-PAGE using serially diluted bovine serum albumin (BSA, Bio-Rad) as a standard on the same gel

[46] .

[0052] Gel filtration chromatography. This was performed with an AKTA Fast Performance Liquid Chromatography System (AKTA Pure 25L, GE Healthcare Life Sciences) using a size exclusion column (Superdex 200, 10 / 300 GL, GE Healthcare Life Sciences) to analyze the size distribution of proteins and protein complexes as previously described [11, 53, 57]. The column was calibrated with a gel filtration calibration kit (GE Healthcare Life Sciences) and purified NoV P particles (~830 kDa)

[57] , small P particles (~420 kDa)

[20] , and P dimer (~69 kDa)

[11] as previously described [53, 55]. Proteins from the elution peak were analyzed by SDS-PAGE.

[0053] Cesium chloride (CsCl) density gradient ultracentrifugation. 0.5 mL of resin-purified S60-VP8 particles were mixed with 11 mL of CsCl solution with a density of 1.300 g / mL and packed into a 12 ml centrifuge tube. After centrifugation for 45 h at 288,000 g in an Optima L-90K ultracentrifuge (Beckman Coulter) using a SW41Ti rotor, the gradient was fractionated into 23 fractions of 0.5 mL each by bottom puncture. The CsCl density of the fractions was determined based on the refractive index. S60-VP8 particles in the fractions were analyzed by ELISA, in which individual fractions were diluted 20-fold in PBS and coated onto microtiter plates. The coated proteins were detected by NoV VLP-specific and RV VP8-specific antibodies.

[0054] Electron microscopy. Protein samples were prepared for electron microscopy (EM) examination of particle formation using 1% ammonium molybdate as a staining solution

[22] . Specimens were observed under an EM10 C2 microscope (Zeiss, Germany) at 80 kV and magnifications ranging from 10,000 to 40,000.

[0055] Electrospray ionization mass spectrometry (ESI-MS). All ESI-MS measurements were performed in positive ion mode using a Synapt G2S quadrupole ion mobility separation time-of-flight (Q-IMS-TOF) mass spectrometer (Waters, Manchester, UK) equipped with a nanoflow ESI (nanoESI) source. Each sample solution was prepared in 200 mM ammonium acetate buffer (pH 6.8, 25 °C) and loaded into a nanoESI tip generated from a borosilicate capillary (1.0 mm outer diameter, 0.68 mm inner diameter) stretched to approximately 5 μm using a P-1000 micropipette puller (Sutter Instruments, Novato, CA). To perform ESI, a platinum wire was inserted into the nanoESI tip and a voltage of 1.10 kV was applied. A source temperature of 60 °C was used. Cone, trap, and transfer voltages were 50 V, 5 V, and 2 V, respectively, trap gas flow rate was 60 mL min-1, and all other parameters were set to default values. Data collection and processing were performed using Waters MassLynx software (version 4.1).

[0056] N-Terminal Amino Acid Sequencing. SDS-PAGE gel slices containing the cleaved S domain proteins were excised and subjected to N-terminal amino acid sequencing at the Protein Facility of Iowa State University (http: / / www.protein.iaState.edu / ) using a 494 Procise Protein Sequencer / 140C Analyzer from Applied Biosystem, Inc.

[0057] Immunization of mice. Three to four week old BALB / c mice (Harlan-Sprague-Dawley, Indianapolis, IN) were divided into four groups (N=6) for immunization with the following immunogens (10 μg / mouse): 1) S60-VP8 chimeric particles, 2) free VP8 protein, 3) S60 particles without VP8 antigen, and 4) an equal volume of diluent (phosphate buffered saline, PBS, pH 7.4). Immunizations were performed intramuscularly three times at two week intervals with Inject Alum adjuvant (Thermo Scientific, 50 μl / mouse). Blood was collected two weeks after the third immunization, and serum was prepared from the blood samples using standard protocols.

[0058] Enzyme immunoassay (EIA). EIA was performed to measure antibody titers in mouse sera after immunization with different immunogens as previously described

[46] . 1 μg / mL of gel filtration purified free VP8 antigen was coated onto 96-well microtiter plates and incubated with serially diluted mouse sera

[47] . Bound antibodies were detected by goat anti-mouse secondary antibody-HRP conjugate (MP Biomedicals, Inc). Antibody titers were defined by limiting dilution with a cutoff signal intensity of OD450=0.1.

[0059] HBGA binding assays. Synthetic oligosaccharide and saliva-based HBGA binding assays were performed to measure the binding function of VP8-displaying S60 particles to their HBGA ligands

[45] . Briefly, synthetic oligosaccharides (2 μg / mL) representing H1, Leb, and Ley antigens, respectively, or boiled saliva samples (1:1000 dilution) that were H1 and / or Leb positive or H1 and Leb negative were coated onto 96-well microtiter plates and incubated with various S60-VP8 particles or S60 particles without RV VP8 at the indicated concentrations. Bound protein was measured by incubation with guinea pig anti-VP8 antiserum (for S60-VP8 particles) or guinea pig hyperimmune serum against GII.4Nov VLPs (for S60 particles) followed by HRP-conjugated goat anti-guinea pig IgG (ICN Pharmaceuticals).

[0060] Serum blocking titers for RV VP8-HBGA ligand attachment. This was performed as a surrogate neutralization assay, as previously described

[47] . Boiled human saliva samples, diluted (1:1000) with positive H1 and Lewis b (Leb) antigens, ligand for P[8]RV [45, 58], were coated onto microtiter plates. After immunization with various immunogens (S60-VP8 particles, free VP8 antigen, S60 particles, PBS), 625 ng / mL of P particle-presenting RV VP8 (PP-VP8)

[46] was preincubated at different dilutions with postimmunization serum before adding PP-VP8 to the coated saliva samples. The 50% blocking titer (BT50) was defined as the lowest serum dilution that caused at least a 50% reduction in the binding of PP-VP8 particles to HBGA / saliva samples compared to the unblocked positive control.

[0061] RV neutralization assay: This was performed as previously described

[53] . Briefly, MA104 cells were cultured in 6-well plates and tissue culture-adapted RV Wa strain (G1P[8]) was used as inoculum at a titer of approximately 50 PFU / well. Trypsinized Wa RV was incubated with mouse serum after immunization with the indicated immunogens (see above) for 1 h and then added to the cells. The plates were overlaid with medium containing trypsin (Invitrogen) and 0.8% agarose. After 4 days of incubation, plaques were stained and counted. The neutralization rate (%) of sera was calculated by the reduction in the number of plaques in wells treated with antisera relative to the number in untreated control wells.

[0062] Structural modeling of S60 particles. The structures of S60 particles and S60-VP8 chimeric particles with or without Hisx6 peptide were modeled using the crystal structure of 60-valent feline calicivirus (FCV) VLP

[59] (PDB#: 4PB6) as a template using the software PyMOL Molecular Graphics System, version 1.8.2.0 (Schroinger, LLC). All crystal structure-based images were generated by this software.

[0063] Structural reconstruction of S60-VP8 chimeric particles by cryoEM. This was performed using a similar cryo-EM approach described in the applicant's previous studies [20, 21, 46]. Briefly, an aliquot (3–4 μL) of gel filtration purified S60-VP8 chimeric particles was flash frozen on a Quantifoil grid, which was then loaded onto the microscope. Low electron (e) dose images (approximately 20 e / Å2) were recorded on film using a CM200 cryomicroscope at a nominal magnification of ×50,000, and a defocus range of 2.0–4.0 μm. Micrographs were selected and digitized using a Nikon Super CoolScan 9000ED scanner with a step size of 6.35 μm / pixel. Scanned images were binned, resulting in a final sampling of images at 2.49 Å / pixel. Images of S60-VP8 chimeric particles were selected using the Boxer program in EMAN. Selected images were manually filtered to exclude false positives. Contrast transfer function (CTF) parameters associated with sets of particle images derived from the same micrograph were determined manually using the ctfit program in EMAN. An initial model of the particle was created using the startoct program in EMAN. The raw particle centers and orientations were then determined iteratively using the refine program in EMAN, and 3D maps were reconstructed from the 2D images by the make3d program in EMAN until convergence. Icosahedral symmetry was imparted during the reconstruction of the S60-VP8 chimera particles. Analysis of the cryo-EM models, including fitting of the S60 particle model (see above) and P[8]RV VP8(2DWR), was performed using UCSF Chimera software (version 1.12: http: / / www.rbvi.ucsf.edu / chimera).

[0064] Statistical analysis. Statistical differences between data sets were calculated using unpaired nonparametric t-tests with the software GraphPad Prism 6 (GraphPad Software, Inc.). P-values ​​were set at 0.05 for significant differences (P<0.05), 0.01 for highly significant differences (P<0.01), and 0.001 for extremely significant differences (P<0.001).

[0065] Ethical statement. This study was carried out in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (23a). The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Cincinnati Children's Hospital Research Foundation (Animal Welfare Assurance Number A3108-01).

[0066]

[0067] result Low particle formation efficiency of native NoV S domain. Applicant's study started with the production of native S domain with hinge of GII.4 NoV (VA387) in E. coli using the expression vector pGEX-4T-1, resulting in a GST-S domain fusion protein with a molecular weight (MW) of about 51 kDa (Figure 1, A and B). Free S domain protein of about 25 kDa without GST (Figure 1C) was obtained by thrombin cleavage while GST remained bound to the Sepharose beads. EM observation of the S protein revealed few ring-like structures with a thin layer of about 20 nm in diameter, which are most likely equivalent to assembled S particles (Figure 1D). To determine the S particle formation efficiency, gel filtration chromatography of the S domain protein was performed, revealing two broad peaks (Figure 1E). SDS PAGE (Fig. 1F) followed by Western analysis using NoV VLP hyperimmune serum

[15] (data not shown) and N-terminal sequencing (Fig. 2, see below) confirmed that both peaks were S proteins. Peak 1, with a high MW of >800 kDa, should represent self-assembled S particles or complexes, whereas peak 2 should be S domain monomers (~25 kDa) and / or dimers (~50 kDa).

[0068] Applicants also observed minor protein bands with lower MW that co-occurred with the GST-S fusion protein (Fig. 1B, 42 kDa) and free S protein (Fig. 1C, 16 kDa), respectively, and these minor protein bands reacted with NoV VLP-specific antibodies (data not shown) and exhibited S domain sequences, so they must be proteinase-cleaved forms of the S protein (Fig. 2, see below). Applicants further noted that the S domain protein assembled into S particles or complexes was mostly digested into smaller S domain proteins (Fig. 1E and F, peak 1, fractions #15 and #16). In contrast, the unassembled S protein remained intact (Fig. 1E and F, peak 2, fractions #28 and #29), suggesting that assembled S particles or complexes were sensitive to proteinases, whereas unassembled S protein was not. From the fact that peak 1 represents only a small portion (<25%) of the total S protein, Applicants conclude that native NoV S domain protein assembled into particles with low efficiency.

[0069] Identification of an exposed protease cleavage site in the S protein. The above findings prompted us to identify the protease cleavage site. This was accomplished by N-terminal sequencing of two cleaved S protein bands of approximately 16 kDa (Fig. 1, C and F), which yielded the same five-residue NAPGE sequence (Fig. 2A). This penta-residue matches the S domain sequence N70–E74, indicating that the cleavage site is between R69 and N70, which is a trypsitrypsin / clostripaina recognition site (Fig. 2B). Genetic analysis of the NoV VP1 sequence showed that this protease site is highly conserved among all GII NoVs (Fig. 2C). Structural analysis of the GII NoV shell structure (Wen Jiang, unpublished data) showed that this protease site is exposed on the shell surface (Fig. 2D).

[0070] Disruption of the protease site for high S particle formation efficiency. Based on the above data, Applicant introduced the R69A mutation to disrupt the proteinase cleavage site, resulting in high S particle formation efficiency.R69A The protein was purified using a Hisx6 peptide linked to the C-terminus to replace the GST tag and avoid the thrombin cleavage step for a simplified purification procedure (Figure 3A). Applicants also inserted a short linker (GGGG) between the hinge and the Hisx6 peptide to allow flexibility in the Hisx6 peptide to prove the concept of antigen presentation of S particles.

[0071] S R69A The protein (~25 kDa) was produced well in E. coli and purified by Hisx6-conjugated TALON CellThru Resin with very high yield (>40 mg / L bacterial culture) and high stability (Figure 3B). EM observation showed many ring-like structures with uniform size, representing self-assembled S particles with a diameter of ~22 nm (Figure 3C). Gel filtration revealed one large peak and two small peaks (Figure 3, D and E), which should represent S particles (>1 mDa), S dimers (~50 kDa), and S monomers (~25 kDa), respectively, based on their MW, supported by EM observation and ESI-MS analysis (below). SDS-PAGE revealed a small peak at ~50 kDa (Figure 3, B and E), which frequently reacted with NoV VLP-specific antibodies, indicating that these are fully undenatured S domain dimers in SDS-PAGE analysis. This was particularly evident in the S particle fractions (peak 1: fractions #8 and #9) from gel filtration chromatography (Figure 3, D and E) compared to the dimer (peak 2: #16) and monomer (peak 3: #19) fractions. These data support the conclusion that S R69A We showed that the majority of the protein assembled into unified S particles.

[0072] The SR68A protein self-assembles into S60 particles with 60 valences. Applicant then performs ESI-MS analysis to determine the S R69AThe protein complexity was determined, revealing three protein forms: 1) S monomer at 25.047 kDa, 2) S dimer at 50.095 kDa, and 3) S particles at approximately 1.47 mDa (Figure 3F). Since the calculated MW of the recombinant S domain protein is 24585.89 daltons (Figure 2B), the observed self-assembled S particles must be 60-valent, denoted S60 particles accordingly. The fact that no signal was observed above approximately 1.47 mDa indicates that conventional 180-valent S particles did not assemble, consistent with the uniform particle size observed by EM (Figure 3C). These 60-valent S60 particles were further confirmed by structural reconstruction of S60-VP8 chimeric particles by cryoEM techniques (Figure 7, see below).

[0073] Structural modeling of S60 particles. Although the detailed structure of the hexavalent NoV capsid or its inner shell remains unknown, a crystal structure of a hexavalent feline calicivirus (FCV) VLP has been reported

[59] , providing a way to model and understand the structure of S60 particles. A structural model of S60 particles was constructed using the crystal structure of a hexavalent FCV VLP (PDB#: 4PB6) as a template (Figure 4, B–D). The modeled S60 particles exhibited somewhat pentagonal (Figure 4C) and hexagonal (Figure 4D) shapes with five-fold or two-fold axes, respectively. These pentagonal and hexagonal shapes are easily recognizable among S60 particles in EM micrographs (Figure 4A and Figure 3C). In addition, the S60 particle model fits well into the S60 particle region of the cryoEM density map of the S60-VP8 particle (Figure 7, D–F), supporting the structural similarity between the hexavalent FCV shell and NoV S60 particles. As expected, 60 C-terminal hinges are exposed on the surface of each S60 particle ( Fig. 4, B to D ), providing excellent fusion points for foreign antigens displayed by S60 particles.

[0074] Applicants also modeled S60 particles with a C-terminal linker (GGGG) and Hisx6 peptide to mimic antigen presentation by S60 particles (Figure 4, E-G). The resulting model showed that 60 Hisx6 peptides were presented on the surface of each S60 particle, supporting the fact that S60 particles with C-terminally bound Hisx6 peptides were efficiently purified by Hisx6-bound resin (Figure 3B). Thus, it is quite conceivable that various antigens from other pathogens could also be presented by S60 particles by fusing them to the exposed C-terminus of the S protein.

[0075] Production and characterization of S60-VP8 chimeric particles. To demonstrate the feasibility of S60 particles as a platform for antigen presentation to enhance immunogenicity, Applicants produced S60-VP8 chimeric particles that display the major RV neutralizing antigen, RV VP8 protein. This was achieved by linking the RV VP8 protein to the S60-VP8 chimeric particle via a linker. R69A This was achieved by fusing the Hisx6 peptide to the C-terminus of the VP8 protein (Figure 5A). A Hisx6 peptide was added to the C-terminus of the VP8 protein for purification purposes. R69A The S-VP8 chimeric protein (approximately 45 kDa) was successfully expressed in the E. coli system with high yields of >30 mg / liter of bacterial culture (Figure 5B). R69A Gel filtration analysis of the S-VP8 proteins revealed that they were R69A The results revealed three typical peaks, most likely representing S-VP8 particles (peak 1), dimers (peak 2), and monomers (peak 3) (Figure 5C). Retention comparison of the three peaks revealed that S R69A -Indicating that approximately half of the VP8 protein self-assembled into particles (peak 1 vs. peak 2 and peak 3).

[0076] EM observation of proteins from peak 1 revealed many S proteins of uniform size whose surfaces had recognizable protrusions to display VP8 proteins. R69AESI-MS analysis of peak 1 protein revealed the expected 60-valent S60-VP8 particles with a molecular weight of approximately 3.4 mDa (Figure 5E). Again, consistent with the uniform size of S60-VP8 particles on the micrograph of the same protein, no signal for 180-valent particles was observed (Figure 5D). However, ESI-MS analysis did not reveal any signal for the S60-VP8 particles (Figure 5D), which results in a rough surface that differs from the relatively smooth surface of the S60 particles (Figures 3C and 4A). ESI-MS analysis of peak 1 protein revealed the expected 60-valent S60-VP8 particles with a molecular weight of approximately 3.4 mDa (Figure 5E). Again, consistent with the uniform size of S60-VP8 particles on the micrograph of the same protein, no signal for 180-valent particles was observed (Figure 5D). However, ESI-MS analysis did not reveal any signal for the S R69A -VP8 protein monomer (44.950 kDa) and a trace degradation product of 19.90 kDa were revealed, indicating that S60-VP8 particles can be degraded to monomers.

[0077] Further stabilization of S60-VP8 particles. R69A The relatively low particle formation efficiency by the S60-VP8 protein indicated that there was room for improvement by increasing the intermolecular interactions between adjacent S domains in the S particle. Inspection of the GII.4 shell structure (WJ, unpublished data) showed that V57 and Q58 of the S domain are sterically close to M140 and S136 of the adjacent S domain, respectively, with distances of 5.7–5.9 Å (Fig. 6, A and B). This suggested that the two paired residues are suitable positions for introducing inter-S domain disulfide bonds to further enhance S60-VP8 particle formation.

[0078] S R69A When V57 and M140 of the -VP8 protein were mutated to cysteine ​​(Figure 6C), S 69A / 58C / 140C The S60-VP8 protein was well expressed with very high yields of >50 mg / liter of bacterial culture (Figure 6D). Gel filtration analysis showed that the majority of the protein (~70%) was assembled into S60-VP8 particles (Figure 6E), which was confirmed by EM observations (data not shown). It was noted that peak 3, representing the S monomer, was completely absent, confirming an increase in inter-S domain interactions after the introduction of disulfide bonds.

[0079] S R69AWhen V57, Q58, and S136 of the -VP8 protein were mutated to cysteine ​​(Figure 6F), S 69A / 57C / 58C / 136C The S-VP8 protein could be produced in high yields of >40 mg / liter of bacterial culture (Figure 6G). Gel filtration analysis (Figure 6, H and J) demonstrated that the S-VP8 protein was 69A / 57C / 136C Our results showed that the majority (>90%) of the S60-VP8 protein self-assembled into S60-VP8 particles, as confirmed by EM observation (Figure 6I). Notably, both peaks 2 and 3, representing S dimers and monomers, respectively, disappeared (Figure 6, H and J), supporting the notion that the efficiency of S60-VP8 particle formation was dramatically increased as a result of S interdomain disulfide bonds. Applicants also demonstrated that the S R69A Quadruple cysteine ​​mutations were performed at V57, Q58, S136, and M140 in all four S60-VP8 proteins, and the results for protein yield and S60-VP8 particle formation efficiency were consistent with those of S60-VP8. 69A / 57C / 58C / 136C -VP8 protein (data not shown), indicating that the quadruple cysteine ​​mutation was sufficient to produce highly stable S60-VP8 particles.

[0080] Structure of S60-VP8 particle. Applicant constructed the three-dimensional (3-D) structure of S60-VP8 particle at 14 Å resolution by cryo-EM technique (see Materials and Methods), showing T=1 symmetry with 60 S-VP8 proteins (Figure 7). Surface structure of S60-VP8 particle (Figure 7A) showed that VP8 antigens were presented on the surface of S60-VP8 particle, forming protrusions extending from the internal S60 particle. The central slice (Figure 7B) and the later slice structure (Figure 7C) of S60-VP8 particle showed the structure of external VP8 antigens (cyan and partial green) and internal S60 particle (red, yellow and partial green). The five-fold axis of icosahedral S60 particle can be recognized (Figure 7C). The diameter of S60-VP8 chimeric particle is about 28 nm.

[0081] When the crystal structure of the 60-valent FCV shell (PDB#: 4PB6) was fitted to the S60 particle portion of the S60-VP8 particle cryoEM density map, both structures fitted very well (Figure 7, D-F). Transparent cryoEM density maps with the fitted FCV shell structure of the first half (Figure 7D), central slice (Figure 7E), and second half (Figure 7F) of the S60-VP8 particle demonstrated an excellent fit between the 60-valent shell structure of FCV and the NoV S60 particle region of the S60-VP8 particle, confirming the icosahedral structure of the 60-valent shell of Applicant's S60 particle (Figure 4) and the 60-valent of the S60-VP8 particle.

[0082] Next, Applicant fitted the fitted 60 copies of the VP8 crystal structure of P[8]RV Wa strain (PDB code: 2DWR) to the protruding region of the S60-VP8 particle cryoEM density map (Figure 7, G and H). The clear cryoEM density maps of the first half (Figure 7G) and the central slice (Figure 7H) of the S60-VP8 particle with the fitted VP8 crystal structure showed an excellent fit between the protruding region of the S60-VP8 particle and the 60 VP8 structure, further confirming the structure and orientation of the VP8 antigen on the surface of the S60 particle. Based on this fitting result, Applicant used the crystal structure of the 60 copies of the FCV shell and the 60 VP8s of P[8]RV to create an S60-VP8 particle model (Figure 7I).

[0083] The VP8-displayed S60 particles retained the ligand-binding function. Applicant's previous study has shown that VP8 of P[8]RV bound to H1 antigens but not to Ley antigens

[45] . Saliva-based binding assays showed that S60-VP8 particles bound to saliva samples positive for H1 and / or Leb antigens, but not to saliva samples negative for H1 and Leb antigens. These data indicate that the VP8 antigens displayed on S60 particles are correctly packaged with the ligand-binding function, making S60-VP8 particles effective as RV vaccine candidates.

[0084] Improved immunogenicity of S60 particles against presented VP8 antigen. Mice were immunized with S60-VP8 particles (N=6) and VP8-specific immune responses were measured using free VP8 antigen as a control for comparison. After three immunizations, the VP8-specific IgG response after immunization with S60-VP8 particles was 11.6-fold higher than the response induced by free VP8 (P=0.0004) (Figure 10A). S60 particles as a negative control did not induce a VP8-specific IgG response. These data indicated that S60 particles could improve the immunogenicity of presented RV VP8 antigen.

[0085] S60-VP8 particle-elicited antisera enhanced blocking against VP8 ligand binding. Binding of VP8 to RV host ligands or receptors is a critical step in RV infection

[43] . Therefore, an in vitro blocking assay for binding of RV VP8 protein to HBGAs was developed as a surrogate RV neutralization assay

[47] . Applicant performed such a blocking assay using previously developed P-VP8 particles

[47] and Leb-positive saliva samples

[45] as RV ligands. Applicant found that mouse antisera after immunization with S60-VP8 particles exhibited 50% blocking titers (BT50) 22.8-fold higher than those of antisera after immunization with free VP8 antigen (P=0.0003) (Figure 10B), further supporting the view that S60 particles significantly improve the immunogenicity of presented RV VP8 antigen. As a negative control, mouse serum after immunization with S60 particles without VP8 antigen clearly did not exhibit such blocking.

[0086] S60-VP8 particle-induced antisera enhanced neutralization against RV infection. Applicants also performed a conventional cell culture-based neutralization assay to determine the neutralizing activity of S60-VP8 particle-induced antisera against infection with the cell culture-adapted (P[8]) RV Wa strain. Consistent with BT50 (see above), mouse antisera after immunization with S60-VP8 particles exhibited significantly higher neutralizing activity than that of antisera after immunization with free VP8 antigen at three different serum dilutions (1:75, 1:150, and 1:300) (P=0.0003, P=0.0001, and P=0.0016, respectively) (Figure 10C). Mouse sera after immunization with S60 particles without VP8 antigen did not apparently exhibit such neutralizing activity. These data further supported the notion that S60 particles are a potential vaccine platform for increasing the immunogenicity of displayed RV VP8 antigens and that S60-VP8 particles are promising vaccine candidates against RV infection.

[0087] S60 particles as a multifunctional vaccine platform. In addition to the RV VP8 antigen, applicants were able to fuse several other epitopes and antigens to the S60 particles through the same exposed S domain C-terminus via linkers including the M2e epitope of influenza A virus, the TSR antigen of the sporozoite surface protein (CSP) of the malaria parasite Plasmodium falciparum, and the P domain of hepatitis E virus (Table 1). Thus, the artificially developed S60 particles serve as a multifunctional platform for novel vaccine development. [Table 1]

[0088] 1 The M2e epitope is the ectodomain of the matrix-2 (M2) protein that forms a proton-selective ion channel of influenza A viruses. 2The TSR / CSP antigen is a C-terminal antigen of the major surface protein of the sporozoite surface protein (CSP), which plays an important role in host cell invasion by the malaria parasite Plasmodium falciparum. 3 Complete RV VP8 is the full-length VP8 domain of the spike protein of human P[8] rotavirus. 3 Murine RV VP8 is the core portion of the spike protein of murine rotavirus. 4 The HEV P domain is the protruding domain of the Hepatitis E virus capsid.

[0089] Consideration In this study, applicants developed a novel technology to produce highly efficient, uniform, 60-valent NoV S60 particles via a simple bacterial expression system. This was achieved by exploiting the homotypic interactions of the NoV VP1 S domain, which naturally builds the inner shell of the NoV capsid, as well as several modifications to stabilize the S domain proteins and strengthen S domain-S domain interactions. Specifically, applicants introduced the R69A mutation to disrupt an exposed protease cleavage site on the native shell that would otherwise result in facile degradation of the S protein. In addition, applicants introduced triple (V57C / Q58C / S136'C) or quadruple (V57C / Q58C / S136'C / M140'C) staining mutations into two pairs of sterically close residues between two adjacent S domains (V57 / M140' and Q58 / S136', FIG. 6) to establish inter-S domain disulfide bonds and stronger inter-S domain interactions than those exhibited by the native NoV shell. Finally, the bioengineered S domains were easily produced in high yields by a simple E. coli system, resulting in the efficient self-assembly of S60 particles.

[0090] Self-assembled multivalent S60 particles with 60 flexibly exposed S domains C-terminally are an ideal platform for antigen presentation to improve immunogenicity against presented antigens for vaccine development. This idea was largely proven in this study by constructing chimeric S60 particles displaying 60 RV VP8 proteins, the major RV neutralizing antigens. S60-VP8 particles can be easily produced with high stability. They elicited significantly higher IgG responses in mice against the presented VP8 antigen than those induced by free VP8 protein. Mouse antisera after vaccination with S60-VP8 particles showed significantly stronger blocking against binding of RV VP8 to its glycan ligands and showed significantly higher neutralizing activity against RV infection and replication in cultured cells than sera after immunization with free VP8 antigen. Although the protective efficacy of the S60-VP8 particle vaccine has been determined using a murine RV challenge model in Applicant's laboratory, the data presented in this report strongly support the view that S60-VP8 particles are promising vaccine candidates against RV infection and therefore that S60 particles are an excellent platform for antigen presentation for the development of novel vaccines.

[0091] The native NoV capsid is made by 180 VP1, a single major structural protein of NoV. In vitro expression of NoV VP1 via eukaryotic systems often resulted in a mixture of 180- and 60-valent VLPs, and the two VLP formats were interchangeable by artificial denaturation and renaturation procedures

[60] . Although not yet fully studied, previous expression of a truncated S domain via a baculovirus / insect cell system appeared to self-assemble 180-valent S particles [11, 24]. However, unified 60-valent NoV VLPs or S particles via an expression system have never been produced before. Thus, Applicant's production technology of unified NoV S60 particles via a simple E. coli system represents an advancement in biotechnology. Self-assembly of unified S60 particles may result from the combinatorial effect of the extensively modified S domain and the unique folding of the prokaryotic E. coli expression system. Homogeneous complexity and size of vaccine candidates are important considerations for quality control, since variability in vaccine complexity and size leads to variability in vaccine vaccination outcomes.

[0092] Artificially introduced intermolecular disulfide bonds can be used as a general approach to stabilize viral protein particles or complexes. During the applicant's previous construction of NoV P particles, the applicant found that the addition of a cysteine-containing peptide to the end of the NoV P domain promoted and stabilized P particle formation via inter-P dimer disulfide bonds [20-23]. In this current study, S60 particles efficiently self-assembled (Figure 3D), but with the addition of VP8 antigen, the formation efficiency of the original version of S60-VP8 particles was relatively low (Figure 5C). Notably, the self-assembly efficiency of S60-VP8 particles was significantly improved by introducing inter-S domain disulfide bonds. This was achieved in two fundamental steps. First, applicants analyzed the shell structure of GII.4 NoV (Wen Jiang, unpublished data) and identified two pairs of sterically close (5.7-5.9 Å) residues (V57 / M140' and Q58 / S136') between two adjacent S domains (Figure 6, A and B). Applicants then simultaneously mutated two to four of these residues to cysteines in different combinations: 1) V57C / M140'C, 2) Q58C / S136'C, 3) V57C / Q58C / S136'C, 4) V57C / Q58C / S140'C, and 5) V57C / Q58C / S136'C / S140'C, and subsequently produced them and measured the self-assembly efficiency of the resulting S60-VP8 particles.

[0093] Among these mutations, S60-VP8 particles with triple cysteine ​​mutations (V57C / Q58C / S136'C) showed the highest particle formation efficiency of over 95% for S-VP8 protein to self-assemble into S60-VP8 particles (Figure 6, F-J). The dimer and monomer forms of the mutated S-VP8 protein were completely abolished (compare Figure 6H with Figure 5C and Figure 5E). Applicant also noted that S60-VP8 particles with quadruple cysteine ​​mutations (V57C / Q58C / S136'C / S140'C) showed nearly the same high efficiency of S60-VP8 particle formation as those with triple cysteine ​​mutations (data not shown). However, the detailed structural basis or mechanism behind these different results among various cysteine ​​mutation combinations remains elusive. These results and our previous work on P particles [20-23] suggested that the introduction of intermolecular disulfide bonds could be used as a general approach to promote and stabilize viral protein particle or complex formation. Following these data, downstream experiments will be performed using S60-VP8 particles with the R69A and V57C / Q58C / S136'C mutations, and simultaneously downstream experiments will be performed using modified S domains with the same mutations to be used to produce stable S60 particles as a platform for presenting other antigens.

[0094] The S60 and S60-VP8 particles in this study were purified via a small Hisx6 peptide attached to the exposed C-terminus of the S domain or S-VP8 protein. Applicant's data indicates that the GST tag is not suitable for the production of S60 and S60-VP8 particles because it is large (220 residues), interferes with the formation of S60 particles, and needs to be removed by an extra thrombin cleavage step, complicating the purification procedure. In addition, the possibility of a tag-free purification method was also tested. Applicant found that both S60 and S60-VP8 particles can be selectively precipitated by ammonium sulfate and degraded in PBS and other buffers (data not shown). Finally, Applicant found that S60 and S60-VP8 particles eluted as a single peak in gel filtration size exclusion columns and anion exchange chromatography (data not shown). These data collectively indicate that S60 and S60-VP8 particles, and most likely other S60 antigen chimeric particles, can be purified with a tag-free approach.

[0095] The freely exposed C-terminus of S60 is another feature that promotes S60 particles to be a useful vaccine platform. Foreign antigens or epitopes can be easily fused to the end of the S domain via a flexible linker by recombinant DNA technology. This study clearly demonstrated that Hisx6 peptide and RV VP8 antigen can be well presented by S60 particles, as shown by the structural stability of S60-Hisx6 and S60-VP8 particles, and by their excellent binding ability to TALON CellThru Resin (Hisx6) and H1 and Leb ligand (RV VP8). In addition, the fact that several other tested antigens or epitopes can be well presented by S60 particles indicates that S60 particles are a multifunctional vaccine platform.

[0096] Modeling of S60 particles, S60-Hisx6 using the crystal structure of 60-valent FCV VLPP, and reconstruction of the 3D structure of S60-VP8 particles by cryoEM techniques provide new insights into the structural basis of how S60 particles present Hsix6 peptides and RV VP8 antigens. Fitting the structure of the S60 particle model to the S60 particle region, as well as fitting 60 copies of VP8 antigen to the protruding region of the cryoEM density map of S60-VP8 particles, should further clarify the structural relationship of S60 particles and their presented antigens. These structural data will be useful in the design and understanding of future presentation of other foreign antigens by S60 particles. Finally, these structural studies also confirmed the T=1 icosahedral symmetry of the 60-valent S60 and S60-VP8 particles.

[0097] In summary, we developed self-assembled multivalent protein nanoparticles that feature easy production, high stability, and high immunogenicity, and serve as an ideal platform for antigen presentation. As a proof of concept, chimeric S60 particles presenting 60 copies of RV neutralizing VP8 antigen were constructed. Applicants' data demonstrated that the highly immunogenic S60-VP8 particles are promising vaccine candidates against RV infection, and that the S60 particles are a multifunctional platform to enhance the immunogenicity of various antigens for the development of novel vaccines against different pathogens.

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[0159] All percentages and ratios are calculated by weight unless otherwise specified.

[0160] All percentages and ratios are calculated based on the total composition unless otherwise specified.

[0161] It is to be understood that every maximum numerical limit given throughout this specification will include every lower numerical limit, as if such lower numerical limit were expressly written herein. Every maximum numerical limit given throughout this specification will include every higher numerical limit, as if such higher numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0162] The dimensions and values ​​disclosed herein should not be understood to be strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm."

[0163] All documents cited herein, including any cross-references or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in combination with any other reference(s), teaches, suggests, or discloses such invention. Further, to the extent that a meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document applies.

[0164] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A multivalent icosahedral composition for antigen presentation comprising an S particle, the S particle comprising: a) a Norovirus (NoV) S domain protein; b) a linker protein domain operably linked to the Norovirus S domain protein; c) an antigen protein domain operably linked to said linker.

2. The polyvalent icosahedral composition of claim 1 , wherein the composition has an icosahedral symmetric structure.

3. 3. The multivalent icosahedral composition of claim 1 or 2, wherein the composition comprises 60 sites for antigen presentation.

4. 4. The multivalent icosahedral composition of claim 1, wherein the Norovirus S domain protein comprises a mutation at a proteinase cleavage site of the NoV S domain protein, the mutation rendering the site resistant to trypsin cleavage.

5. 5. The multivalent icosahedral composition of any of claims 1-4, wherein the Norovirus S domain protein comprises a mutation to the proteinase cleavage site, the mutation being at position 69 or 70, and the mutation rendering the site resistant to trypsin cleavage.

6. 6. The multivalent icosahedral composition of any of claims 1 to 5, wherein the Norovirus S domain protein comprises a mutation to the proteinase cleavage site, said mutation occurring at position R69, preferably said mutation being any amino acid other than K sufficient to destroy the proteinase cleavage site, and more preferably said mutation being R69A.

7. 7. The multivalent icosahedral composition of any of claims 1 to 6, wherein the Norovirus S domain protein comprises a mutation to the proteinase cleavage site, said mutation occurring at position N70, preferably said mutation being any amino acid other than P sufficient to destroy the proteinase cleavage site.

8. The multivalent icosahedral composition of any one of claims 1 to 7, wherein the Norovirus S domain protein contains sufficient mutations to provide a binding site for a non-native disulfide bond.

9. 9. The multivalent icosahedral composition of claim 1, wherein the Norovirus S domain protein comprises mutations of at least two amino acids sterically close to each other to cysteine ​​residues sufficient to provide at least one non-native disulfide bond binding site, or at least two non-native disulfide bond binding sites, or at least three non-native disulfide bond binding sites between adjacent S domain proteins of the multivalent icosahedral S particle.

10. 10. The multivalent icosahedral composition of any of claims 1-9, wherein the Norovirus S domain protein comprises sufficient mutations to provide at least one non-native disulfide bond formation site, the mutations being selected from V57C, Q58C, S136C, M140C, or a combination thereof.

11. The polyvalent icosahedral composition of any one of claims 1 to 10, wherein the Norovirus S domain protein is from a calicivirus, the calicivirus having 180 copies of a single capsid protein.

12. The multivalent icosahedral composition of claim 1 , wherein the linker comprises an amino acid sequence of sufficient length to provide space and certain flexibility between the S domain protein particle and the presented antigen.

13. The polyvalent icosahedral composition of any one of claims 1 to 12, wherein the linker comprises 3 to 6 amino acids.

14. 14. The multivalent icosahedral composition of any of claims 1 to 13, wherein the antigen protein domain comprises an antigen having a size of from 8 amino acids up to about 300 amino acids, or from 8 amino acids up to about 400 amino acids, or from 8 amino acids up to about 500 amino acids.

15. The multivalent icosahedral composition of any one of claims 1 to 14, wherein the antigenic protein domain comprises a rotavirus (RV) antigen.

16. The multivalent icosahedral composition of any one of claims 1 to 15, wherein the antigenic protein domain comprises an RV spike protein antigen (VP8 antigen).

17. 17. The multivalent icosahedral composition of any of claims 1 to 16, wherein the antigenic protein domain comprises an antigen selected from a TSR antigen of the sporozoite surface protein (CSP) of the malaria parasite Plasmodium falciparum, a receptor binding domain of the HA1 protein and M2e epitope of influenza A virus, a P domain antigen of hepatitis E, a surface spike protein of astrovirus, and combinations thereof.

18. 1. A recombinant fusion protein comprising: a) a Norovirus (NoV) S domain protein, preferably having one or more mutations to destroy a protease site, more preferably having one or more mutations to destroy a protease site and introduce one or two or three cysteine ​​residues; b) a linker protein domain operably linked to the Norovirus S domain protein; c) an antigen protein domain operably linked to said linker.

19. 18. A method of making a multivalent icosahedral structure according to any one of claims 1 to 17, comprising the steps of: a) making a first region comprising a modified NoV S domain protein, said modification comprising a mutation sufficient to destroy an exposed protease cleavage site, said mutation preventing proteolysis, preferably an R69A mutation, and introducing one or more mutations into the NoV S domain protein sufficient to form an S domain interprotein disulfide bond, preferably selected from V57C, Q58C, S136C and M140C, and combinations thereof; and b) recombinantly expressing the first region using a linker and an antigen.

20. 20. The method of claim 19, wherein the composition is produced in E. coli.

21. A method of inducing an immune response in an individual in need thereof, comprising administering a composition according to any one of claims 1 to 17, preferably administering said composition two or more times, or three or more times, or four or more times.

22. A container comprising at least one dose of the composition of any of claims 1 to 17.

23. 23. A kit comprising one or more containers of claim 22, a delivery device, and instructions for administration of the composition.

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