NSP10 self-assembling fusion proteins for vaccines, therapeutics, diagnostics and other nanomaterial applications

Abstract

A fusion protein is provided which is based on a self-assembling gene-regulatory NSP10 protein and a protein or peptide capable of being fused to NSP10 without interfering with the assembly or aggregation of the resulting fusion protein. The disclosure also relates to any nanoparticle formed thereby whether complete or not, and methods for the use of the NSP10 fusion protein are also disclosed, including use as vaccines for any indication in humans or animals, therapeutic methods involving the use of the fusion proteins such as using the protein to targeted an antibody or receptor, such as for treating or diagnosing cancer, biosensors using the fusion protein, or the use of the fusion proteins in cell sorting or any imaging application.

Claims

1. A method of eliciting an immunogenic reaction in a human or animal comprising administering to said human or animal an immunologically effective amount of a fusion protein comprising a self-assembling coronavirus NSP10 protein and an immunogenic protein or peptide capable of being fused to the NSP10 protein without interfering with the assembly or aggregation of the resulting fusion protein, wherein the fusion protein forms a capsid assembly, and wherein the capsid is a dodecameric capsid exhibiting 32 point symmetry.

2. The method according to claim 1 wherein the immunogenic protein or peptide is from a target selected from the group consisting of Malaria, Dengue Fever, Chikungunya, Yellow fever, Zika Virus, Leishmaniasis, Chagas, Tick-borne encephalitis, hemorragic disease, Japanese encephalitis, Influenza virus, rotavirus, common cold virus, coronaviruses, HIV, Ebola, hoof and mouth disease, polio virus, rhinovirus, semliki forest virus, Herpesvirus, tuberculosis, staphylococcus, viral pneumonia, and hepatitis virus.

3. The method according to claim 1 wherein the immunogenic protein or peptide is a viral protein or peptide.

4. The method according to claim 1 wherein the immunogenic protein or peptide is from a virus family selected from the group consisting of Poxviridae, Asfariviridae, Iridoviridae, Herpesviridae, Baculoviridae, Adenoviridae, Polyomaviridae, Papillomaviridae, Parvoviridae, Reoviridea, Birnaviridae, Coronavridae, Arteriviridea, Togaviridae, Flaviviridae, Picornaviridae, Astroviridae, Caliciviridae, Paramyxoviridae, Filiviridae, Rhabdoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Retroviridae, Hepadnaviridae, and Caulimoviridae.

5. The method of claim 1 wherein the NSP10 protein has the sequence of SEQ ID NO: 7.

6. The method of claim 1 wherein the immunogenic peptide or protein fused to NSP10 is a viral protein, fragment, or peptide; a bacterial protein, fragment or peptide; a virus-like particle (VLP); an immune regulatory protein; or a microbial protein.

7. The method of claim 1 wherein the immunogenic peptide or protein fused to NSP10 is a peptide or protein selected from the group consisting of hemoglobin, silver condensing peptide, an HIV Tat protein, an HIV Tat peptide, an HIV-1 P24 protein, an HIV gp120 protein, an HIV gp120 peptide, a coronavirus S gene peptide or protein, an Influenza hemagglutinin, and a peptide or protein from a virus selected from the group consisting of Ebola virus, MERS virus, SARS virus, coronavirus, Zika virus, Dengue fever virus, and yellow fever virus.

8. The method of claim 1 wherein the immunogenic protein or peptide is immunogenic against a target selected from the group consisting of Malaria, Dengue Fever, Chikungunya, Yellow fever, Zika Virus, Leishmaniasis, Chagas, Tick-borne encephalitis, hemorragic disease, Japanese encephalitis, Influenza virus, rotavirus, common cold virus, coronavirus, HIV virus, Ebola virus, hoof and mouth disease, polio virus, rhinovirus, semliki forest virus, Herpesvirus, and hepatitis virus.

9. The method of claim 1 wherein the fusion protein comprises an NSP10 protein fused to the immunogenic protein or peptide that was prepared using a DNA segment or expression vector so as to be injectable in a DNA vaccine, or wherein the NSP10 protein and the immunogenic protein or peptide of the fused protein are co-expressed in a DNA vaccine to enhance production of the immunogenic protein or peptide.

10. A method of vaccinating a human or animal comprising administering to said human or animal a vaccine comprising an immunologically effective amount of a fusion protein comprising a self-assembling coronavirus NSP10 protein and an immunogenic protein or peptide capable of being fused to the NSP10 protein without interfering with the assembly or aggregation of the resulting fusion protein, wherein the fusion protein forms a capsid assembly, and wherein the capsid is a dodecameric capsid exhibiting 32 point symmetry.

11. The method of claim 10 wherein immunogenic protein or peptide is immunogenic against a target selected from the group consisting of Malaria, Influenza virus, coronavirus, Ebola virus, hoof and mouth disease, polio virus, Herpesvirus, and hepatitis virus.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Each ferritin protein (subunit) shown in separate colors above is comprised of five principal helices (A,B,C,D & E). The N terminus (located on the A helix) and C terminus (located on the E helix) of each 17 kilo Dalton subunit, terminate in the completed quaternary structure on the outer surface and inner core respectively. A typical 24 subunit ferritin will have a diameter of 120 Å and a hollow 80 Å diameter core.

(2) FIG. 2. A graphical image of an example of a ferritin fusion at the 3-fold axes with an influenza hemagglutinin. Individual hemagglutinins and ferritin monomeric units are individually colored.

(3) FIG. 3. NSP10 Dodecamer/dodecahedron Viewed down the three-fold showing the close association of the three n-terminal helices. The surface directly opposite has the three c-termini surrounding the three-fold.

(4) FIG. 4. A space filling surface rendering of the NSP10 capsid illustrating the patches of positive electrostatic charge (shown in blue) on the capsid surface. An individual capsid is approximately ˜80 Å in diameter with a hollow ˜30 Å hydrophobic core.

(5) FIG. 5. A view of the NSP10 capsid looking down the two-fold axis. Note the prominent antiparallel beta sheet top surface shown in blue.

(6) FIG. 6. The trigonal pyramidal structure illustrates the capsid symmetry. The apex of each corner of the trigonal pyramid represents the tetrahedral 3-dimensional arrangement of three-fold axes and the position of one group of the amino acid termini (N or C). For example, arrows denote an antigen display. The position of each three fold axis is indicated by colored arrows. Different colors represent the n or c terminal regions and the tetrahedral arrangement of the capsid three-fold axes. Each three-fold penetrates the capsid, three-fold surfaces on each axis are non-identical (c or n-terminal three-fold axes).

(7) FIG. 7. A graphical depiction of an NSP fusion with a viral stem and receptor. In the example the receptor sequence is fused through the C to N fusion creating 4 spikes which are tetrahedral in arrangement. The remaining visible NSP N-terminal three-fold axes in this orientation, are colored in blue. Note the Sigma C receptor is depicted as a ribbon diagram and the capsid is depicted as a space filling/surface rendering for clarity. Note the significant and unrestricted access to the N-terminal fusion area, shown in blue.

(8) FIG. 8. An atomic model of an influenza hemagglutinin viral receptor and stem illustrating a fusion through the n-terminus of an NSPL capsid and the tetrahedral arrangement of the receptors. Note the hemagglutinin receptor is depicted as a ribbon diagram and the capsid is depicted as a space filling/surface rendering for clarity. Note that even with a large fusion protein, there remains a significant and unrestricted access to the C-terminal fusion area of NSP10, shown in red.

(9) FIG. 9. An atomic model illustrating the combination fusions at both the c and n-termini of NSP10 (Sigma C and hemagglutinin). The receptors are depicted as a ribbon diagram and the capsid is depicted as a space filling/surface rendering for clarity. Note the absence of steric clashes even with the combination of large protein fusions.

(10) FIG. 10. An atomic model illustrating the application of presenting the same viral stem system from the same family of viruses fused through both termini to create an octamer arrangement. The receptor example shown is from a paramyxovirus where the fusions through the n or c-termini can be designed to utilize the n-terminal helices or modified c-terminal fusion core.

(11) FIG. 11(A) is an example of an adenovirus tri-fold stem and receptor with C to N terminus fusion requirement and FIG. 11(B) the corresponding primary amino acid sequence with observed secondary structure (SEQ ID NO: 9).

(12) FIG. 12 is a photographic image of a TEM revealing numerous NSPL VLPs of the native IMP fusion material confirming the formation of the large oligomeric structures as indicated by native PAGE electrophoresis.

(13) FIG. 13(A) shows the SDS PAGE of the GST fusion isolated NSP-IMP fusion protein showing the high relative purity and the significant yield of native material produced by E. coli expression. FIG. 13(B) shows native PAGE of the sample shown in “A”: Lane 1: protein standard ferritin monomer (˜500 kd) and dimer (˜1000 kd); Lane 2 NSP-IMP showing two dominant oligomeric forms, one of approximate 300 kd and the other of approximate 600-700 kd.

(14) FIGS. 14(A) and 14(B). The fusion as set forth in Example 6 herein was successfully expressed in both E coli and Bacillus. FIG. 14(A) shows the TEM image of the Bacillus material as isolated by His tag affinity chromatography and FIG. 14(B) is a demonstration of hemagglutination activity-biological function, of the assembled nanoparticle.

(15) FIG. 15. This figure shows the remainder of the Reoviral Fibrous Stem and Receptor as described in Example 3 (SEQ ID NO: 9).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(16) In conjunction with the present invention, a fusion protein is provided which comprises a self-assembling gene-regulatory NSP10 protein as described above which can be utilized as a fusion protein including the NSP10 protein and a protein or peptide capable of being fused to NSP10 without interfering with the assembly or aggregation of the resulting fusion protein. In addition, the fusion protein may comprise the NSP protein with a small molecule or other therapeutic material which may be contained within a hollow interior hydrophobic core of the NSP protein. These fusion proteins may be made in a variety of ways as set forth below, so as these methods allow the protein to undergo self-assembly of the protein form. By self-assembling, it is indicated that the fusion protein that forms may be a polymer aggregate, and the self-assembly can be partial or full. The fusion protein of the present disclosure may also be formed into a capsid assembly, such as a dodecameric capsid exhibiting 32 point symmetry. The fusion protein may be formed recombinantly or in a number of suitable chemical or physical ways that would be well known to one skilled in the art. The fusion protein may also have the protein or peptide fused to NSP10 at the n or c-termini positioned at the surface of the NSP10.

(17) As indicated above, the NSP10 protein of the present invention may have SEQ ID NO:7, or certain sequence homologies thereof, or can also be other proteins that have the same topology or folding pattern of the NSP10 molecule and thus have the same assembly properties as NSP10. In particular, NSP10 self-assembles into a spherical dodecamer having trigonal 32 point symmetry with an outer diameter of approximately 84 Å and an inner hollow hydrophobic chamber of 36 Å in diameter (FIGS. 3 & 4) (see Su et al., 2006; PDB identifier: 2G9T, sequence identifier POC6U8). The folding topology of NSP10 is a mixed alpha helical and beta sheet structure which can be further described as having two pseudo-subdomains, a small alpha-helical bundle, we denote as subdomain I (residues and helical regions 1-39; 70-91; 104-115) and a small beta sheet domain, we denote as subdomain II (residues 40-70; 90-105). The helical subdomains I self-associate to form a trimer interaction at the four capsid n-terminal three-fold axes and subdomains II self-associate as trimeric units on the four c-terminal three-fold axes. One zinc binding site occurs at the interface between the two subdomains and the three other zinc sites are located within subdomain II near the c-terminus. Accordingly, any protein containing the same folding topology as NSP10 is meant to be encompassed by the NSP10 proteins as described herein. Further, the NSP10 fusion protein can be further stabilized by adding intermolecular cross-linking disulfide bridges so as to reduce or eliminate the zinc binding features of the self-assembly.

(18) The NSP fusion protein as described above may be configured so that the peptide or protein fused to the NSP10 at the n or c-termini positioned at the surface of the NSP10. In addition, the present fusion protein may also be configured wherein the NSP10 has an n-terminus and a c-terminus, and wherein at least one of the two termini are positioned at the surface so as to become available for peptide or protein fusion. The peptide or protein fused to the NSP10 (via recombinant or other means) can be any suitable protein or peptide which can be fused to NSP10 without affecting the self-assembly and/or folding of the molecule as described above.

(19) Accordingly, the peptide or protein fused to NSP10 can be any of a large variety of useful biomolecules, including antigens, viral proteins, fragments, or peptides, bacterial proteins, fragments or peptides, microbial proteins, peptides or fragments, or virus-like particles (VLP). Specific peptides or proteins are discussed below, including proteins or fragments thereof from an HIV gp120, a coronavirus S gene, HIV gp120, an Influenza hemagglutinin, proteins from an Ebola virus, a MERS virus, a SARS virus, a Zika virus, Dengue fever virus, yellow fever virus, or fragments of proteins thereof. The viral protein, fragment, or peptide may be selected from a wide variety of virus families, including but not limited to Poxviridae, Asfariviridae, Iridoviridae, Herpeseviridae, Baculoviridae, Adenoviridae, Polyomaviridae, Papillomaviridae, Parvoviridae, Reoviridea, Birnaviridae, Coronavridae, Arteriviridea, Togaviridae, Flaviviridae, Picornaviridae, Astroviridea, Caliciviridae, Paramyxoviridae, Filiviridae, Rhabdoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Retroviridae, Hepadnaviridae, and Caulimoviridae.

(20) In the present invention, one application of the fusion protein described herein is as a vaccine composition, or in a method of enhancing immunogenicity or generating antibodies. In one exemplary embodiment, a vaccine may be formed by the fusion protein of NSP10 with a suitable antigen. Vaccine compositions may also be formed from this fusion protein and may include ingredients well known for use in injectable or otherwise administrable vaccines, include conventional vehicles, carriers or excipients that would be well known in the art. The vaccines can be utilized against a wide variety of pathogenic conditions, and may constitute, e.g., anti-parasitic vaccines, anti-insect vaccines, anti-microbial vaccines, anti-protozoan vaccines, cancer vaccines and/or viral vaccines. For example, immunogenic compositions may be prepare which comprise an immunogenic amount of the fusion protein according to claim 1 and a pharmaceutically acceptable vehicle, carrier, or excipient. A list of potential vaccine targets for the present invention include those responsible for Malaria, Dengue Fever, Chikungunya Yellow fever, Zika Virus, Leishmaniaisis, Chagas, Tick-borne encephalitis, hemorragic disease, Japanese encephalitis, Influenza virus, rotavirus, common cold virus, coronaviruses. HIV, Ebola, hoof and mouth disease, polio virus, rhinovirus, semliki forest virus, Herpesvirus, tuberculosis, staphylococcus, viral pneumonia, and hepatitis virus.

(21) The NSP10 protein of the invention may also be fused to a protein or fragment from the Apicoplexan or protozoan family of parasites such as Malaria or Chagas disease. The NSP10 protein may also be fused to a viral protein or fragment from a coronavirus S gene. The NSP10 protein may also be configured where the residues lining the inner core, such as the loop containing residues 80-90, are modified or new amino acids are inserted for new functionality. It may also be used as a diagnostic agent or tool in numerous fields, including medical, pharmaceutical, industrial, and numerous other applications.

(22) A method of eliciting an immunogenic reaction in a human or animal comprising administering to said human or animal an immunologically effective amount of the NSP10 fusion protein as described herein. By reference to “effective amount”, whether immunologically, pharmaceutically, or in other contexts, is intended to mean any non-toxic but sufficient amount of the compound, composition or agent that produces the desired prophylactic, immunogenic, therapeutic or other effect. Thus, as one skilled in the art would readily understand, the exact amount of the composition or a particular agent that is required will vary from subject to subject, depending on a number of factors including specific condition treated or diagnosed, and age, general condition, and other factors concerning the subject or the treatment, and any dosing regimen will also be determined to suit the individual and the purpose of the treatment. Accordingly, the “effective amount” of any particular compound, composition or agent will vary based on the particular circumstances, and an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation.

(23) In another exemplary embodiment of the present invention, the fusion protein of the invention may include an internalized therapeutic payload wherein the payload is situated within a hollow hydrophobic core of the NSP10. Other suitable small molecules, such as imaging agents or other therapeutic molecules that are sized to fit in the hydrophobic core of NSP10 may also be utilized in conjunction with the invention. In general, the hollow cavity in the inner hydrophobic core of the NSP10 protein has a diameter of roughly about 20 to 40 Angstroms and a volume of roughly about 20,000 to 30,000 Å.sup.3, thus generally housing materials having widths of about 40 Angstroms or less. It is possible to utilize the hollow central core to trap therapeutics for targeted therapeutic delivery through antibody or receptor directed fusions. This can be done by adjusting the pH and/or buffer properties to cause disassembly of the capsid. Once disassembled, the capsid can be re-assembled in the presence of a therapeutic agent by adjusting the pH and buffer back to the optimum conditions for re-assembly. Therapeutic or diagnostic agents can range from a small protein to peptides or small molecules such as anticancer agents like doxorubicin, cis-platinum, camptothecin, irinotecan, etc. The capacity of the core is limited by the volume and could contain from dozens of large heterocyclic anticancer or other chemical agents, to up to several hundred (400) for smaller anticancer chemotherapeutic agents, such as cisplatin, carboplatin, oxaliplatin, etc. In addition to trapping chemicals during re-assembly, surface mapping reveals a series of pores on the capsid surface that communicate with the central cavity, which suggests that it should be possible to diffuse small molecular agents into the capsid core by establishing the appropriate concentration gradient.

(24) The present fusion proteins of the invention may also be formed into pharmaceutical compositions comprising the fusion proteins with any of a number of well-known suitable, pharmaceutically acceptable vehicles, carrier or excipients. As would be evident to one skilled in the art, such vehicles, carriers or excipients may be any of a wide variety of physical forms in which the fusion protein may be administered when needed for therapeutic or diagnostic purposes. Such suitable forms may include solvents, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents and the like. By “pharmaceutically acceptable” is generally understood to mean that said forms are substantially compatible with the fusion protein or active ingredient therein and/or other ingredients that may be in the composition and is substantially not deleterious to a patient undergoing treatment thereof. General examples of suitable forms include phosphate buffered saline (PBS) and other biologically acceptable buffers, maltodextrin, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, cellulose, methylcellulose, silicified microcrystalline cellulose, mannitol, such as mannitol 400, glycolate, such as sodium starch glycolate, carboxymethylcellulose, such as sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Other suitable forms include those materials by which the present composition may be formed as a solution, gel, cream, lotion, ointments, drops, and the like.

(25) These compositions may be administered in any of a wide variety of methods, e.g., parenteral, oral, intranasal, subcutaneous, aerosolized or intravenous administration in a human or animal. Other modes of administration, such as enteral, topical, sublingual, intravenous, subcutaneous, intramuscular, percutaneous, or via inhalation may also be used when so determined by one of ordinary skill in the art. In general, when so desired, such pharmaceutical compositions are administered in effective amounts as described above.

(26) The fusion proteins of the present invention may be isolated or purified by any means conventionally used in the art. In addition, isolated nucleic acid sequences coding for the fusion protein are contemplated by the invention. The NSP proteins of the invention may be prepared in a variety of ways using any suitable means well known in the art, including recombinant, chemical or physical means. Recombinant methods of expressing the proteins are well known and can be carried out readily by those of ordinary skill in the art. Such expression methods may be prokaryotic or eukaryotic processes, with or without additional steps such as glycosylation. Other physical or chemical means for the attachment of the fusion protein to the NSP10 would also be well known in the art of fusing proteins.

(27) In accordance with the invention, the NSP protein as described above may be used as an antigen display system for the production of antibodies or the development of vaccines. The proteins of the invention may also be used to display antibody or affinity directing proteins or peptides at either or both termini. With regard to the display of antigens, presentation of antigens to the immune system, or antigen presentation, such are possible using the NSP proteins of the invention. In typical immunogenic formulations, the use of smaller monomeric proteins or peptides that are combined with adjuvants, such as the well-known immunopotentiator known as Freund's Adjuvant which are mineral oil mixtures that promote a strong immune response to the desired antigen. VLPs, which are much larger than the small monomeric protein or peptides, independently serve as immunopotentiators generating a strong immune response by their presence. By displaying the desired antigen on their surface, this serves to focus or direct the immune response to these antigens. For example, small antigenic peptides present greater challenges in eliciting the desired immune response. By fusing and displaying them on the surface of a VLP, a significant improvement in both titer and type of desired immune response can be gained (Li, Soistman & Carter 2006). As a further refinement in the antigen display, when nanoparticles or VLPs can promote the natural display of more complex oligomeric structures on their surface, such as viral receptors or other receptors this is of tremendous value in creating a neutralizing immune response. NSP10 allows the fusion and display of up to 24 peptides or up to 8 trimeric receptors, and allowing for these fusions in the C—N or N—C polarity, a major improvement over the prior art. In addition, two separate sets of trimeric receptors can be readily created and displayed on the surface. In general, the display of antigens on nanoparticles such as NSP10 can be regarded an “antigen display system” or “antigen presentation system.”

(28) As indicated above, the NSP proteins of the invention may be fusion proteins, or may be proteins wherein a self-assembling NSP10 protein is formed with a hollow hydrophobic core, and this core may be used to house a variety of small therapeutic or diagnostic materials that can be situated within this hydrophobic hollow core of NSP10. In addition, the NSP10 fusion protein of the invention may comprise an NSP protein which self assembles into a dodecahedron or higher oligomeric protein form having both the n and c-termini positioned at the surface to which peptide and protein fusions can be made, and a peptide or protein that can fuse to said NSP10 without interfering with the assembly or aggregation of the protein.

(29) Still other exemplary methods and uses of the NSP10 protein as described above are possible. For example, a method of enhancing the immunogenicity of an antigen is provided wherein the antigen is fused to an NSP10 protein, wherein the antigen can fuse to NSP10 without interfering with the assembly or aggregation of the protein. A method of cell sorting is also provided comprising introducing the NSP protein of the invention into a cell sorting apparatus for a time sufficient to allow the fusion protein to bond with a specific type of cell, and then sorting cells based on said bonding. A method of imaging a target material is also provided comprising introducing the above NSP protein having an imaging agent to a medium containing said target material and obtaining imaging of said target based on bonding between the fusion protein and said target.

(30) As indicated herein, numerous uses are contemplated for the NSP proteins as described herein, including as antigen display systems for the production of antibodies or the development of vaccines, in order to display antibody or affinity directing proteins or peptides at either or both termini, or to carry an internalized imaging agent within its hydrophobic core. The NSP10 proteins as described herein may also be used as a peptide or protein display systems for applications in biosensors, or for applications in target directed therapeutics. The NSP10 proteins as described above may be used as an attachment scaffold whereby the peptide, small molecule or protein can be attached to the NSP10 protein by a chemical or physical process. Additionally, the NSP10 fusion protein of the invention may be fused or incorporated with a vaccine or other therapeutic in a DNA segment or expression vector for use as a DNA-based injectable. In addition, it will also be possible to co-express NSP10 in a DNA vaccine to enhance production of the recombinant protein of interest

(31) As shown above and in the attached examples, It has been demonstrated here that the NSP10 fusion proteins of the invention can be successfully expressed and self-assembled into polymeric forms including dodecamers or higher (e.g., dimeric forms) structures. Both small and large fusions have been successfully demonstrated as illustrated in the examples. Further, these have been demonstrated in two different prokaryotic systems and one eukaryotic system to date. In cases where the complexity or post translational modifications are desired or required for the proper activity or antigenicity, these systems can also be expressed in systems such as yeast, CHO cells, HK293 cells, insect cells or transgenic plants. The choice of system would be necessitated by the application and thus easily anticipated by one skilled in the art. Accordingly, it would be understood that the expression vectors or GMO viruses could be used directly in animals to express the nanoparticles in vivo for the same purposes outlined herein. Such applications and others would be considered within the scope of this invention.

(32) It is also possible to utilize sterile filtration for NSP10 nanoparticles. Because of the slightly lower micron size as compared with other nanoparticles, these particles are more readily filterable with 0.2 micron filtration to sterilize the final formulation. Sterile formulations with 10% glycerol can be frozen at −80° C. for long term storage.

(33) NSP10 may also be utilized as a host cell protein suppressor. As indicated above, NSP10 is a viral gene regulatory/replicase-inhibitor protein that binds to the host cell 40S ribosomal unit and inhibits translation of host proteins. By suppressing host cell expression, NSP10 facilitates the production of its own viral gene expression.

(34) The Co-expressing the NSP-10 family of proteins, by itself or together with other proteins for therapeutic purposes or as a inclusion in a DNA vaccine or therapeutic for the express purpose of suppressing the translation of the host proteins is thus contemplated in the present invention Suppression of host cell proteins by a properly constructed DNA vaccine would ensure a greater amount of the antigen or VLP was produced, lowering the DNA required for effective dose and lowering the cost of production. The Table below shows the sequence of one Nonstructural protein 10 in accordance with the present invention:

(35) TABLE-US-00001 TABLE I Nonstructural protein 10, NSP10 (d2g9td1) (SEQ ID NO: 1) AGNATEVPANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHT GTGQAITVTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDKGKYVQI PTTCANDPVGFTLRNTVCTVCGMWKGYGCSCDQLREPLMQSADASTLFN GFAV 

(36) The amino acid sequence of NSP-10, the underlined sequence indicates the required amino acids for capsid construction. The core capsid encompasses 122 amino acids (˜14 kd), vs 151 total (˜17 kd). The underlined sequence itself is shown below:

(37) TABLE-US-00002 (SEQ ID NO: 7) PANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTGQAIT VTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDKGKYVQIPTTCAND PVGFTLRNTVCTVCGMWKGYGCS

(38) Still further, the present NSP-10 proteins as described herein by be useful in all applications of nanomaterial synthesis and plasmon resonance. With regard to recombinant expression, suitable methods can be employed as described above, and can include transgenic production in plants, (e.g., rice, tobacco, etc.,) and animals. The NSP10 proteins can also be used in a number of diagnostic applications as well, including diagnoses relating to disease conditions or other applications involving small molecules, e.g., in the medical, pharmaceutical and industrial fields.

EXAMPLES

Example 1

(39) The sequence of a hemagglutinin H5 fusion protein is shown below with the fusion at the N-terminus of NSP10. In the sequence below, the NSP10 sequence is underlined, and the linking residues are shown in bold.

(40) TABLE-US-00003 Hemagglutinin H5 Fusion at N-terminus of NPS10 (SEQ ID NO: 2) DQICIGYHANNSTKQIDTIMEKNVTVTHAQDILEKKHNGKLCSLKGVKP LILKDCSVAGWLLGNPMCDEFLNAPEWSYIVEKNNPINGLCYPGDFNDY EELKHLVSSTNLFEKIRIIPRNSWTNHDASSGVSSACPHLGRSSFFRNV VWLIKKNNVYPTIKRTYNNTNVEDLLILWGIHHPNDAAEQAKLYQNLNA YVSVGTSTLNQRSIPKIATRPKVNGQSGRMEFFWTILRPNDTISFESTG NFIAPEYAYKIVKKGDSAIMRSELEYGNCDTKCQTPLGAINSSMPFHNV HPLTIGECPKYVKSDKLVLATGMRNVPQKKKRGLFGAIAGFIEGGWQGM VDGWYGYHHINGQGSGYAADKKSTQKAIDGITNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFIDVWTYNAELLVLMENERTLDLHDSNV KNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYNYPKYSE SGGSPANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTG QAITVTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPT TCANDPVGFTLRNTVCTVCGMWKGYGCS 

(41) (About 617 residues or 83.6 kd)

Example 2

(42) The sequence of a Gp41 component fusion via the N-terminus of NSP10 is shown below. In the sequence below, the NSP10 sequence is underlined, and the linking residues are shown in bold.

(43) TABLE-US-00004 Gp41 component Fusion via the N-terminus of NPS10 (SEQ ID NO: 3) EAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGLMHN QDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQPANS TVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTGQAITVTPE ANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPTTCANDPVG FTLRNTVCTVCGMWKGYGCS 

(44) Additional Examples of fusions are provided in Examples 3 and 4 below.

Example 3

(45) TABLE-US-00005 Reoviral Fibrous Stem and Receptor: PANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTGQAIT VTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPTTCAN DPVGFTLRNTVCTVCGMWKGYGCSGGS
with the remainder as shown in FIG. 15.

Example 4

(46) Sigma-C capsid protein Fusion OS=Avian reovirus (strain S1133) (including trimeric helical stem).

(47) TABLE-US-00006 (SEQ ID NO: 4) PANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTGQAIT VTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPTTCAN DPVGFTLRNTVCTVCGMWKGYGCSGGSMAGLNPSQRREVVSLILSLTSN VNISHGDLTPIYERLTNLEASTELLHRSISDISTTVSNISANLQDMTHT LDDVTANLDGLRTTVTALQDSVSILSTNVTDLTNRSSAHAAILSSLQTT VDGNSTAISNLKSDISSNGLAITDLQDRVKSLESTASHGLSFSPPLSVA DGVVSLDMDPYFCSQRVSLTSYSAEAQLMQFRWMARGTNGSSDTIDMTV NAHCHGRRTDYMMSSTGNLTVTSNVVLLTFDLSDITHIPSDLARLVPSA GFQAASFPVDVSFTRDSATHAYQAYGVYSSSRVFTITFPTGGDGTANIR SLTVRTGIDT

(48) 451 residues—about 61 kd

Example 5

(49) Demonstration of Practical Application without Undo Experimentation

(50) TABLE-US-00007 NSP-IMP (SEQ ID NO: 5) PANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTGQAIT VTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPTTCAN DPVGFTLRNTVCTVCGMWKGYGCSMGAACGKSQRAAAAVEPPLSTAEKA EAAAVAAAEHSQKAEEAAEVAAACATKASAEAAVLTGVEPGAEPAAEAE EAPKQNEIEEQQTTTSPAQTHATEEQPAAPPVVPLSDADAQVLAAAEAA KQEAASSNMPRAYLFYACELNEGSLMMQWTTTQITEEDMHAKNLILLAS FVPAKHKTVSKSKLTQNGGITYFLQEMKYKWEVWSKVQRQAYYQGWIKF VKAADEMEASFTLHHFAAPAPPAKLFLLHTGPIENKVLPAKEEEPFNVS VFGLAAVTPPSPPYKPGANITPKRFGEIATGAGGAYMQLSRRGGDAAFD EKEVQKWLAADGLQMKKGEGITLDAAGGYERRSEKKGGDAAAATAAVEA EPTKVSQD 

(51) Expression in bacteria using an expression vector with a removable GST fusion protein for simplification of purification. Two viral fusion proteins were made through the c terminus, both were clearly expressed and captured by GST or His tag affinity chromatography yielding relatively pure protein. SDS gel electrophoresis of the isolated GST fusion protein were in accordance with the predicted molecular weights and Native PAGE electrophoresis indicated approximately 50% in fully assembled capsid and the remaining 50% in a single band representing a smaller oligomeric form. In the case of His tag expression which had a smaller fusion partner, 100% of the monomeric form was incorporated—self-assembled into the capsid. These gels are shown in FIGS. 13(A) and 13(B), wherein in A, the SDS PAGE of the GST fusion isolated NSP-IMP fusion protein showing the high relative purity and the significant yield of native material produced by E. coli expression is provided. In B, the native PAGE of the sample shown in “A” is provided wherein Lane 1 is a protein standard ferritin monomer (˜500 kd) and dimer (˜1000 kd); and Lane 2 is NSP-IMP showing two dominant oligomeric forms, one of approximate 300 kd and the other of approximate 600-700 kd. A TEM revealing numerous NSPL VLPs of the native IMP fusion material confirming the formation of the large oligomeric structures as indicated by native PAGE electrophoresis is shown in FIG. 12.

Example 6

(52) Demonstration of Practical Application without Undo Experimentation:

(53) TABLE-US-00008 NSP-EDS (SEQ ID NO: 6) PANSTVLSFCAFAVDPAKAYKDYLASGGQPITNCVKMLCTHTGTGQAIT VTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPTTCAN DPVGFTLRNTVCTVCGMWKGYGCSGGGSDGELTLAYDSTDFQVTENGLA LKVSPTQTPLTRIISMGNNLFDSGYEIFASCPQNKAAKVAGYVYLTSVG GLVHGTIQIKATAGYWFTGGNSVQESIRFGLVLCPFSARDPTANLSGWP APVVWSGDSNTPLYFAANAISYTNNRVNLAVTGNFYKEETELPGYTRHS FCPTGTTGMNFTGGNLYVCPCTVNTGATTLNAIYMVFVITQSALGTNFF ASNTPPNTFFLTPPIPFTYVGAQ 

(54) The Fusion protein above was successfully expressed in both E coli and Bacillus. This is shown in FIG. 13 (A) which is the TEM image of the Bacillus material as isolated by His tag affinity chromatography and in FIG. 13(B) which is the demonstration of hemagglutination activity-biological function, of the assembled nanoparticle.

(55) In summary, Described herein is a self-assembling gene regulatory protein NSP-10 which assembles into a dodecameric capsid exhibiting 32 point symmetry. In the assembled capsid the specialized positions of the N and C termini occur at points of 3-fold capsid symmetry properly disposed with the correct distances from the triad to anchor the fusion peptide a t the three-fold and promote nucleation of the correct folding for complex helical or fibrous trimeric assemblies, such as those found on viral receptors responsible for tropism and cell infection. Native formation of these viral receptor assemblies are essential properties of antigens (and vaccines) which prompt the immune system to create highly potent and broadly neutralizing antibodies. Such scaffolds can also serve as points of fusion for cellular receptors for targeting the delivery of therapeutics for cancerous cells or other therapeutically important targets. Here we have shown that complex fusions can be made which overcome protein fusion sequence polarity restrictions that limit the applications of other vaccine nanoparticle display systems. The invention described herein is one of the most unique and versatile nanoparticle fusion systems created to date, allowing for surface displaying fusions from both the c and n-termini. Complex divalent functionalities easily achieved in a single nanoparticle and with advantages in purification and other properties desirable for vaccine, therapeutic or other nanoparticle development.

(56) It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description and examples are for the purpose of illustration only, and not for the purpose of limitation.