Self-assembling nanostructured vaccine

Abstract

The present disclosure provides nanostructures and nanostructure-based vaccines. Some nanostructures of the present disclosure display antigens capable of eliciting an immune response to infectious agents such as bacteria, viruses, and pathogens. Some vaccines of the present disclosure are useful for preventing or reducing the severity of infection by infectious agents including, but not limited to, Lyme disease, pertussis, herpes virus, orthomyxovirus, paramyxovirus, pneumovirus, filovirus, flavivirus, reovirus, retrovirus, meningococcus, or malaria. The antigens can be attached to the core of the nanostructure non-covalently or covalently, including as a fusion protein or by other means disclosed herein. Multimeric antigens can optionally be displayed along the symmetry axis of the nanostructure. Also provided are proteins and nucleic acid molecules encoding such proteins, vaccine compositions, and methods of administration.

Description

[0001] Cross-referencing

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 636,757, filed February 28, 2018, and U.S. Provisional Patent Application Serial No. 62 / 724,721, filed August 30, 2018, each of which is incorporated herein by reference in its entirety.

[0003] Instructions for submitting text files electronically

[0004] This application was submitted electronically via EFS-Web and includes a sequence list in .txt format submitted electronically. The .txt file contains a sequence list named "ICVX_001_02WO_SeqList_ST25.txt" created on February 27, 2019, and is approximately 373 kilobytes in size. Technical Field

[0005] This disclosure generally relates to vaccines and methods of administration thereof. Specifically, this disclosure relates to nanostructure-based vaccines capable of evoking an immune response to antigens, such as antigenic proteins of various infectious agents, including bacteria, viruses, and parasites. Background Technology

[0006] Vaccination is a treatment used to prevent or reduce the severity of infections caused by various infectious agents, including bacteria, viruses, and parasites. Developing new vaccines has significant commercial and public health implications. Specifically, Lyme disease, pertussis, herpesviruses, orthomyxoviruses, paramyxoviruses, pneumoviruses, filoviruses, flaviviruses, reoviruses, retroviruses, and malaria are infectious agents for which vaccines already exist, are under development, or will require vaccines.

[0007] Subunit vaccines are vaccines made from isolated antigens, typically proteins recombinantly expressed in bacterial, insect, or mammalian cell hosts. Typically, the antigenic components of a subunit vaccine are selected from proteins of an infectious pathogen that are observed to elicit a natural immune response following infection, although in some cases other components of an infectious agent can be used. Typical antigens for subunit vaccines include proteins expressed on the surface of the target infectious agent, such as surface-expressed envelope glycoproteins of viruses. Preferably, the antigens are targets of neutralizing antibodies. More preferably, the antigens are targets of broadly neutralizing antibodies, whereby an immune response to the antigen encompasses immunity to multiple strains of the infectious agent. In some cases, N-linked or O-linked glycans of subunit vaccines can also be important in vaccination by contributing to epitopes of the antigen or by directing an immune response to specific epitopes on the antigen through steric hindrance. The immune response that occurs in response to vaccination can be to the protein itself, to the glycan, or to both the protein and the attached glycan. Subunit vaccines have various advantages, including that they contain no live pathogens, thereby eliminating concerns of vaccine infection of patients; they can be designed using standard genetic engineering techniques; they are more homogenous than other forms of vaccines; and they can be produced in standardized, recombinant protein expression production systems using well-characterized expression systems. In some cases, antigens can be genetically engineered to favor the generation of desired antibodies, such as neutralizing or broadly neutralizing antibodies. In particular, structural information about antigens of interest obtained through X-ray crystallography, electron microscopy, or nuclear magnetic resonance experiments can be used to guide the rational design of subunit vaccines.

[0008] A known limitation of subunit vaccines is that the immune response elicited can sometimes be weaker than the immune response to other types of vaccines, such as whole virus, live vaccines or attenuated live vaccines. The present inventors have recognized and disclosed herein that a nanostructure-based vaccine has the advantages of utilizing a subunit vaccine, while at the same time having the potential to increase the potency and breadth of the vaccine-induced immune response by multivalent display of antigens in a symmetrically ordered array. A nanostructure-based vaccine is a form of a "nanoparticle vaccine." In the present disclosure, a nanostructure-based vaccine is not the same as a nanoparticle vaccine, as the term nanoparticle vaccine has been used in the art to refer to protein-based or glycoprotein-based vaccines (see, e.g., U.S. Patent No. US 9,441,019), polymeric liposomes (see, e.g., U.S. Patent No. 7,285,289), surfactant micelles (see, e.g., U.S. Patent Publication No. US 2004 / 0038406 Al), and synthetic biodegradable particles (see, e.g., U.S. Patent No. US 8,323,696). Nanostructure-based vaccination represents a paradigm of vaccination with significant commercial and public health implications. Thus, there is a need for nanostructure-based vaccines and methods of their use for eliciting an immune response to infectious agents such as bacteria, viruses, and parasites; and for preventing or reducing the severity of infection by infectious agents including, but not limited to, Lyme disease, pertussis, herpes viruses, orthomyxoviruses, paramyxoviruses, pneumoviruses, filoviruses, flaviviruses, reoviruses, retroviruses, meningococci, and malaria. SUMMARY

[0009] Described herein are nanostructures, vaccines, methods of their use, and methods of making the nanostructures.

[0010] In one aspect, the present disclosure provides a nanostructure comprising a first plurality of polypeptides, wherein the first plurality of polypeptides is arranged according to at least one symmetry operator; the nanostructure comprises a first plurality of antigens; each of the first plurality of antigens has a proximal end and a distal end; and the proximal ends of the antigens are each attached to a member of the first plurality of polypeptides.

[0011] In another aspect, the present disclosure provides a vaccine comprising any of the nanostructures of the present disclosure, wherein the vaccine is capable of eliciting a neutralizing antibody response to an infectious agent. In one embodiment, the vaccine is provided as a pharmaceutical composition.

[0012] In another aspect, the present disclosure provides a method of generating immunity to an infectious agent in a subject, the method comprising administering any of the vaccines of the present disclosure.

[0013] In another aspect, the present disclosure provides a method of making any of the nanostructures of the present disclosure by in vitro assembly from components purified from one or more recombinant expression systems. In another aspect, the present disclosure provides a method of making any of the nanostructures of the present disclosure by co-expressing all components in a recombinant expression system, thereby producing the nanostructure, and purifying the nanostructure.

[0014] In one embodiment of the nanostructures of the present disclosure, the nanostructure further comprises a second plurality of polypeptides, wherein the second plurality of polypeptides is attached to the first plurality of polypeptides. In one embodiment, the nanostructure further comprises a second plurality of antigens. In one embodiment, the nanostructure further comprises a second plurality of antigens, each of the second plurality of antigens has a proximal end and a distal end, and the proximal end of the second antigen is attached to a member of the second plurality of polypeptides; and optionally, the proximal end of the antigen is the N-terminus of the antigen or the C-terminus of the antigen.

[0015] In one embodiment of the nanostructures of the present disclosure, the plurality of antigens is a plurality of antigenic proteins or antigenic fragments thereof. In one embodiment, the antigenic protein of the nanostructure is selected from the group consisting of SEQ ID NOs: 52-88 and 90-113 or a variant thereof; or the antigenic protein is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 52-88 and 90-97; or the antigenic protein is any one of HIV Env, RSV F, influenza HA, EBV gp350, CMV gB, CMV UL128, CMV UL130, CMV UL131A, CMV gH, CMV gL, Lyme OspA, pertussis toxin, Dengue E, SARS S, MERS, Zaire ebolavirus GP, Sudan ebolavirus GP, Marburg virus GP, Hanta virus Gn, Hanta virus Gc, HepB surface antigen, measles H, Zika envelope domain III, malaria CSP, malaria Pfs25, MenB fHbp, MenB NadA, MenB NHBA, Nipah virus F, Nipah virus G, rotavirus VP4, rotavirus VP8*, hMPV F, hMPV G, PV F, or PV HN 8.

[0016] In one embodiment, the nanostructure is configured to display a target epitope of the antigen; and optionally, the target epitope is accessible to an antibody as defined below. In any embodiment in which the nanostructure comprises a plurality of antigenic proteins, optionally, the nanostructure is configured to elicit an immune response to the first plurality of antigenic proteins that is preferential to a target epitope of the antigenic proteins. In embodiments of the disclosure, the target epitope is a conserved epitope, it is an epitope to neutralizing antibodies, it is an epitope to cross-reacting antibodies, or it is an epitope to broadly neutralizing antibodies.

[0017] In one embodiment of the nanostructure of the disclosure, the plurality of antigens comprises at least one mutation selected from the group consisting of a mutation that stabilizes the interface, a cysteine mutation configured to result in a disulfide bond, a deletion of a loop, an addition of an N-linked glycosylation site, a removal of an N-linked glycosylation site, a mutation that disrupts an epitope, and a mutation that creates an epitope. In one embodiment, the plurality of antigens comprises an antigenic oligosaccharide.

[0018] In one embodiment of the vaccine of the disclosure, the neutralizing antibody response is protective against infection by an infectious agent. In one embodiment, the neutralizing antibody response is broadly neutralizing against different strains of an infectious agent. In one embodiment, the infectious agent is any one of Lyme disease, pertussis, a herpes virus, an orthomyxovirus, a paramyxovirus, a pneumovirus, a filovirus, a flavivirus, a reovirus, a retrovirus, meningococcus, or malaria. In one embodiment, the infectious agent is a virus selected from HIV, RSV, influenza, EBV, CMV, dengue, severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, Ebola virus, Marburg virus, Hanta virus, hepatitis B, HPV, measles, Nipah virus, rotavirus, Metapneumo virus, parainfluenza virus, and Zika virus. In one embodiment, the infectious agent is Lyme disease or pertussis. In one embodiment, the infectious agent is malaria. In one embodiment, the infectious agent is meningococcus.

[0019] In one embodiment of the method of generating immunity of the present disclosure, the method further comprises administering an adjuvant. In one embodiment, the method further comprises repeatedly administering the vaccine. In one embodiment, the method further comprises administering a second vaccine selected from the group consisting of a nanoparticle-based vaccine, a protein-based vaccine, a live vaccine, a live attenuated vaccine, a whole cell vaccine, a DNA vaccine, or an RNA vaccine; and optionally, the first vaccine is a priming vaccine and the second vaccine is a boosting vaccine, or optionally, the second vaccine is a priming vaccine and the first vaccine is a boosting vaccine. In one embodiment, the method induces directed affinity maturation. In one embodiment, the method results in a broadly neutralizing immune response.

[0020] In one embodiment of the method of making any of the nanostructures of the present disclosure, the method effects in vitro assembly of the nanostructure by: expressing the first plurality of polypeptides sequentially or non-sequentially in a first recombinant expression system, expressing the first plurality of antigens in a second recombinant expression system, purifying the first plurality of polypeptides, purifying the first plurality of antigens, with the proviso that expression of each component precedes purification of that component; and then mixing the first plurality of polypeptides and the first plurality of antigens; thereby producing the nanostructure.

[0021] In one embodiment of the method of making a nanostructure, the method effects in vitro assembly of the nanostructure by: expressing the first plurality of polypeptides sequentially or non-sequentially in a first recombinant expression system, expressing the first plurality of antigens in a second recombinant expression system, expressing the second plurality of polypeptides in a third recombinant expression system, purifying the first plurality of polypeptides, purifying the first plurality of antigens, purifying the second plurality of polypeptides, with the proviso that expression of each component precedes purification of that component; and mixing the first plurality of polypeptides, the first plurality of antigens, and the second plurality of polypeptides; thereby producing the nanostructure. Optionally, the first recombinant expression system and the second recombinant expression system are the same, and the first plurality of polypeptides and the first plurality of antigens are purified together.

[0022] In one embodiment of the method of making a nanostructure, the method comprises expressing the first plurality of polypeptides and the first plurality of antigens in a single recombinant expression system, thereby producing the nanostructure, and purifying the nanostructure. In one embodiment, the method comprises expressing the first plurality of polypeptides, the first plurality of antigens, and the second plurality of polypeptides in a single recombinant expression system, thereby producing the nanostructure, and purifying the nanostructure. In one embodiment, optionally, the first plurality of polypeptides and the first plurality of antigens are encoded by a single open reading frame; and, optionally, the single open reading frame encodes a fusion protein of the polypeptides and the antigens; and, optionally, the single open reading frame encodes a self-cleaving peptide.

[0023] The foregoing paragraph is not intended to define every aspect of the application, and additional aspects are described in other sections, such as the detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if such combinations are not found in the same sentence, clause, or paragraph of this document. As an additional aspect, the application includes all embodiments of the application that are narrower in scope in any way than the variations defined by the specific paragraph above. For example, where aspects of the application are described as belonging to a genus, it should be understood that each member of the genus individually is an aspect of the application. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1A A schematic showing the production of a nanostructure with antigens by in vitro assembly. The two components or building blocks of a given nanostructure can be expressed and purified separately, which allows for the initiation of assembly of the nanostructure by mixing the purified components in vitro, a process referred to as in vitro assembly. In some embodiments, the two components of a nanostructure can be expressed in different expression hosts (e.g., human HEK293F cells or bacterial Escherichia coli cells). The figure schematically depicts the assembly of a 120 subunit nanostructure with 20 trimeric antigens (60 antigen subunits) via in vitro assembly of an antigen nanostructure trimer fusion protein produced in HEK293F cells and a nanostructure pentamer protein produced in E. coli.

[0025] Figure 1B Depicted are exemplary nanostructure architectures.

[0026] Figures 2A-2C A schematic showing the illustration of DS-Cavl (SEQ ID NO: 1) (A), DS-Cavl-foldon-T33-31A (SEQ ID NO: 2) (B), and DS-Cavl-T33-31A (SEQ ID NO: 3) (C) secreted in tissue culture supernatant. Figure 2A Figure 2B Figure 2C ​​) Detection plot for fusion proteins. ELISA analysis was performed on tissue culture supernatants of cells expressing DS-Cavl (top), DS-Cavl -Foldon-T33-31A / T33-31B (lower left), and DS-Cavl -T33-31A / T33-31B (lower right). Four different monoclonal antibodies that bind RSV F were used to assess the presence of DS-Cavl or DS-Cavl fusion proteins in the supernatants. The results confirm the secretion of proteins containing well-folded RSV F antigens.

[0027] Figure 3 Size exclusion chromatography of DS-Cavl -I53-50A is shown. Protein purified from tissue culture supernatant by immobilized metal affinity chromatography was applied to a Superose 6 10 / 300 GL size exclusion column. Protein eluted as a single monodisperse species.

[0028] Figure 4 Size exclusion chromatography of in vitro assembled DS-Cavl -I53-50 nanostmctures is shown. Purified DS-Cavl -I53-50A and I53-50B.4PT1 proteins were mixed in approximately 1 : 1 molar ratio, incubated overnight at 4°C, and then applied to a Sephacryl S-500 16 / 60 HR size exclusion column. Assembled nanostmctures eluted as a single monodisperse peak at approximately 65 mL, while excess DS-Cavl -I53-50A trimer component eluted at approximately 90 mL.

[0029] Figure 5 Negative stain electron micrographs and two-dimensional class averages of in vitro assembled DS-Cavl -I53-50 nanostmctures are shown. In vitro assembled DS-Cavl -I53-50 nanostmctures purified by size exclusion chromatography were imaged by negative stain electron microscopy (top). Averaging of many nanostmctures produced two-dimensional class averages (bottom) that indicate that the I53-50 portion of the nanostmcture is highly ordered and uniform, while the precise three-dimensional position of the displayed antigen varies slightly due to the flexible nature of the linker between the DS-Cavl and I53-50A domains of the DS-Cavl -I53-50A fusion protein.

[0030] Figures 6A-6C A series of plots depicting the antigenicity of DS-Cavl -I53-50 nanostmctures is shown. Analysis of purified DS-Cavl -I53-50 nanostmctures by ELISA using four RSV F-specific monoclonal antibodies, including pre-fusion specific antibodies MPE8, D25, and RSD5, demonstrates that the antigenicity of the displayed RSV F is preserved in the nanostmcture. Figure 6AThis indicates that the DS-Cav1 antigen correctly folds and remains in a pre-fusion state when multivalently displayed on the DS-Cav1-I53-50 nanostructure. This finding was confirmed by surface plasmon resonance measurements using various RSV F-specific antibodies, when combined with the trimer DS-Cav1 ( Figure 6C Compared to this, it further demonstrates the multivalent display of DS-Cav1. Figure 6B This leads to an affinity effect that reduces the rate of antibody dissociation.

[0031] Figure 7 This is a graph depicting the DS-Cav1-specific serum antibody titers from mice immunized with the DS-Cav1-I53-50 nanostructure. Mouse groups were immunized with I53-50 nanostructures lacking the additional antigen, trimer DS-Cav1, or I53-50 nanostructures containing the DS-Cav1 antigen at 33%, 66%, or 100% valence. DS-Cav1-specific serum antibody titers were determined by ELISA on DS-Cav1-coated plates. The serum antibody titers for each mouse are plotted as circles, with the geometric mean within each group plotted as a horizontal line, and reported numerically at the bottom.

[0032] Figure 8 This is a graph depicting the serum neutralizing activity induced by immunization with the DS-Cav1-I53-50 nanostructure. Mouse groups were immunized with the I53-50 nanostructure lacking the additional antigen, the trimer DS-Cav1, or the I53-50 nanostructure with the DS-Cav1 antigen at 33%, 66%, or 100% valence. The neutralizing titer for each mouse is plotted as a circle, with the geometric mean within each group plotted as a horizontal line.

[0033] Figures 9A-9B This diagram depicts the immunogenicity of the primate immune system induced by immunization with the DS-Cav1-folded I53-50 nanostructure. At weeks 0 and 4, rhesus monkeys were intramuscularly injected with the DS-Cav1-folded I53-50 nanostructure, in which either the free DS-Cav1 trimer or the DS-Cav1-folded I53-50 nanostructure represented DS-Cav1 in 100% valence. In both cases, the dose of DS-Cav1 antigen was 50 μg, and the immunogen was formulated with MF59-like, squalene-based oil-in-water emulsion adjuvant SWE. Anti-DS-Cav1 antibody titers in serum obtained from the animals at weeks 6 and 16 were evaluated. Figure 9A ) and RSV neutralizing antibody titer ( Figure 9A ).

[0034] Figure 10This is a graph depicting the physical stability of DS-Cav1 when fused with I53-50A and / or further assembled into icosahedral nanostructures. Samples containing equal concentrations (50 nM) of DS-Cav1, including trimer DS-Cav1, trimer DS-Cav1-sheet-I53-50A, and DS-Cav1-sheet-I53-50 nanostructures, were divided into four aliquots and incubated at 20 °C, 50 °C, 70 °C, or 80 °C for 1 hour. After cooling to room temperature, D25 binding was determined by surface plasmon resonance (SPR).

[0035] Figures 11A-11J This is a graph depicting the physical stability of the nanostructure. Chemical denaturation in guanidine hydrochloride (GdnHCl), monitored by intrinsic tryptophan fluorescence, was used as a second antibody-independent technique to evaluate the trimer DS-Cav1 (…). Figure 1A and Figure 1B ), DS-Cav1-Folder-I53-50A (Figures 1C and 1D), DS-Cav1-Folder-I53-50 (Figures 1E and 1F), I53-50 (Figures 1G and 1H), and I53-50A (Figures 1I and 1D). Figure 11J The data indicate the excellent physical stability of the DS-Cav1 antigen when fused with the I53-50A nanostructure component gene. Detailed Implementation

[0036] This disclosure relates to nanostructures and nanostructure-based vaccines. Some of the nanostructures disclosed herein display antigens capable of evoking an immune response to infectious agents such as bacteria, viruses, and parasites. Some of the vaccines disclosed herein can be used to prevent or reduce the severity of infection by infectious agents, including but not limited to Lyme disease, pertussis, herpesviruses, orthomyxoviruses, paramyxoviruses, pneumoviruses, filoviruses, flaviviruses, reoviruses, retroviruses, meningococci, and malaria. The antigens can be non-covalently or covalently attached to the core of the nanostructure, including as fusion proteins or by other means disclosed herein. Multimeric antigens can optionally be displayed along the symmetry axis of the nanostructure. Proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use are also provided.

[0037] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, and therefore the embodiments can, of course, vary. It should also be understood that, because the scope of the invention will be limited only by the claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0038] 1. Overview of Nanostructures

[0039] The nanostructures of the present invention may include polyprotein assemblies suitable for displaying antigens or antigenic fragments. The nanostructures of the present invention include at least a first plurality of polypeptides. The first plurality of polypeptides may be derived from a naturally occurring protein sequence by substituting at least one amino acid residue or by adding to the N or C terminus of one or more residues. In some cases, the first plurality of polypeptides comprises a gene sequence determined de novo by computational methods. The first plurality of polypeptides may form the entire nanostructure; or the nanostructure may comprise one or more additional polypeptides, thereby making the nanostructure include two, three, four, five, six, seven, or more polypeptides. In some cases, the first plurality of polypeptides will form a trimer associated by 3x rotational symmetry, and the second plurality of polypeptides will form a pentamer associated by 5x rotational symmetry. These one or more polypeptides may be arranged together such that members of each plurality of polypeptides are associated with each other by a symmetry operator. A general computational method for designing self-assembled protein materials is disclosed in US Patent Publication No. 2015 / 0356240A1, which includes symmetrically docking protein structural units into a target symmetry architecture.

[0040] The term "core" in this document refers to the central portion of a nanostructure that links together the antigen or antigenic fragments displayed by the nanostructure. In one embodiment, the core and the displayed antigen are the same polypeptide, meaning the antigen itself is capable of self-assembling into the nanostructure. The advantage of designing the antigen itself to self-assemble is that the entire nanostructure serves as an antigenic component of the vaccine. However, in one embodiment, the core of the nanostructure of this disclosure is a universal platform suitable for displaying any of the various antigens that might be chosen to be included in a vaccine. The advantage of designing the core as a universal platform is that one or more polypeptides constituting the core can be pre-designed and optimized and then applied to different antigens. It should be understood that in some cases, the same polypeptide can form part of the "core" and then extend outward as both a connector for attaching the antigen and as the antigen itself (i.e., a fusion protein with the antigen). In embodiments of this disclosure, the antigen is an infectious protein, glycoprotein, or oligosaccharide.

[0041] In some cases, even if the core is capable of self-assembly independently in the absence of antigen, self-assembly can be further promoted by antigen polymerization. This is the case, for example, when a homotrimeric antigen (such as HIV gp140, influenza HA, or RSVF protein) is an antigen displayed on the particle, or one of several antigens. In some cases, trimeric antigens positioned along the triple axis of the nanostructure promote proper folding and conformational stability of the antigen, and make the self-assembly of the nanostructure a cooperative process, because the antigen is properly trimerized partly due to its display along the triple axis of the nanostructure core, and the nanostructure is stably in its assembled form at least partially through non-covalent or covalent interactions between the trimer units. In some cases, introducing mutations into the antigen or nanostructure components can optionally further stabilize the assembly, especially if the cysteine ​​residues are located at positions that generate intramolecular disulfide bonds. In some cases, dimeric, trimeric, tetrameric, pentameric, or hexameric antigens are displayed on a core designed to have matching 2-, 3-, 4-, 5-, or 6-fold axes of symmetry, thereby allowing the core to accommodate an arrangement of multimeric antigens with the natural symmetry of the antigen.

[0042] 2. Various non-limiting examples of nanostructures

[0043] Non-limiting examples of implementation methods are in Figure 1AThe figure depicts an RSV F protein recombinantly expressed in 293F cells, fused with the genes of a component of the nanostructure (a first plurality of polypeptides); together with a pentamer protein assembly recombinantly expressed in E. coli cells (a second plurality of polypeptides), these two plurality of polypeptides self-assemble into a nanostructure displaying 20 F protein trimers around an icosahedral core (“designed nanoparticle immunogen”). In this embodiment, the core has a generic design. As explained below, in other embodiments, the RSV F protein is replaced with another antigen protein, such as a trimer glycoprotein from another virus. In some embodiments, the nanostructure includes trimer glycoproteins of HIV-1, HIV-2, EBV, CMV, RSV, influenza, Ebola virus, Marburg virus, dengue fever, SARS, MERS, Hantavirus, or Zika virus. In some embodiments, the nanostructure comprises a trimeric glycoprotein of a virus that is evolutionarily or sequentially associated with any of these exemplary viruses, including but not limited to herpesviruses, orthomyxoviruses, paramyxoviruses, pneumoviruses, filamentous viruses, flaviviruses, reoviruses, or retroviruses. In one embodiment, the nanostructure comprises one or more extracellular domains of a transmembrane protein or glycoprotein, or antigenic fragments thereof. In some embodiments, the nanostructure comprises antigenic proteins or protein fragments or antigenic oligosaccharides of bacterial pathogens, including but not limited to Neisseria meningitides (also known as "meningococcus"), Haemophilus influenzae type B, Streptococcus pneumoniae, and Listeria monocytogenes.

[0044] In some cases, the trimeric antigens that can be used with this or similar nanostructures are limited to proteins such as HIV gp140, influenza HA, dengue E protein, or Ebola sGP. When other trimeric antigens are used, they can optionally be positioned on the triplet axis of symmetry of the nanostructure. In some cases, the selected antigen is monomeric but still positioned on the triplet axis. Therefore, Figure 1A The nanostructures depicted are capable of displaying 20 trimer antigens or 60 monomer antigens. Alternatively, a pentamer complex of the nanostructure may be used to display 12 pentamer antigens or 70 monomer antigens. In one embodiment, the nanostructure comprises 20 copies of the trimer antigen and 12 copies of the pentamer antigen.

[0045] 2.1. Nanostructure Core

[0046] Other potential arrangements of the polypeptide disclosed herein are Figure 1BAs shown in the diagram. In some embodiments, the nanostructure is adapted to display up to 8 trimers; 8 trimers and 12 dimers; 6 tetramers and 12 dimers; 6 tetramers and 8 trimers; 20 trimers and 30 dimers; 4 trimers and 6 dimers; 4 first trimers and 4 second trimers, or 8 trimers; 12 pentamers and 20 trimers; or 12 pentamers and 30 dimers; or 4 trimers. In some cases, one of the axes of symmetry is not used for antigen display; therefore, in some embodiments, the nanostructure is adapted to display up to 8 trimers; 12 dimers; 6 tetramers; 20 trimers; 30 dimers; 4 trimers; 6 dimers; 8 trimers; or 12 pentamers. In some cases, monomeric antigens are displayed; therefore, the nanostructure is suitable for displaying up to 12, 24, 60, or 70 monomeric antigens. In other cases, the nanostructure comprises a mixture of multiple peptides, such that the originally identical peptide at the core of the nanostructure displays different antigens or does not display any antigen. Thus, depending on the ratio of peptides, in some cases, the nanostructure is suitable for displaying 1 to 130 antigens (e.g., on I52 particles), wherein each of the displayed antigens may be identical or may be different members of a mixed population proportional to any chosen ratio. Antigens may be co-expressed in a recombinant expression system and self-assembled prior to purification. Alternatively, antigens may be expressed separately and then mixed together before or after purification from the expression host and associated contaminants. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more antigens are displayed. Non-limiting exemplary nanostructures are provided in the following literature: Bale et al. Science 353:389-94 (2016); Heinze et al. J. Phys. Chem B. 120:5945-5952 (2016); King et al. Nature 510:103-108 (2014); and King et al. Science 336:1171-71 (2012).

[0047] 2.2. Hybrid Nanostructures

[0048] In some embodiments, the nanostructure displays two or more antigens from the same organism on the same nanostructure, such as, but not limited to, HIV gp140 and HIV gp41; or Ebola virus GP1 and GP2; or measles H and F proteins; or CMV gB and CMV UL128, UL130, UL131A, gH (UL75), and gL (UL115). In some cases, the nanostructure displays two antigenic proteins or glycoproteins generated by post-transcriptional cleavage, such as by cleavage of RSV F protein or influenza HA protein by a recombinant expression system endogenous protease or by exogenously provided protease.

[0049] In some cases, the nanostructure is suitable for displaying the same antigens from two or more different strains of pathogens. In non-limiting examples, the same nanostructure displays a mixed population of homotrimeric protein antigens or a mixed heterotrimeric protein antigen of different strains of infectious agents. In one embodiment, the nanostructure displays the HA protein of H1N1 influenza A and H3N2 influenza A proteins. In one embodiment, the nanostructure displays the HA protein of influenza A and influenza B. In one embodiment, the gp140 protein of HIV from different strains is displayed on a single nanostructure. Two, three, four, five, or six strains of HIV can be displayed by the same nanostructure. Unbound by theory, the advantage of such mixed nanostructures is that they promote the generation of cross-reactive or broadly neutralizing immune responses. In some cases, the nanostructure-based vaccine of this disclosure is a universal influenza vaccine. In some cases, the nanostructure-based vaccine of this disclosure is an HIV vaccine. In some cases, the nanostructure-based vaccine of this disclosure provides durable protection against HIV. In some cases, the nanostructure-based vaccine of this disclosure provides durable protection against influenza. In one embodiment, the nanostructure is adapted to display the E protein of dengue serotypes 1, 2, 3, and 4. In one embodiment, the nanostructure-based vaccine comprises a nanostructure that individually displays the E protein from each of dengue serotypes 1, 2, 3, and 4. In one embodiment, the nanostructure-based vaccine of this disclosure provides immunity against dengue virus without increasing the risk of dengue hemorrhagic fever or dengue shock syndrome.

[0050] When preparing mixed nanostructures, it may be advantageous to ensure homogeneity in a strain-specific manner rather than allowing heterodimerization, such that, for example, all H1N1 influenza A HA proteins are displayed on one triplet axis of the T33 particle, while all H3N2 influenza A HA proteins are displayed on another triplet axis. This can be achieved by using nanostructures comprising two or more polypeptides as the core of the nanostructure, each polypeptide being attached to a different antigen. Alternatively, the nanostructure can be engineered with one or more symmetry-breaking mutations (such as buttonhole mutations or intramolecular disulfide bond mutations) that prevent trimerization between different antigens. In that case, the nanostructure displays multimeric antigens from different strains at symmetrically equivalent positions on the nanostructure, but each position on the nanostructure is occupied by homomers from the same strain, with only a small proportion of interstrain heteromers. In some cases, the antigens themselves can be genetically engineered to prevent interstrain heterodimerization. In one embodiment, the nanostructure is engineered to prevent heteropolymerization of two antigenic proteins with conserved structures but different antigenicities, such as, for example, the HA protein from 2009 H1N1 California influenza and the HA protein from 1999 H1N1 New Caledonia influenza. Furthermore, when a hybrid nanostructure is prepared and the antigen is displayed as a fusion protein, the nanostructure will contain three or more different proteins because the fusion proteins will share the same (or equivalent) domains for forming the core of the nanostructure with different antigenic domains targeting each antigen displayed on the nanostructure.

[0051] 2.3. Attachment method

[0052] The nanostructures disclosed herein display antigens in various ways, including as gene fusions or through other means disclosed herein. As used herein, “attach to” means any means known in the art for inducing association between two peptides. Association can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or non-selective.

[0053] In some embodiments, attachment is achieved by genetic engineering to generate antigens that fuse with the N- or C-terminus of one of a plurality of polypeptides constituting the nanostructure. Thus, the nanostructure can consist of or be substantially composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides, thereby exhibiting 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigens, wherein at least one of the multiple antigens is fused to the gene of at least one of the multiple polypeptides. In some cases, the nanostructure consists substantially of a plurality of polypeptides capable of self-assembly and containing multiple antigens fused to their genes. In some cases, the nanostructure consists substantially of a first plurality of polypeptides and a second plurality of polypeptides, the first plurality of polypeptides containing multiple antigens fused to their genes; the second plurality of polypeptides being capable of co-assembling into a two-component nanostructure, one plurality of polypeptides attaching the antigen to the nanostructure, while the other plurality of polypeptides facilitates the self-assembly of the nanostructure.

[0054] In some embodiments, attachment is achieved through post-translational covalent attachment between one or more polypeptides and one or more antigens. In some cases, chemical cross-linking is used to non-specifically attach antigens to nanostructured polypeptides. In some cases, chemical cross-linking is used to specifically attach antigens to nanostructured polypeptides. Various specific and non-specific cross-linking chemistry methods are known in the art, such as click chemistry and other methods. Generally, any cross-linking chemical used to link two proteins can be applied to the nanostructures disclosed in this invention. Specifically, chemistry for generating immunoconjugates or antibody-drug conjugates can be used. In some cases, cleavable or non-cleavable linkers are used to generate antigen-nanostructure conjugates (ANCs). Processes and methods for conjugating antigens with carriers are provided, for example, U.S. Patent Publication No. 2008 / 0145373A1. In one embodiment, the antigen is a polysaccharide. In some cases, the antigen is a polysaccharide, and the nanostructure acts as a hapten. In one embodiment, the target antigen is a protein, and the conjugation of the target antigen with the polysaccharide is used to enhance the immune response. Processes for preparing protein-polysaccharide conjugates are provided, for example, in U.S. Patent No. 6,248,334. In some cases, the conjugation of proteins with polysaccharides transforms the polysaccharide from a weakly immunogenic, T-cell-independent antigen into a T-cell-dependent antigen that recruits T-cell helpers, thereby stimulating a heightened immune response. See J.M. Curuse et al. (eds.), Conjugate Vaccines, Karger, Basel, (1989); and R.W. Willis et al. (eds.), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994).

[0055] In one embodiment, attachment is achieved through non-covalent attachment between one or more peptides and one or more antigens. In some cases, the antigen is engineered to carry a negative charge on at least one surface, and the peptide is engineered to carry a positive charge on at least one surface, or both positive and negative charges, respectively. This promotes intermolecular association between the antigen and the peptide of the nanostructure via electrostatic forces. In some cases, shape complementarity is employed to induce the connection between the antigen and the nanostructure. Shape complementarity may be pre-existing or rationally designed. In some cases, computational design of protein-protein interfaces is used to achieve attachment. In one embodiment, the antigen is biotin-labeled, and the peptide contains streptavidin, or vice versa. In one embodiment, streptavidin is displayed as a tetramer on the four axes of the nanostructure via gene fusion or otherwise, and the biotin-labeled antigen is a monomer, dimer, or tetramer, thereby allowing association with the nanostructure in a configuration suitable for the native multimerization of the antigen. In some cases, a protein-based adaptor can be used to capture the antigen. In some cases, the peptide is fused with a protein capable of binding a complementary protein, which is fused with the antigen. In one embodiment, the peptide is fused to the rotavirus VP6 protein to form a trimer, and the N-terminus of the antigen is fused to the N-terminal peptide of the rotavirus VP7, thereby allowing the antigen to associate with the trimer-trimer of the nanostructure. See Chen et al. Molecular interactions in rotavirus assembly and uncoatingseen by high-resolution cryo-EM. PNAS 2009 Jun 106(26)10644-10648.

[0056] In one embodiment, each of the first plurality of antigenic proteins has a proximal end and a distal end, and the proximal end of each antigenic protein is attached to a member of the first plurality of polypeptides. Therefore, the distal end of the antigenic protein is defined as the antigenic portion furthest from the center of the nanostructure. In one embodiment, the antigenic protein contains a target epitope, and the nanostructure is configured to display the target epitope. In some cases, the antigenic protein may contain more than one target epitope, and the nanostructure is configured to display each target epitope. In some cases, epitopes progressively closer to the distal end (not bound by theory) are preferentially accessible to the immune system. The distal end of the antigenic protein may be its N-terminus, its C-terminus, or neither. Therefore, depending on how the antigenic protein attaches to the nanostructure, the antigenic protein can be displayed in any orientation. In some cases, the antigenic protein is displayed such that one or more known epitopes are oriented at or toward the distal end of the antigenic protein, such that these one or more epitopes are preferentially accessible to the immune system. In some cases, the orientation will reproduce the orientation of a viral protein relative to a virus. Therefore, in the case of influenza HA, the antigenic protein HA can be oriented such that, similar to the orientation of HA throughout the virus, the receptor binding site is located distal to the protein; or alternatively, the influenza HA protein can be oriented such that the stem epitope is preferentially accessible to the immune system. The choice of orientation can direct the immune system to one epitope or another. In this example, the immune response to influenza can be directed to either the receptor binding site or the stem by orientation selection. Similarly, the orientation of other antigens may affect the immune response. In some embodiments, the orientation of the antigen results in an immune response targeting the preferred epitope. In the case of HIV, in some embodiments, the antigenic protein is the Env protein of HIV-1 or HIV-2, or an antigenic fragment thereof. The orientation of the Env protein or its fragment will, in some cases, reproduce the orientation of the Env protein relative to the HIV virion, thereby making the proximal end the proximal membrane end of the Env protein or its fragment. In some cases, preferred epitopes are selected from the group consisting of: CD4 binding sites (CD4bs); the V2 proteoglycan moiety on the top of the trimer of the Env; the V3 proteoglycan moiety on the high-mannose patch of the Env; the membrane proximal external region (MPER) of the transmembrane domain of the Env; and the gp120-gp41 interface with or without the fusion peptide. In some cases, epitope priority is controlled by other means, such as localizing the glycan on the nanostructure by adding or subtracting the N-linked glycan sequence motif NX-[T / S] at predetermined positions in the amino acid sequence of any polypeptide including the antigen of the nanostructure.In some cases, epitopes located at an intermediate distance from proximal to distal are preferred over epitopes located more distally, based on various considerations, including but not limited to the overall geometry of the nanostructure, surface hydrophobicity, surface charge, and competitive binding to proteins endogenously present in the subject or exogenously provided in the vaccine composition. This disclosure covers all known methods of rationally designing protein structures, and the foregoing is not intended to be limiting.

[0057] 2.4. Nanostructured polypeptide sequences

[0058] One or more polypeptides disclosed herein may have any of a variety of amino acid sequences. U.S. Patent Publication No. US 2015 / 0356240A1 describes various methods for designing nanostructures. As disclosed in U.S. Patent Publication No. US 2016 / 0122392A1 and International Patent Publication No. WO 2014 / 124301A1, the ability to design isolated polypeptides of SEQ ID NO:1-51 to achieve their pairwise self-assembly to form nanostructures (such as icosahedral nanostructures) is described. This design involves the design of suitable interface residues for each member of a polypeptide pair that can assemble to form a nanostructure. The nanostructures thus formed comprise symmetrically repeating non-natural, non-covalent polypeptide-peptide interfaces that orient the first and second components as nanostructures, such as nanostructures having icosahedral symmetry. Therefore, in one embodiment, the first and second polypeptides are selected from the group consisting of SEQ ID NO:1-51. In each case, the N-terminal methionine residue is optional.

[0059] Table 1

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Table 1 provides the amino acid sequences of a first and second polypeptide from embodiments of the present disclosure. In each case, the sequence pairs together form an I53 icosahedron. The right column in Table 1 identifies residue numbers in each exemplary polypeptide that are identified as present at the interface of the resulting assembled nanostructure (i.e., “identified interface residues”). As can be seen, the number of interface residues in the exemplary polypeptides of SEQ ID NO: 1-34 ranges from 4 to 13. In various embodiments, the first and second polypeptides comprise amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in length to those of the polypeptides selected from the group consisting of SEQ ID NO: 1-34, and identical at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface positions (depending on the number of interface residues of the given polypeptide). SEQ ID NO:35-51 represents other amino acid sequences of the first and second polypeptides from embodiments of the present disclosure. In other embodiments, the first and second polypeptides comprise amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in length to those of polypeptides selected from the group consisting of SEQ ID NO:1-51, and identical at least 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% of the identified interface positions.

[0068] Typically, as with proteins, peptides are expected to tolerate certain variations in the designed sequence without disrupting subsequent assembly into nanostructures: especially when such variations involve conserved amino acid substitutions. As used herein, “conserved amino acid substitution” means that: hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) can only be substituted by other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) can only be substituted by other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (Arg, His, Lys) can only be substituted by other amino acids with positively charged side chains; amino acids with negatively charged side chains (Asp, Glu) can only be substituted by other amino acids with negatively charged side chains; and amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) can only be substituted by other amino acids with polar, uncharged side chains.

[0069] In various embodiments of the nanostructures of the present invention, the first polypeptide and the second polypeptide comprise polypeptides having an amino acid sequence selected from the following pairs or modified forms thereof (i.e., permitted modifications as disclosed for the polypeptides of the present invention: polypeptides comprising an amino acid sequence that is at least 75% identical in length to the amino acid sequence indicated by SEQ ID NO. and / or identical at at least one identified interface site):

[0070] SEQ ID NO:1 and SEQ ID NO:2 (I53-34A and I53-34B);

[0071] SEQ ID NO:3 and SEQ ID NO:4 (I53-40A and I53-40B);

[0072] SEQ ID NO:3 and SEQ ID NO:24 (I53-40A and I53-40B.1);

[0073] SEQ ID NO:23 and SEQ ID NO:4 (I53-40A.1 and I53-40B);

[0074] SEQ ID NO:35 and SEQ ID NO:36 (genus I53-40A and genera I53-40B);

[0075] SEQ ID NO:5 and SEQ ID NO:6 (I53-47A and I53-47B);

[0076] SEQ ID NO:5 and SEQ ID NO:27 (I53-47A and I53-47B.1);

[0077] SEQ ID NO:5 and SEQ ID NO:28 (I53-47A and I53-47B.1NegT2);

[0078] SEQ ID NO:25 and SEQ ID NO:6 (I53-47A.1 and I53-47B);

[0079] SEQ ID NO:25 and SEQ ID NO:27 (I53-47A.1 and I53-47B.1);

[0080] SEQ ID NO:25 and SEQ ID NO:28 (I53-47A.1 and I53-47B.1NegT2);

[0081] SEQ ID NO:26 and SEQ ID NO:6 (I53-47A.1NegT2 and I53-47B);

[0082] SEQ ID NO:26 and SEQ ID NO:27 (I53-47A.1NegT2 and I53-47B.1);

[0083] SEQ ID NO:26 and SEQ ID NO:28 (I53-47A.1NegT2 and I53-47B.1NegT2);

[0084] SEQ ID NO:37 and SEQ ID NO:38 (genus I53-47A and genera I53-47B);

[0085] SEQ ID NO:7 and SEQ ID NO:8 (I53-50A and I53-50B);

[0086] SEQ ID NO:7 and SEQ ID NO:32 (I53-50A and I53-50B.1);

[0087] SEQ ID NO:7 and SEQ ID NO:33 (I53-50A and I53-50B.1NegT2);

[0088] SEQ ID NO:7 and SEQ ID NO:34 (I53-50A and I53-50B.4PosT1);

[0089] SEQ ID NO:29 and SEQ ID NO:8 (I53-50A.1 and I53-50B);

[0090] SEQ ID NO:29 and SEQ ID NO:32 (I53-50A.1 and I53-50B.1);

[0091] SEQ ID NO:29 and SEQ ID NO:33 (I53-50A.1 and I53-50B.1NegT2);

[0092] SEQ ID NO:29 and SEQ ID NO:34 (I53-50A.1 and I53-50B.4PosT1);

[0093] SEQ ID NO:30 and SEQ ID NO:8 (I53-50A.1NegT2 and I53-50B);

[0094] SEQ ID NO:30 and SEQ ID NO:32 (I53-50A.1NegT2 and I53-50B.1);

[0095] SEQ ID NO:30 and SEQ ID NO:33 (I53-50A.1NegT2 and I53-50B.1NegT2);

[0096] SEQ ID NO:30 and SEQ ID NO:34 (I53-50A.1NegT2 and I53-50B.4PosT1);

[0097] SEQ ID NO:31 and SEQ ID NO:8 (I53-50A.1PosT1 and I53-50B);

[0098] SEQ ID NO:31 and SEQ ID NO:32 (I53-50A.1PosT1 and I53-50B.1);

[0099] SEQ ID NO:31 and SEQ ID NO:33 (I53-50A.1PosT1 and I53-50B.1NegT2);

[0100] SEQ ID NO:31 and SEQ ID NO:34 (I53-50A.1PosT1 and I53-50B.4PosT1);

[0101] SEQ ID NO:39 and SEQ ID NO:40 (genus I53-50A and genera I53-50B);

[0102] SEQ ID NO:9 and SEQ ID NO:10 (I53-51A and I53-51B);

[0103] SEQ ID NO:11 and SEQ ID NO:12 (I52-03A and I52-03B);

[0104] SEQ ID NO:13 and SEQ ID NO:14 (I52-32A and I52-32B);

[0105] SEQ ID NO:15 and SEQ ID NO:16 (I52-33A and I52-33B)

[0106] SEQ ID NO:17 and SEQ ID NO:18 (I32-06A and I32-06B);

[0107] SEQ ID NO:19 and SEQ ID NO:20 (I32-19A and I32-19B);

[0108] SEQ ID NO:21 and SEQ ID NO:22 (I32-28A and I32-28B);

[0109] SEQ ID NO:23 and SEQ ID NO:24 (I53-40A.1 and I53-40B.1);

[0110] SEQ ID NO:41 and SEQ ID NO:42 (T32-28A and T32-28B);

[0111] SEQ ID NO:43 and SEQ ID NO:44 (T33-09A and T33-09B);

[0112] SEQ ID NO:45 and SEQ ID NO:46 (T33-15A and T33-15B);

[0113] SEQ ID NO:47 and SEQ ID NO:48 (T33-21A and T33-21B);

[0114] SEQ ID NO:49 and SEQ ID NO:50 (T33-28A and T32-28B); and

[0115] SEQ ID NO:51 and SEQ ID NO:44 (T33-31A and T33-09B (also known as T33-31B)).

[0116] In one embodiment, one or more proteins or antigenic fragments thereof are expressed as a fusion protein with a first polypeptide and / or a second polypeptide. In these embodiments, one or more proteins or antigenic fragments thereof are present at the N-terminus of the fusion protein, provided that this conformation is conducive to the presentation of one or more proteins or antigenic fragments thereof on the exterior of the nanostructure. Preferably, the protein is present at the N-terminus of the fusion protein, as long as it is located proximal to the C-terminus of the protein. In these embodiments, one or more proteins or antigenic fragments thereof are present at the C-terminus of the fusion protein, provided that this conformation is conducive to the presentation of one or more proteins or antigenic fragments thereof on the exterior of the nanostructure. Preferably, the protein is present at the C-terminus of the fusion protein, as long as it is located proximal to the M-terminus of the protein.

[0117] Non-limiting examples of nanostructures that can be used in vaccines disclosed herein include those disclosed in U.S. Patent No. 9,630,994 and U.S. Provisional Patent Application No. 62 / 481,331, which are incorporated herein in their entirety.

[0118] 3. Antigen

[0119] This disclosure provides nanostructure-based vaccines for any of a variety of known bacteria, viruses, or parasites associated with human or animal diseases. Specifically, this disclosure relates to vaccines for Lyme disease, pertussis, herpesviruses, orthomyxoviruses, paramyxoviruses, pneumoviruses, filoviruses, flaviviruses, reoviruses, retroviruses, malaria, viral meningitis, fungal meningitis, and bacterial meningitis (including Neisseria meningitidis (also known as "meningococcus"), Haemophilus influenzae type B, Streptococcus pneumoniae, and Listeria monocytogenes). For each of these organisms, antigens (proteins or polysaccharides) are known to elicit a protective immune response. This disclosure relates to incorporating any of these antigens (particularly antigenic proteins) into nanostructure-based vaccines. Guidance can be specifically derived from studies of the immune response to infection or vaccination, such as the isolation of binding or neutralizing antibodies, genetic analysis of antigen sequences, structural studies of antigenic proteins and antibodies, and, most particularly, clinical and veterinary experience with subunit vaccines. With virtually no limitations, any known subunit vaccine can be adapted for use with the nanostructures of this disclosure by employing the presentation methods provided above. In some embodiments, the nanostructure-based vaccines of this disclosure comprise oligosaccharides (e.g., meningococcal oligosaccharides) conjugated directly to the nanostructure, either through an intermediate protein (e.g., diphtheria toxoid, tetanus toxoid, or CRM197). In some embodiments, the nanostructure-based vaccines of this disclosure comprise antigens or antigenic fragments from the list provided in Table 2.

[0120] Table 2 Non-restrictive list of antigens

[0121]

[0122]

[0123]

[0124] In some embodiments, the antigen is an antigenic protein of a polypeptide or a variant thereof selected from SEQ ID NO:52-88 and 90-113 provided in Table 3.

[0125] Table 3

[0126] Non-restrictive list of antigen sequences

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140] 4. Component structural domains and connectors

[0141] In one embodiment, the nanostructure includes a trimer component. The trimer component includes a protein-protein interface that induces three copies of a first polypeptide to self-associate to form a trimer structural unit. Each copy of the first polypeptide further includes a surface-exposed interface that interacts with a complementary surface-exposed interface on a second component domain. As described in King et al. (Nature 510, 103-108, 2014), Bale et al. (Science 353, 389-394, 2016), and patent publications WO2014124301A1 and US20160122392A1, the complementary protein-protein interface between the trimer component domain and the second component domain drives the assembly of multiple copies of the trimer component domain and the second component domain into the target nanostructure. In some embodiments, each copy of the trimer component domain of the nanostructure serves as a gene fusion with an antigenic protein or an antigenic fragment thereof; these nanostructures exhibit proteins in their full valence state. In other embodiments, the nanostructures of the present invention comprise one or more copies of a trimer component domain carrying an antigenic protein or an antigenic fragment thereof as a gene fusion, and one or more trimer component domains not carrying an antigenic protein as a gene fusion; these nanostructures exhibit the F protein in a partially valenced state. The trimer component domain can be any polypeptide sequence that forms a trimer and interacts with a second component domain to drive assembly into the target nanostructure. In some embodiments, the nanostructures comprise a first polypeptide and a second polypeptide selected from those disclosed in US 20130274441 A1, US 2015 / 0356240 A1, US 2016 / 0122392 A1, and WO 2018 / 187325 A1, each of which is incorporated herein by reference in its entirety.

[0142] In the nanostructures disclosed herein, the antigenic protein and the core of the nanostructure can be genetically fused, thereby enabling them to reside within a single polypeptide. Preferably, the connection between the protein and the core of the nanostructure allows the protein or its antigenic fragments to be displayed on the exterior of the nanostructure. Thus, the connection point with the core of the nanostructure should be located on the exterior of the resulting nanostructure core. As those skilled in the art will understand, a variety of polypeptide sequences can be used to connect the protein or its antigenic fragments to the core of the nanostructure. These polypeptide sequences are referred to as adapters. Any suitable adapter can be used; there are no amino acid sequence requirements for serving as a suitable adapter. Besides enabling the protein or its antigenic fragments to be displayed on the exterior of the nanostructure, the adapter does not need to impose a rigid relative orientation of the protein or its antigenic fragments relative to the core of the nanostructure. In some embodiments, the adapter includes additional trimerizing domains (e.g., folding domains of T4 fibritin) that contribute to stabilizing the trimeric form of the F protein.

[0143] >T4 fibritin folded substructure domain (optional in the joint region) GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:89)

[0144] In some embodiments, the linker may comprise a Gly-Ser linker of any suitable length (i.e., a linker composed of glycine and serine residues). In some embodiments, the length of the Gly-Ser linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the Gly-Ser linker may comprise or consist of the amino acid sequence GSGGSGSGSGSGSGSG, GGSGGSGS, or GSGGSGSGSG.

[0145] 5. Assembly of nanostructures

[0146] In some embodiments, one or more purified samples of various peptides used to form nanostructures are mixed in approximately equimolar molar ratios under aqueous conditions. The peptides interact with each other to drive the assembly of the target nanostructure. Successful assembly of the target nanostructure can be confirmed by analyzing the in vitro assembly reaction using common biochemical or biophysical methods for assessing the physical size of the protein or protein assembly, including but not limited to size exclusion chromatography, native (non-denaturing) gel electrophoresis, dynamic light scattering, multi-angle light scattering, analytical ultracentrifugation, negative staining electron microscopy, cryo-electron microscopy, or X-ray crystallography. If necessary, the assembled nanostructure can be purified from other substances or molecules present in the in vitro assembly reaction using preparative techniques commonly used to separate proteins based on their physical size, including but not limited to size exclusion chromatography, preparative ultracentrifugation, tangential flow filtration, or preparative gel electrophoresis. The presence of antigenic proteins in the nanostructure can be assessed using techniques commonly used to identify protein molecules in aqueous solutions, including but not limited to SDS-PAGE, mass spectrometry, protein sequencing, or amino acid analysis. The accessibility of a protein outside a particle, as well as its conformation or antigenicity, can be assessed using techniques commonly used to detect the presence and conformation of antigens, including but not limited to binding via monoclonal antibodies, conformation-specific monoclonal antibodies, or antigen-specific antiserum.

[0147] In other embodiments, the nanostructures of the present invention comprise two or more different first polypeptides carrying different antigenic proteins as gene fusions; these nanostructures collectively display multiple different proteins on the same nanostructure. These multi-antigen nanostructures are generated by in vitro assembly with a mixture of first polypeptides, wherein each first polypeptide, as a gene fusion, carries one of two or more different proteins. The fraction of each first polypeptide in the mixture determines the average valence state of each antigenic protein in the resulting nanostructure. The presence and average valence state of each protein-carrying first polypeptide in a given sample can be assessed by quantitative analysis using the techniques described above for evaluating the presence of antigenic proteins in fully valenced nanostructures.

[0148] In various embodiments, the diameter of the nanostructure is between about 20 nanometers (nm) and about 40 nM, wherein the internal cavity span is between about 15 nM and about 32 nM, and the longest dimension of the pore size in the protein shell is between about 1 nM and about 14 nM.

[0149] In one embodiment, the nanostructure exhibits icosahedral symmetry. In this embodiment, the nanostructure may include 60 copies of a first polypeptide and 60 copies of a second polypeptide. In one such embodiment, the number of identical first polypeptides in each first component differs from the number of identical second polypeptides in each second component. For example, in one embodiment, the nanostructure includes twelve first components and twenty second components; in this embodiment, each first component may, for example, include five copies of the same first polypeptide, and each second component may, for example, include three copies of the same second polypeptide. In another embodiment, the nanostructure includes twelve first components and thirty second components; in this embodiment, each first component may, for example, include five copies of the same first polypeptide, and each second component may, for example, include two copies of the same second polypeptide. In a further embodiment, the nanostructure includes twenty first components and thirty second components; in this embodiment, each first component may, for example, include three copies of the same first polypeptide, and each second component may, for example, include two copies of the same second polypeptide. All these embodiments are capable of forming synthetic nanomaterials with regular icosahedral symmetry.

[0150] In various further embodiments, the oligomeric states of the first and second polypeptides are as follows:

[0151] I53-34A: Trimer + I53-34B: Pentamer;

[0152] I53-40A: Pentomer + I53-40B: Trimer;

[0153] I53-47A: Trimer + I53-47B: Pentamer;

[0154] I53-50A: Trimer + I53-50B: Pentomer;

[0155] I53-51A: Trimer + I53-51B: Pentamer;

[0156] I32-06A: Dimer + I32-06B: Trimer;

[0157] I32-19A: Trimer + I32-19B: Dimer;

[0158] I32-28A: Trimer + I32-28B: Dimer;

[0159] I52-03A: Pentomer + I52-03B: Dimer;

[0160] I52-32A: Dimer + I52-32B: Pentomer and

[0161] I52-33A: Pentomer + I52-33B: Dimer

[0162] 6. Nucleic acid

[0163] On the other hand, this disclosure provides isolated nucleic acids encoding the fusion proteins of this disclosure. The isolated nucleic acid sequences may comprise RNA or DNA. As used herein, “isolated nucleic acids” are those that have been removed from their normal surrounding nucleic acid sequences in a genome or cDNA sequence. Such isolated nucleic acid sequences may contain additional sequences to facilitate the expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, output and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be apparent to those skilled in the art that which nucleic acid sequences will encode the proteins of this disclosure.

[0164] On the other hand, this disclosure provides recombinant expression vectors comprising isolated nucleic acids of any embodiment or combination of embodiments of this disclosure operably linked to a suitable control sequence. A “recombinant expression vector” includes a vector that operably links a nucleic acid coding region or gene to any control sequence capable of enabling expression of a gene product. A “control sequence” operably linked to a nucleic acid sequence of this disclosure is a nucleic acid sequence capable of influencing the expression of a nucleic acid molecule. The control sequence need not be adjacent to the nucleic acid sequence, as long as the control sequence functions to guide the expression of the nucleic acid sequence. Thus, for example, an intermediate untranslated but transcribed sequence may exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited to plasmids and virus-based expression vectors. The control sequence used to drive the expression of the disclosed nucleic acid sequence in a mammalian system can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, and EF) or inducible (driven by any of a number of inducible promoters, including but not limited to tetracycline, ecdysone, and steroid-responsive promoters). The construction of expression vectors for transfecting prokaryotic cells is also well known in the art and can therefore be performed using standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, edited by EJ Murray, The Humana Press Inc., Clifton, NJ); and the Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be replicable in the host organism as an episome or by integration into the host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, this disclosure is intended to include other expression vectors that perform equivalent functions, such as viral vectors.

[0165] On the other hand, this disclosure provides host cells transfected with the recombinant expression vector disclosed herein, wherein the host cells may be prokaryotic or eukaryotic. The cells may be transiently or stably transfected. Such transfection of the expression vector into prokaryotic and eukaryotic cells can be achieved via any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation or liposome-mediated, DEAE-glucan-mediated, polycation-mediated, or virus-mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd edition (RIFreshney. 1987. Liss, Inc. New York, NY).) A method for producing a polypeptide according to this disclosure is another part of this disclosure. The method comprises the steps of: (a) culturing a host according to this disclosure under conditions favorable to polypeptide expression, and (b) optionally, recovering the expressed polypeptide.

[0166] 7. Vaccines and administration

[0167] This disclosure also provides vaccines comprising the nanostructures described herein. Such compositions can be used to generate antibodies in mammals, such as humans. The vaccine compositions of this disclosure typically comprise pharmaceutically acceptable carriers, and a detailed discussion of such carriers is available from Remington: The Science and Practice of Pharmacy.

[0168] The pH of the composition is typically between about 4.5 and about 11, such as between about 5 and about 11, between about 5.5 and about 11, between about 6 and about 11, between about 5 and about 10.5, between about 5.5 and about 10.5, between about 6 and about 10.5, between about 5 and about 10, between about 5.5 and about 10, between about 6 and about 10, between about 5 and about 9.5, between about 5.5 and about 9.5, between about 6 and about 9.5, between about 5 and about 9, between about 5.5 and about 9, between about 6 and about 9, between about 5 and about 8.5, between about 5.5 and about 8.5, between about 6 and about 8.5, between about 5 and about 8, between about 5.5 and about 8, between about 6 and about 8, between about 4.5, about 5, about 6.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, etc. A stable pH can be maintained by using buffers (such as Tris buffer, citrate buffer, phosphate buffer, or histidine buffer). Therefore, compositions typically contain buffers.

[0169] The composition may be sterile and / or pyrogen-free. The composition may be isotonic for human use.

[0170] The vaccine composition comprises an immunologically effective amount of one or more of its antigens. An “immunologically effective amount” is an amount that, when administered to a subject, effectively elicits an antibody response against the antigen. This amount can vary depending on the individual’s health and physical condition, age, the individual’s immune system’s ability to synthesize antibodies, the desired level of protection, the formulation of the vaccine, the treating physician’s assessment of the medical condition, and other relevant factors. It is contemplated that the amount will be within a relatively wide range that can be determined through routine testing. The antigen content of the compositions disclosed herein is typically expressed as protein mass per dose. Doses of 10-500 μg (e.g., 50 μg) per antigen can be useful.

[0171] Vaccine compositions may contain immunological adjuvants. Exemplary adjuvants include the following: 1. mineral-containing compositions; 2. oil emulsions; 3. saponin formulations; 4. virions and virus-like particles; 5. bacterial or microbial derivatives; 6. bioadhesives and mucosal adhesives; 7. liposomes; 8. polyoxyethylene ethers and polyoxyethylene ester formulations; 9. polyphosphazene (PCPP); 10. muramyl peptides; 11. imidazoquinone compounds; 12. thiourea compounds; 13. tryptamine compounds; 14. human immunomodulators; 15. lipopeptides; 16. benzonaphthyl; 17. microparticles; 18. immunostimulatory polynucleotides (such as RNA or DNA; for example, oligonucleotides containing CPG).

[0172] For example, the composition may contain an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion containing squalene such as MF59 or AS03), a TLR7 agonist (such as imidazoquinoline or imiquimod), or a combination thereof. Suitable aluminum salts include hydroxides (e.g., hydroxyoxides), phosphates (e.g., hydroxyphosphates, orthophosphates) (see, for example, Chapters 8 and 9 of Vaccine Design... (1995) edited by Powell and Newman. ISBN: 030644867X. Plenum). The salt may be in any suitable form (e.g., gel, crystals, amorphous, etc.), and the adsorption of antigen to the salt is an example. The aluminum salt in the composition for administration to the patient... +++ The concentration can be less than 5 mg / ml, for example <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. The preferred range is between 0.3 and 1 mg / ml. A maximum value of 0.85 mg / dose is preferred. Aluminum hydroxide and aluminum phosphate adjuvants are suitable for use in conjunction with this disclosure.

[0173] Exemplary adjuvants include, but are not limited to, Adju-Phos, Adjumerlm, albumin-heparin microparticles, seaweed dextran, Algammulin, alum, antigen formulations, AS-2 adjuvants, autologous dendritic cells, autologous PBMCs, and Avridine. TM B7-2, BAK, BAY R1005, Bupivacaine, Bupivacaine Hydrochloride, BWZL, Calcitriol, Calcium Phosphate Gel, CCR5 Peptide, CFA, Cholera Whole Toxin (CT) and Cholera Toxin B Subunit (CTB), Cholera Toxin A1-Subunit-Protein AD Fragment Fusion Protein, CpG, CRL1005, Liposomes Containing Cytokines, D-Murapalmitine, DDA, DHEA, Diphtheria Toxoid, DL-PGL, DMPC, DMPG, DOC / Alum Complex, Fowlpox, Freund's Disease 's) complete adjuvant, γ-inulin, Gerbu adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-α, IFA, IFN-γ in pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, imiquimod, Immotherapeutic agent TM Immunoliposomes containing antibodies against co-stimulatory molecules, interferon-γ, interleukin-1β, interleukin-12, interleukin-2, interleukin-7, and ISCOM(s) TM Iscoprep 7.0.3 TMKeyhole hemocyanin, lipid adjuvants, liposomes, loxoribine, LT (R192G), LT-OA or LT oral adjuvant, LT-R192G, LTK63, LTK72, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPL.TM., MPL-SE, MTP-PE, MTP-PE liposomes, Murametide, Murapalmitine, NAGO, nCT natural cholera toxin, nonionic surfactant vesicles, nontoxic mutant E112K of cholera toxin mCT-E112K, methylparaben, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, Peptomer-NP, Pleuran, PLG, PLGA, PGA and PLA, Pluronic L121, PMMA, PODDS TM PolyrA: PolyrU, Polysorbate 80, Protein Cochleates, QS-21, Quadri A Saponins, Quil-A, Rehydragel HPA, Rehydragel LV, RIBI, Ribilike Adjuvant Systems (MPL, TMD, CWS), S-28463, SAF-1, Sclavo Peptide, Sendai Liposomes, Lipid Matrix Containing Sendai, Span 85, Spector, Squalane 1, Squalene 2, Stearoyl Tyrosine, Tetanus Toxoid (TT), Theramide TM Threonyl muramyl dipeptide (TMDP), Ty particles, and Walter Reed liposomes are all options. The choice of adjuvant depends on the subject to be treated. Preferably, a pharmaceutically acceptable adjuvant is used.

[0174] A suitable immunological adjuvant comprises a compound of formula (I) as defined in WO2011 / 027222 or a pharmaceutically acceptable salt thereof adsorbed onto an aluminum salt. Many other adjuvants may be used, including any of those disclosed in Powell and Newman (1995).

[0175] The composition may contain antimicrobial agents, especially when packaged in multiple doses. Antimicrobial agents such as thimerosal and 2-phenoxyethanol are common in vaccines, but sometimes it may be desirable to use mercury-free preservatives or no preservatives at all.

[0176] The composition may contain detergents such as polysorbates, such as polysorbate 80. Detergents are typically present at low levels (e.g., <0.01%).

[0177] The composition may contain a sodium salt (e.g., sodium chloride) to provide tension. A concentration of 10 ± 2 mg / ml NaCl is typical, for example, about 9 mg / ml.

[0178] In some embodiments, the buffer in the vaccine composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The composition may also contain a lyophilization protectant, such as sucrose, sorbitol, or trehalose. In some embodiments, the composition contains a preservative, such as benzalkonium chloride, benzyl ammonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition contains a extender, such as glycine. In other embodiments, the composition comprises a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or combinations thereof. The composition may also comprise a tonic modifier, such as a compound that makes the formulation substantially isotonic or isotonic with human blood. Exemplary tonic modifiers include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the composition further comprises a stabilizer, such as a lyophilized or liquid molecule that substantially prevents or reduces the chemical and / or physical instability of the nanostructure. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0179] In another aspect, this disclosure provides a method for inducing an immune response against an infectious agent, the method comprising administering to a subject in need an immunologically effective amount of the immunogenic composition described herein, the immunogenic composition comprising a nanostructure as described herein.

[0180] In some implementations, the immune response includes the production of neutralizing antibodies against the infectious agent. In some implementations, the neutralizing antibodies are complement-independent.

[0181] An immune response may include a humoral immune response, a cell-mediated immune response, or both. In some embodiments, an immune response is induced against each delivered antigenic protein. A cell-mediated immune response may include a helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both. In some embodiments, the immune response includes a humoral immune response, and the antibody is a neutralizing antibody. The neutralizing antibody blocks viral infection of the cells. The virus infects epithelial cells and fibroblasts. In some embodiments, the immune response mitigates or prevents infection of both cell types. The neutralizing antibody response may be complement-dependent or complement-independent. In some embodiments, the neutralizing antibody response is complement-independent. In some embodiments, the neutralizing antibody response is cross-neutralizing; that is, the antibody generated against the administered composition neutralizes viral strains other than those used in the composition.

[0182] A useful measure of antibody efficacy in the art is a “50% neutralizing titer.” To determine a 50% neutralizing titer, serum from immunized animals is diluted to assess which diluted serum still retains the ability to block 50% of the virus from entering cells. For example, a titer of 700 means that the serum retains the ability to neutralize 50% of the virus after being diluted 700-fold. Therefore, a higher titer indicates a more potent neutralizing antibody response. In some embodiments, the titer is in the range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The 50% neutralizing titer range can include approximately 400, approximately 600, approximately 800, approximately 1000, approximately 1500, approximately 2000, approximately 2500, approximately 3000, approximately 3500, approximately 4000, approximately 4500, approximately 5000, approximately 5500, approximately 6000, approximately 6500, approximately 7000, approximately 8000, approximately 9000, approximately 10000, approximately 11000, and approximately 1... The upper limits are approximately 2000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000, or 30000. For example, a 50% neutralization titer could be approximately 3000 to approximately 25000. "Approximately" means ±10% of the stated value.

[0183] The compositions disclosed herein are typically administered directly to the subject. Direct delivery can be achieved via parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of tissues) or any other suitable route. For example, intramuscular administration, such as to the thigh or upper arm, can be used. Injection can be via a needle (e.g., a subcutaneous needle), but needle-free injection can be used alternatively. A typical intramuscular dose volume is 0.5 mL.

[0184] Dosage can be administered via a single-dose or multiple-dose regimen. Multiple doses can be used in primary immunization and / or booster immunization regimens. In a multiple-dose regimen, different doses can be administered via the same or different routes, such as parenteral primary immunization and mucosal booster, mucosal primary immunization and parenteral booster, etc. Multiple doses will typically be administered at intervals of at least 1 week (e.g., approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, approximately 16 weeks, etc.).

[0185] Subjects may be animals, preferably vertebrates, and more preferably mammals. Exemplary subjects include, for example, humans, cattle, pigs, chickens, cats, or dogs, as the infectious agents covered herein can cause problems across a wide range of species. When the vaccine is used for preventative purposes, the human subject is preferably a child (e.g., a toddler or infant), adolescent, or adult; when the vaccine is used for therapeutic purposes, the human subject is preferably an adolescent or adult. Vaccines intended for children may also be administered to adults, for example, to assess safety, dosage, immunogenicity, etc.

[0186] The vaccines disclosed herein can be preventive (i.e., to prevent disease) or therapeutic (i.e., to reduce or eliminate the symptoms of disease). The term preventive can be understood as reducing the severity of a particular symptom or preventing the onset of a particular symptom. To avoid confusion, the term preventive vaccine can also refer to a vaccine that improves the effects of future infections, for example, by reducing the severity or duration of such infections.

[0187] The isolated and / or purified nanostructures described herein can be administered alone or in mixed-modality protocols as a priming or booster, such as protein booster following RNA priming. The benefits of RNA priming / boosting strategies compared to protein priming strategies include, for example, increased antibody titers, a more balanced IgG1:IgG2a subtype profile, induction of TH1-type CD4+ T cell-mediated immune responses similar to those of viral particles, and reduced production of non-neutralizing antibodies. RNA priming can increase the immunogenicity of compositions, whether or not they contain adjuvants.

[0188] In RNA primacy-protein enhancement strategies, RNA and protein target the same antigen. Examples of suitable RNA delivery modalities include virus-like replicon particles (VRPs), alphavirus RNA, replicons encapsulated in lipid nanoparticles (LNPs), or formulated RNA, such as replicons formulated with cationic nanoemulsions (CNEs). Suitable cationic oil-in-water nanoemulsions are disclosed in WO2012 / 006380, for example, comprising an oil core (e.g., containing squalene) and cationic lipids (e.g., DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.).

[0189] In some embodiments, the RNA molecule is encapsulated in, bound to, or adsorbed onto the following substances: cationic lipids, liposomes, helices, virions, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsions, polycationic peptides, cationic nanoemulsions, or combinations thereof.

[0190] This document also provides kits and instructions for use for administering the nucleic acids (e.g., RNA), purified proteins, and purified nanostructures described herein. This disclosure also provides delivery devices pre-filled with the compositions or vaccines disclosed herein.

[0191] The pharmaceutical compositions described herein may be administered in combination with one or more other therapeutic agents. These other therapeutic agents may include, but are not limited to: antibiotics or antibacterial agents, antiemetics, antifungals, anti-inflammatory agents, antiviral agents, immunomodulators, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumor antibiotics, antimitotic agents, topoisomerase inhibitors, cell inhibitors, anti-invasive agents, anti-angiogenic agents, growth factor function inhibitors, viral replication inhibitors, viral enzyme inhibitors, anticancer agents, alpha-interferon, beta-interferon, ribavirin, hormones and other Toll-like receptor modulators, immunoglobulins (Ig), and antibodies that modulate Ig function (such as anti-IgE (omalizumab)).

[0192] In some embodiments, the compositions disclosed herein can be used as medicines, for example, to induce or enhance an immune response in a subject (such as a mammal) in need.

[0193] In some embodiments, the compositions disclosed herein can be used to manufacture medicaments for inducing or enhancing immune responses in subjects (such as mammals) in need.

[0194] One way to assess the efficacy of therapeutic treatments includes monitoring for infection with the infectious agent after administration of the compositions or vaccines disclosed herein. One way to assess the efficacy of prophylactic treatments includes monitoring the immune response against the antigen systemically (e.g., monitoring IgG1 and IgG2a production levels) and / or mucosally (e.g., monitoring IgA production levels). Typically, antigen-specific serum antibody responses are determined post-immunization but before challenge, while antigen-specific mucosal antibody responses are determined post-immunization and post-challenge.

[0195] 8. Terminology

[0196] All publications and patents mentioned herein are hereby incorporated in their entirety by reference, as if each individual publication or patent were expressly and individually indicated to be incorporated by reference. In case of conflict, this application (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or suggestion of any kind that they constitute valid prior art or are part of common general knowledge in any country of the world.

[0197] In this application, unless otherwise stated, the techniques used can be found in several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, ColdSpring Harbor Laboratory Press); Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, CA); "Guide to Protein Purification" in Methods in Enzymology (edited by MP Deutshcer, (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA); Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI. Freshney, 1987, Liss, Inc., New York, NY); Gene Transfer and Expression Protocols, pp. 109-128, edited by E.J. Murray, The Humana Press Inc., Clifton, NJ; and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0198] In this specification, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the stated range, and, where appropriate, to include fractions thereof (such as one-tenth and one-hundredth of an integer). The term “about” when immediately preceding a number or value means a range of ±10% of that number or value. It should be understood that, unless otherwise specified, the term “a / an” as used herein means “one or more” of the listed components. The use of alternatives (e.g., “or”) shall be understood to mean one, two, or any combination thereof of the alternatives. The term “and / or” shall be understood to mean one or two alternatives. As used herein, the terms “comprising” and “including” are used synonymously.

[0199] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0200] Unless otherwise expressly defined, the following terms and phrases common to the various embodiments disclosed herein are defined as follows:

[0201] As used in this article, the term protein refers to either protein or glycoprotein.

[0202] As used herein, the term immunogenicity refers to the ability of a particular protein or a particular region thereof to elicit an immune response against said particular protein or against a protein containing an amino acid sequence that is highly identical to said particular protein. According to this disclosure, two highly identical proteins have amino acid sequences that are at least 80% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical.

[0203] As used herein, an “immune response” to the vaccine or nanostructure of this disclosure is a humoral and / or cellular immune response generated in a subject against antigenic proteins present in the vaccine. For the purposes of this disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a “cellular immune response” is an immune response mediated by T lymphocytes and / or other leukocytes. An important aspect of cellular immunity involves antigen-specific responses mediated by cytolytic T cells (“CTLs”). CTLs are peptide antigen-specific, which are associated with and presented with proteins encoded by the major histocompatibility complex (MHC) and expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected by such microorganisms. Another aspect of cellular immunity involves antigen-specific responses mediated by helper T cells. Helper T cells help stimulate the function of nonspecific effector cells to resist cells displaying peptide antigens associated with MHC molecules on their surface and concentrate the activity of said nonspecific effector cells against said cells. Cellular immune response also refers to the production of cytokines, chemokines and other such molecules by activated T cells and / or other white blood cells (including those derived from CD4+ and CD8+ cells).

[0204] Therefore, an immune response can be an immune response that stimulates the production or activation of CTLs and / or helper T cells. It can also stimulate the production of chemokines and / or cytokines. Vaccines can also elicit antibody-mediated immune responses. Thus, an immune response can include one or more of the following effects: production of antibodies (e.g., IgA or IgG) by B cells; and / or specific inhibitors of hemagglutinin proteins present in the vaccine, cytotoxicity, or activation of helper T cells and / or T cells. These responses can be used to neutralize infectivity and / or mediate antibody-complement or antibody-dependent cell cytotoxicity (ADCC) to provide protection to an immunized individual. Such responses can be measured using standard immunoassays and neutralization assays well known in the art.

[0205] As used herein, the term "antibody" includes complete molecules and their functional fragments, such as Fab, F(ab')2, Fv, scFv, dsFv, or single-domain molecules such as VH and VL capable of specifically binding to antigenic epitopes. The term "antibody" encompasses B cell receptors. The term "antibody" further encompasses camel antibodies.

[0206] As used in this description of viruses, neutralizing antibodies are antibodies that prevent a virus from completing one round of replication. As defined herein, one round of replication refers to the viral life cycle, which begins with viral attachment to a host cell and ends with the budding of newly formed viruses from the host cell. This life cycle includes, but is not limited to, the steps of attaching to the cell, entering the cell, lysis and rearrangement of viral proteins, fusion of the viral membrane with the endosomal membrane, release of viral ribonucleoproteins into the cytoplasm, formation of new viral particles, and budding of viral particles from the host cell membrane.

[0207] As used herein, a broadly neutralizing antibody is an antibody that neutralizes more than one type, subtype, and / or strain of bacteria, viruses, or parasites. For example, a broadly neutralizing antibody against the influenza HA protein from influenza A virus can neutralize influenza B or C virus. As another example, a broadly neutralizing antibody against the influenza HA protein from group I influenza virus can neutralize group 2 virus. As yet another example, a broadly neutralizing antibody against the HA protein from one subtype or strain of virus can neutralize another subtype or strain of virus. For example, a broadly neutralizing antibody against the HA protein from H1 influenza virus can neutralize viruses from one or more subtypes selected from the groups H2, H3, H4, H5, H6, H7, H8, H8, H10, H11, H12, H13, H14, H15, or H16.

[0208] Regarding antigens, those skilled in the art will understand that antigenic proteins from different strains can have different lengths due to mutations (insertions, deletions) in the proteins. Therefore, referring to a corresponding region means a region of another protein that is identical or nearly identical (e.g., at least 95%, at least 98%, or at least 99%) in sequence, structure, and / or function to the region being compared. For example, regarding epitopes, corresponding regions in corresponding proteins from different organismal strains may not have the same number of residues, but will have similar or nearly identical sequences and will perform the same function. To better clarify sequence comparisons between strains, those skilled in the art use a numbering system that associates amino acid positions with reference sequences. Therefore, corresponding amino acid residues in antigenic proteins from different strains may not have the same number of residues relative to their distance from the N-terminal amino acid of the protein. The use of such numbering systems is understood by those skilled in the art.

[0209] According to this disclosure, a trimerizing domain is a series of amino acids that, when linked to a protein or peptide (also known as a fusion), allows the fusion protein to interact with other fusion proteins containing trimerizing domains, thereby resulting in the formation of a trimer structure. Any known trimerizing domain may be used in this disclosure. Examples of trimerizing domains include, but are not limited to, the HIV-1 gp41 trimerizing domain, the SIV gp41 trimerizing domain, the Ebola virus gp-2 trimerizing domain, the HTLV-1 gp-21 trimerizing domain, the T4 fibritin trimerizing domain (i.e., the fold), the yeast heat shock transcription factor trimerizing domain, and the human collagen trimerizing domain.

[0210] As used herein, a variant is a protein or nucleic acid molecule that is sequence-similar but not identical to a reference sequence, wherein the activity of the variant protein (or the protein encoded by the variant nucleic acid molecule) is not significantly altered. These sequence variations can be naturally occurring variations or they can be engineered using genetic engineering techniques known to those skilled in the art. Examples of such techniques can be found in Sambrook J, Fritsch EF, Maniatis T, et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, pp. 9.31–9.57, or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, both of which are incorporated herein by reference in their entirety.

[0211] Regarding variants, any type of change in the amino acid or nucleic acid sequence is permissible, as long as the resulting variant protein retains the ability to elicit neutralizing antibodies against the influenza virus. Examples of such variants include, but are not limited to, deletions, insertions, substitutions, and combinations thereof. For example, with regard to proteins, those skilled in the art will fully understand that one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) can often be removed from the amino and / or carboxyl termini of a protein without significantly affecting its activity. Similarly, one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) can often be inserted into a protein without significantly affecting its activity.

[0212] As noted, the variant proteins of this disclosure may contain amino acid substitutions relative to the nanostructured antigen proteins disclosed herein. Any amino acid substitution is permissible, provided that the protein's activity is not significantly affected. In this regard, it should be understood in the art that amino acids can be grouped based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants containing substitutions are those in which the amino acid is replaced by an amino acid from the same group. Such substitutions are referred to as conservative substitutions.

[0213] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). As used herein, "about" means + / - 5% of the parameter described.

[0214] Naturally occurring residues can be categorized based on shared side-chain properties: 1) hydrophobic: Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr; 3) acidic: Asp, Glu; 4) basic: Asn, Gln, His, Lys, Arg; 5) residues affecting chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. For example, non-conservative substitutions can involve the exchange of members from one category with members from another.

[0215] When altering amino acids, their hydrophilicity index can be considered. Based on hydrophobicity and charge characteristics, a hydrophilicity index was assigned to each amino acid. The hydrophilicity indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydrophilic amino acid index in conferring interacting biological functions to proteins is generally understood in the art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids can be substituted with other amino acids having similar hydrophilic indices or values ​​while still retaining similar biological activities. When changes are made based on the hydrophilic index, substitution of amino acids with a hydrophilic index within ±2 is preferred, substitution of those within ±1 is particularly preferred, and substitution of those within ±0.5 is even more particularly preferred.

[0216] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophobicity, particularly in cases where the aim is to use the resulting biologically equivalent proteins or peptides for immunological inventions (as is the case here). The maximum local average hydrophilicity of a protein (as controlled by the hydrophilicity of its neighboring amino acids) is associated with its immunogenicity and antigenicity, i.e., its biological properties. The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When modifications are made based on similar hydrophilicity values, substitutions of amino acids with hydrophilicity values ​​within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Epitopes derived from primary amino acid sequences can also be identified based on hydrophilicity.

[0217] When such substitutions are required, the desired amino acid substitutions (whether conserved or non-conserved) can be determined by those skilled in the art. For example, amino acid substitutions can be used to identify important residues in HA proteins, or to increase or decrease the immunogenicity, solubility, or stability of the HA proteins described herein. Exemplary amino acid substitutions are shown in Table 4 below.

[0218] Table 4

[0219] Amino acid substitution

[0220]

[0221]

[0222] As used herein, the phrase "significantly affects protein activity" means reducing protein activity by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. For the purposes of this disclosure, such activity can be measured, for example, by the ability of a protein to induce neutralizing antibodies against a virus. This activity can be measured by measuring the titer of such antibodies against a virus, or by measuring the type, subtype, or number of strains neutralized by the induced antibodies. Methods for determining antibody titers and for performing virus neutralization assays are known to those skilled in the art. In addition to the activities described above, other activities that can be measured include the ability to agglutinate erythrocytes and the protein's affinity for cell binding. Methods for measuring such activities are known to those skilled in the art.

[0223] As used herein, a fusion protein is a recombinant protein containing amino acid sequences from at least two unrelated proteins linked together by peptide bonds to form a single protein. Unrelated amino acid sequences may be directly linked to each other, or they may be linked using a linker sequence. As used herein, a protein is considered unrelated if its amino acid sequences are not typically found linked together by peptide bonds in one or more native environments (such as within a cell). For example, the amino acid sequences of the monomeric subunits constituting a polypeptide and the amino acid sequences of an antigenic protein are typically not found linked together by peptide bonds.

[0224] The terms individual, subject, and patient are well known in the art and are used interchangeably herein to refer to any person or other animal susceptible to infection. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species; livestock such as cattle, sheep, pigs, seals, goats, and horses; domesticated mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; and birds, including domesticated birds, wild birds, and game birds such as chickens, turkeys, and other quails, ducks, geese, etc. The terms individual, subject, and patient do not individually indicate a specific age, sex, race, etc. Therefore, an individual of any age (whether male or female) is intended to be covered by this disclosure and includes, but is not limited to, the elderly, adults, children, infants, babies, and toddlers. Similarly, the methods of this disclosure can be applied to any race, including, for example, Caucasians (white), African Americans (black), Native Americans, Native Hawaiians, Hispanics, Latinos, Asians, and Europeans.

[0225] As used in this article, the subjects receiving the vaccine are those who have already been given a vaccine designed to provide protection against bacteria, viruses, or parasites.

[0226] As used in this article, the terms exposed, exposure, etc., indicate that the subject has been in contact with an animal individual known to be infected with bacteria, viruses, or parasites.

[0227] The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing herein should be construed as an admission that this disclosure is not entitled to precede such publications by virtue of prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0228] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described and used herein may also be used in the practice or testing of this disclosure, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials relating to the cited publications.

[0229] It should be understood that certain features of this disclosure described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, different features of this disclosure described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments are expressly included in this disclosure and disclosed herein as if each combination were disclosed separately and expressly herein. Furthermore, all sub-combinations are also expressly included in this disclosure and disclosed herein as if each such sub-combination were disclosed separately and expressly herein.

[0230] This disclosure will be further described in the following embodiments, which do not limit the scope of this disclosure as described in the claims.

[0231] 9. Example

[0232] 9.1. Example 1: Respiratory Syncytial Virus (RSV)

[0233] 9.1.1. Sequence

[0234] In embodiments of this disclosure, the RSV F protein exists as a fusion protein with a first polypeptide and uses a linker; the F protein-linker sequence may include the following:

[0235] >DS-Cav1-folding element (SEQ ID NO:90)

[0236] (MELLILKANAITTILTAVTFCFASG)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNV TLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSE LLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR(KSDELL) GYIPEAPRDGQAYVRKDGEWVLLSTFL

[0237] In various further embodiments, the first polypeptide comprises or consists of a first polypeptide having a sequence selected from the following (optional residues in parentheses):

[0238] >DS-Cav1-Folding-T33-31A(SEQ ID NO:91)

[0239] (MELLILKANVIATILTAVTFCFASS)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR(KSDELL)GYIPEAPRDGQAYVRKDGEWVLLSTFLGGSMEEVVLITVPSALVAVKIAHALVEERLAACVNIVPGLTSIYREEGSVVSDHELLLLVKTTTDAFPKLKERVKELHPYEVPEIVALPIAEGNREYLDWLRENTG

[0240] >DS-Cav1-T33-31A(SEQ ID NO:92)

[0241] (MELLILKANVIATILTAVTFCFASS)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR(KSDELL)GGSMEEVVLITVPSALVAVKIAHALVEERLAACVNIVPGLTSIYREEGSVVSDHELLLLVKTTTDAFPKLKERVKELHPYEVPEIVALPIAEGNREYLDWLRENTG

[0242] >DS-Cav1-Fold-T33-15B (SEQ ID NO:93)

[0243] (MELLILKANVIATILTAVTFCFASS)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR(KSDELL)GYIPEAPRDGQAYVRKDGEWVLLSTFLGGSMVRGIRGAITVNSDTPTSIIIATILLLEKMLEANGIQSYEELAAVIFTVTEDLTSAFPAEAARQIGMHRVPLLSAREVPVPGSLPRVIRVLALWNTDTPQDRVRHVYLSEAVRLRPDLESAQ

[0244] >DS-Cav1-T33-15B(SEQ ID NO:94)

[0245] (MELLILKANVIATILTAVTFCFASS)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR(KSDELL)GGSMVRGIRGAITVNSDTPTSIIIATILLLEKMLEANGIQSYEELAAVIFTVTEDLTSAFPAEAARQIGMHRVPLLSAREVPVPGSLPRVIRVLALWNTDTPQDRVRHVYLSEAVRLRPDLESAQ

[0246] >DS-Cav1-Fold-I53-50A(SEQ ID NO:95)​​(MELLILKANAITTILTAVTFCFASG)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGSHHHHHHHHGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0248] >DS-Cav1-I53-50A(SEQ ID NO:96)

[0249] (MELLILKANVIATILTAVTFCFASS)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNR VFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0250] >DS-Cav1-I32-28A(SEQ ID NO:97)

[0251] (MELLILKANAITTILTAVTFCFASG)QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR(KSDELL)GGSGGSGSDDARIAAIGDVDELNSQIGVLLAEPLPDDVRAALSAIQHDLFDLGGELCIPGHAAITEDHLLRLALWLVHYNGQLPPLEEFILPGGARGAALAHVCRTVCRRAERSIKALGASEPLNIAPAAYVNLLSDLLFVLARVLNRAAGGADVLWDRTRAH

[0252] >DS-Cav1-Tr-Foldon-T33-31A (SEQ ID NO:101)

[0253] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSMEEVVLITVPSALVAVKIAHALVEERLAACVNIVPGLTSIYREEGSVVSDHELLLLVKTTTDAFPKLKERVKELHPYEVPEIVALPIAEGNREYLDWLRENTG

[0254] >DS-Cav1-Tr-T33-31A(SEQ ID NO:102)

[0255] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGGSMEEVVLITVPSALVAVKIAHALVEERLAACVNIVPGLTSIYREEGSVVSDHELLLLVKTTTDAFPKLKERVKELHPYEVPEIVALPIAEGNREYLDWLRENTG

[0256] >DS-Cav1-Tr-Fold-T33-15B (SEQ ID NO:103)

[0257] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSMVRGIRGAITVNSDTPTSIIIATILLLEKMLEANGIQSYEELAAVIFTVTEDLTSAFPAEAARQIGMHRVPLLSAREVPVPGSLPRVIRVLALWNTDTPQDRVRHVYLSEAVRLRPDLESAQ

[0258] >DS-Cav1-Tr-T33-15B(SEQ ID NO:104)

[0259] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGGSMVRGIRGAITVNSDTPTSIIIATILLLEKMLEANGIQSYEELAAVIFTVTEDLTSAFPAEAARQIGMHRVPLLSAREVPVPGSLPRVIRVLALWNTDTPQDRVRHVYLSEAVRLRPDLESAQ

[0260] >DS-Cav1-Tr-Foldon-I53-50A (SEQ ID NO:105)

[0261] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMT SKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGSHHHHHHHHGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0262] >DS-Cav1-Tr-I53-50A(SEQ ID NO:106)

[0263] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATNRRARRELPRFMNYTLNNAKKTNVTLSKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDK QLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPS EVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0264] >DS-Cav1-Tr-I32-28A(SEQ ID NO:107)

[0265] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGGSGGSGSDDARIAAIGDVDELNSQIGVLLAEPLPDDVRAALSAIQHDLFDLGGELCIPGHAAITEDHLLRLALWLVHYNGQLPPLEEFILPGGARGAALAHVCRTVCRRAERSIKALGASEPLNIAPAAYVNLLSDLLFVLARVLNRAAGGADVLWDRTRAH

[0266] 9.1.2. Method

[0267] Expression and screening of a trimeric structural unit comprising an F protein and a trimerization module domain.

[0268] Human codon-optimized sequences, including and lacking DS-Cav1 fusion trimeric structural units, were ordered from Genscript. Structural units for single-component nanostructures (i.e., I3-01) were cloned into a pcDNA3.1 vector (ThermoFisher Scientific) containing one CMV promoter, while structural units for two-component nanostructures (e.g., I53-50) were cloned into a pBudCE4.1 vector (ThermoFisher Scientific) containing both CMV and EF-1α promoters. Recombinant proteins were expressed by transient transfection of Expi293F cells (ThermoFisher Scientific) with polyethyleneimine (PEI). Cell cultures were harvested by centrifugation five days post-transfection. Secreted proteins were analyzed by direct ELISA using cell supernatant or by sandwich ELISA. In short, 96-well MaxiSorp plates (Nunc) were coated with cell supernatant for direct ELISA or mouse anti-His-tag monoclonal antibody (ThermoFisher Scientific) for sandwich ELISA. Secreted proteins were detected using human palizumab, MPE8, RSD5, and D25 monoclonal antibodies. Transfected Expi293F cells were fixed and permeabilized with BD cytofix / cytoperm (BD Biosciences), incubated with human palizumab, MPE8, and D25 monoclonal antibodies, and stained with Alexa Fluor 647-conjugated anti-human IgG antibody (Jackson ImmunoResearch). Stained cells were counted using a FACS Fortessa flow cytometer (BD Biosciences). Analysis was performed using FlowJo software. Mycoplasma contamination of the cell lines was routinely tested.

[0269] Expression and purification of DS-Cav1-I53-50A

[0270] Lentiviral cells were generated by transiently transfecting 293T (ATCC) cells with linear 25-kDa polyethyleneimine (PEI; Polysciences). In short, 4 x 10⁻⁶ cells were transfected with PEI. 6Cells were seeded onto 10 cm tissue culture plates. After 24 h, 3 μg psPAX2, 1.5 μg pMD2G (Addgene plasmids #12260 and #12259, respectively), and 6 μg lentiviral vector plasmid were mixed in 500 μL dilution buffer (5 mM HEPES, 150 mM NaCl, pH 7.05) and 42 μl PEI (1 mg / mL) and incubated for 15 min. The DNA / PEI complex was then added dropwise to the plate. Lentiviral virus was harvested 48 h post-transfection and concentrated 100-fold by centrifugation at 8000 g for 18 h. Transduction of the target cell line was performed in 125 mL shake flasks containing 10 x 10⁻¹⁰ cells in 10 mL of growth medium. 6 Add 100 μL of 100x lentivirus to the flask and incubate the cells at 37°C with shaking (225 rpm) in 8% CO2 for 4–6 h. After 4–6 h, add 20 mL of growth medium to the shake flask.

[0271] The transduced cells were expanded to 1x10 every other day. 6 Cells / ml density was maintained until a final culture size of 4L was achieved. The final cell concentration (approximately 5 x 10⁻⁶ cells / ml) was measured. 6 After achieving cell count / mL and viability (approximately 90% viability), the culture medium was harvested after a total incubation of 17 days. The culture supernatant was harvested by low-speed centrifugation to remove cells. NaCl and NaN3 were added to final concentrations of 250 mM and 0.02%, respectively. The supernatant was loaded onto a 5 mL HisTrap FF Crude column (GE Healthsciences) at 5 mL / min using AKTA Pure (GE Healthsciences). Nickel eluent was applied to a HiLoad 16 / 600 Superdex 200 pg column (GE Healthsciences) for further purification of the target protein by size exclusion chromatography. The size-exclusion purified target protein was flash-frozen in liquid nitrogen and stored at -80°C.

[0272] In vitro assembly of nanostructures with DS-Cav1

[0273] 100% valence particles (20 DS-Cav1 trimers per icosahedral nanostructure) were prepared by mixing 50 μM DS-Cav1-foldon-I53-50A trimers and I53-50B.4PT1 pentamers and incubating overnight at 4°C with shaking. In some cases, the assembled nanostructures were purified from excess components remaining in the in vitro assembly reaction using a GE Sephacryl S-500HR 16 / 60 column in a buffer containing 25 mM Tris pH 8, 250 mM NaCl, and 5% glycerol. Sample loading and SEC fractions were analyzed by SDS-PAGE with and without reducing agents. Peak fractions were combined, concentrated using a GE Vivaspin 20 30 kDa MWCO centrifuge filter, and quantified using an Agilent 8454 spectrophotometer.

[0274] 66% valence particles (approximately 14 DS-Cav1 trimers per icosahedral nanostructure) were prepared by mixing 50, 25, and 75 μM of DS-Cav1-sheet-I53-50A trimers, I53-50A trimers, and I53-50B.4PosT1 pentamers, respectively. 33% valence particles (approximately 7 DS-Cav1 trimers per icosahedral nanostructure) were prepared by mixing 25, 50, and 75 μM of DS-Cav1-sheet-I53-50A trimers, I53-50A trimers, and I53-50B.4PosT1 pentamers, respectively. The in vitro assembly reaction was incubated overnight at 4°C with shaking. In some cases, assembled nanostructures were purified from excess components remaining in the in vitro assembly reaction using a GE Sephacryl S-500HR 16 / 60 column in a buffer containing 25 mM Tris pH 8, 250 mM NaCl, and 5% glycerol. Sample loading and SEC fractions were analyzed by SDS-PAGE in the presence and absence of reducing agents. Peak fractions were combined, concentrated using a GE Vivaspin 2030 kDa MWCO centrifugal filter, and quantified using an Agilent 8454 spectrophotometer after centrifugation at approximately 21,000 g for 10 min at 4 °C. Samples were then transferred in 1 mL aliquots to cryogenic tubes, rapidly frozen in liquid nitrogen, and stored at -80 °C, with a concentration of 1.1 mg / mL for 33% valence particles and 0.6 mg / mL for 66% valence particles.

[0275] Electron microscope with DS-Cav1 nanostructure

[0276] Negative staining samples for EM were prepared by diluting the sample to 0.01 mg / mL with 25 mM Tris pH 8, 250 mM NaCl, and 5% glycerol. 3.5 μL of the solution was incubated for 20 seconds on a glow-discharged copper-carbon coated grid, followed by aspiration with a Whatman No. 1 filter paper. Within seconds of aspiration, a droplet of 3.5 μL of staining agent (2% w / v uranyl formate) was deposited and immediately aspirated, followed by a second staining / absorption cycle.

[0277] Circular dichroism (CD) optical rotation

[0278] Within the wavelength range of 195 to 260 nM, data from F protein (0.5 mg m1) were recorded on a Chirascan polarimeter (Applied Photophysics) with a bandwidth of 1 nM, a step size of 0.5 nM, and a step length of 1 s. -1 The CD spectrum of the far-ultraviolet region was obtained. The spectrum was obtained by an average of three scans, which were then subtracted from the blank spectrum obtained with buffer. Thermal denaturation was monitored by scanning at 1°C intervals after equilibration for 1 min at each temperature. The data were fitted to a simple first-order curve. The value of ΔA222 is expressed as a percentage of the values ​​recorded at 20°C on the y-axis.

[0279] Enzyme-linked immunosorbent assay (ELISA)

[0280] To test the specific binding of antibodies or serum, 96-well MaxiSorp plates (Nunc) were coated with serially diluted tissue culture supernatant from cells expressing trimeric structural units containing F protein and trimeric component domains, or 2 μg / mL of the solution. -1 The following purified proteins were used: Ds-Cav1 with folds, Ds-Cav1 fused to the first trimer polypeptide, or nanostructures displaying DS-Cav1. Plates were blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies (D25, MPE8, palizumab, RSD5) or mouse serum, followed by AP-conjugated goat anti-human IgG (Southern Biotech, 2040-04) or goat anti-mouse IgG (Southern Biotech, 1030-04). Plates were then washed with PBS buffer (Gibco, Invitrogen) and 0.05% Tween-20, and substrate (p-NPP, Sigma) was added. Plates were read at 405 nM.

[0281] Surface plasmon resonance (SPR)

[0282] Experiments were performed at 25°C on a ProteON XPR-36 instrument (Bio-Rad Laboratories) in PBS buffer (Gibco, Invitrogen) and 0.05% Tween-20. D25 mAb was immobilized on the surface of the GLM sensor chip via amine coupling at 1000 response units (RU), and a protein-free blank surface was created as a reference under the same coupling conditions. Monoclonal antibodies (D25, MPE8, palizumab, and 131-2a) were injected into different sensor channels at a flow rate of 100 nl / min and a concentration of 50 nM. Data were processed using Proteon software, with dual references obtained by subtracting the blank surface and buffer-only injections, followed by local fitting of the data.

[0283] Vaccination and serological analysis

[0284] Female BALB / c mice aged 6–9 weeks were obtained from Harlan Laboratories Inc. All procedures were performed according to the guidelines of the Swiss Federal Veterinary Office and after obtaining local ethical approval. Mice were intraperitoneally immunized with 100 μL of immunogen formulation on days 0, 14, and 28. Primary immunization was performed on day 0 with a mouse TLR9 ligand agonist (ODN 1668, InvivoGen). Mice were exsanguinated on days 10, 20, and 40, and antigen-specific and site-specific IgG titers were measured in serum by ELISA. Neutralizing titers were also determined on HEp-2 cells, as described below.

[0285] Virus neutralization assay and microscopic analysis

[0286] HEp-2 cell confluence layers in 96-well plates were infected with a fixed amount of human respiratory syncytial virus (RSV strain A2, Vira Tree#R121) with green fluorescent protein at an MOI of 1. Forty-eight hours post-infection, cells were stained with Hoechst (Sigma#H6024), and images were acquired on a BD Pathway bioimaging system. The percentage of infected cells was automatically calculated using BD AttoVision software. Infected cell counts were plotted as dose-response curves by plotting the relative number of infected cells against antibody dilution.

[0287] Stability of nanostructures with DS-Cav1

[0288] The physical stability of the pre-fusion conformation of the designed DS-Cav1-fold-I53-50 was assessed by incubating different concentrations of protein at 80°C for 1 h in a PCR cycler with a heated lid. Residual pre-fusion conformation was evaluated by directly coating the protein with the pre-fusion specific antibody D25 and by ELISA.

[0289] Statistical analysis

[0290] Sample size was not predetermined using statistical methods. Data were analyzed using Prism 6 (GraphPad software) with the two-tailed, parametric Mann-Whitney U test for comparisons between two groups, or the Kruskall-Wallis test (and Dunn's post-test) for comparisons of three or more groups.

[0291] 9.1.3. Results

[0292] Trimeric structural units containing F protein and trimer component domains

[0293] Several trimeric building blocks, each containing an F protein fused to a trimeric component domain gene, were found to be secreted from HEK293F cells, with their F proteins in a well-folded pre-fusion conformation, as determined by binding to a pre-fusion-specific monoclonal antibody in an ELISA assay. Figure 2 shows an example of ELISA data, analyzing the supernatant of HEK293F cells expressing the DS-Cav1-fold, DS-Cav1-fold-T33-31A, and DS-Cav1-T33-31A. Several other trimeric building blocks produced detectable secretion of well-folded pre-fusion F proteins.

[0294] Expression and purification of DS-Cav1-sheet-I53-50A

[0295] A lentiviral vector encoding DS-Cav1-fold-I53-50A was transduced into HEK293F cells for large-scale expression. Secreted proteins were purified from tissue culture supernatant using immobilized metal affinity chromatography and size exclusion chromatography. Size exclusion chromatogram (...) Figure 3 This indicates that the purified protein forms a single monodisperse substance.

[0296] Expression and purification of I53-50B.4PT1

[0297] I53-50B.4PT1 was expressed and purified as described in Bale et al. and patent publication US20160122392A1. I53-50B.4PT1 is a pentamer protein containing a second domain that interacts with the trimer component domain in I53-50A or DS-Cav1-sheet-I53-50A to drive the assembly of I53-50-based icosahedral nanostructures.

[0298] In vitro assembly and characterization of I53-50 nanostructures with DS-Cav1

[0299] As recently described by Bale et al., I53-50 is a two-component nanostructure with 120 subunits exhibiting icosahedral symmetry, comprising 20 trimer (I53-50A) and 12 pentamer (I53-50B) structural units. The N-terminus of I53-50A is exposed on the exterior of the I53-50 nanostructure, allowing antigens to be displayed on the outside of the nanostructure by fusion with the N-terminal gene of I53-50A. DS-Cav1-foldon-I53-50A and I53-50B.4PT1 were in vitro assembled to form 120-subunit icosahedral nanostructures displaying varying amounts of DS-Cav1 on the outside of the nanostructure by mixing two purified proteins in different molar ratios. In separate preparations, nanostructures displaying DS-Cav1 in valence states of 100% (20 trimers), 66% (approximately 14 trimers), and 33% (approximately 7 trimers) were prepared as described above. Several techniques were used to evaluate substances present in the in vitro assembly reaction after overnight incubation, including size exclusion chromatography-multi-angle light scattering (SEC-MALS), dynamic light scattering, and UV / vis spectroscopy. The assembled 120 subunit nanostructures were purified from the in vitro assembly reaction using size exclusion chromatography (exemplary chromatograms obtained using 100% valence nanostructures are shown in...). Figure 4 The purified nanostructures were characterized by negative staining electron microscopy, which revealed the field of view of monodisperse particles, in which DS-Cav1 was clearly visible as a spike protruding outward from the core icosahedral I53-50 assembly (exemplary micrographs obtained using 100% valence state particles are presented in...). Figure 5(Figure 6). ELISA assays using monoclonal antibodies specific to the pre-fusion conformation confirmed that the DS-Cav1 displayed on the outside of the nanostructure was well-folded and antigenically intact (Figure 6). Surface plasmon resonance experiments evaluating the kinetics of monoclonal antibody binding revealed that antibody dissociation from the 100% valence DS-Cav1-foldon-I53-50 nanostructure was slower than that from the DS-Cav1-foldon trimer, likely due to the affinity effect derived from the multivalent presentation of DS-Cav1 on the outside of the nanostructure (Figure 6). In summary, these experiments confirmed that the DS-Cav1-foldon-I53-50 nanostructure forms a monodisperse icosahedral nanostructure displaying a well-folded, antigenically intact DS-Cav1 trimer on its exterior. These findings prompted experiments to evaluate the utility of the DS-Cav1-foldon-I53-50 nanostructure as an immunogen for inducing humoral immune responses against DS-Cav1 in animals.

[0300] Immunogenicity of DS-Cav1-Fold-I53-50 nanostructure

[0301] Mice were injected with the DS-Cav1-folded I53-50 nanostructure, displaying DS-Cav1 at 33%, 66%, and 100% valence, using the primacy-boost strategy described above. Mice in an additional group were injected with the trimer DS-Cav1-folded nanostructure as a baseline for humoral immune responses against DS-Cav1 induced by the nanostructure or the absence of the displayed DS-Cav1 I53-50 nanostructure (as a negative control for DS-Cav1-specific responses). The titers of DS-Cav1-specific antibodies present in the serum of the injected animals were measured using an ELISA assay of serum extracted from mice at defined time points post-injection. Figure 7 As expected, serum from animals injected with the I53-50 nanostructure lacking the displayed DS-Cav1 did not contain antibodies specific to DS-Cav1. Based on previous results (McClellan et al.), the trimer DS-Cav1-fold induces DS-Cav1-specific antibodies. DS-Cav1 nanostructures at 33%, 66%, and 100% valence all induced higher DS-Cav1-specific antibody titers than the trimer DS-Cav1-fold, with antibody titers increasing with increasing DS-Cav1 valence. In mice injected with the 100% valence DS-Cav1-fold-I53-50 nanostructure, the DS-Cav1-specific titer was on average approximately 2.5 times higher than that of DS-Cav1 alone. These results demonstrate that immunogens in which the paramyxovirus F protein is multivalently displayed on self-assembled protein nanostructures can induce a higher humoral immune response when injected into animals.

[0302] The presence of neutralizing antibody titers in serum from mice injected with the aforementioned series of immunogens was also evaluated using a standard neutralization assay in HEp-2 cells. Figure 8 The trend in serum neutralizing antibody titers was highly correlated with the observed trend in DS-Cav1-specific binding antibody titers. Serum from animals injected with I53-50 nanostructures lacking displayed DS-Cav1 failed to neutralize the virus, consistent with the absence of DS-Cav1-specific antibodies in these sera. Serum from animals injected with the trimer DS-Cav-1-fold had an average titer of 3,030 (1 / ID). 50 Neutralizing the virus. DS-Cav1-I53-50 nanostructures at 33%, 66%, and 100% valence induced higher neutralizing antibody titers than the trimer DS-Cav1-sheet, with average titers of 9,400, 20,000, and 30,500, respectively. These results demonstrate that a higher immunogen-induced response, resulting from the multivalent display of the paramyxovirus F protein on a self-assembled protein nanostructure, leads to more effective virus neutralization.

[0303] Physical stabilization of DS-Cav1 through fusion with I53-50A

[0304] Given the critical antigenic properties of pre-fusion F, we used two orthogonal methods to measure the physical stability of DS-Cav1 upon fusion with I53-50A and / or during further assembly into icosahedral nanostructures. The first assay measured the binding retention of the pre-fusion specific mAb (D25) after thermal stress, a method previously used to characterize the stability of pre-fusion F (McLellan et al. 2013; Joyce et al. 2016; Krarup et al. 2015). Samples containing equivalent concentrations (50 nM) of DS-Cav1 trimer, DS-Cav1-I53-50A trimer, and DS-Cav1-I53-50 nanostructures were divided into four aliquots and incubated for 1 hour at 20°C, 50°C, 70°C, or 80°C. After cooling to room temperature, D25 binding was measured by surface plasmon resonance (SPR). We found that all samples bound D25 equally at 20°C and 50°C, but lost most of their reactivity with D25 after 1 hour at 80°C, as previously reported for DS-Cav1 (McLellan et al. 2013; Joyce et al. 2016). Figure 10 Interestingly, although D25 also failed to bind to the trimer DS-Cav1 incubated at 70°C for 1 hour, the trimer DS-Cav1-I53-50A and DS-Cav1-I53-50 nanostructures retained 50% and 80% of their respective binding signals. Figure 10Although the multivalent nature of the DS-Cav1-I53-50 nanostructure complicates direct quantitative comparisons with the trimer DS-Cav1, these results indicate that gene fusion with the I53-50A trimer further stabilizes the pre-fusion conformation of DS-Cav1 and demonstrates that this increased stability is maintained in the context of assembled nanostructure immunogens.

[0305] We used chemical denaturation in guanidine hydrochloride (GdnHCl) monitored by intrinsic tryptophan fluorescence as a second antibody-independent technique to evaluate physical stability. Analysis of fluorescence emission from DS-Cav1 incubated in 0–6.5 M GdnHCl revealed two subtle but distinct transitions in the protein, one between 0.25 and 2.25 M GdnHCl, and the other between 2.25 and 5.75 M. Figures 11A-11J In contrast, for the trimer DS-Cav1-I53-50A, a single transformation is evident, occurring between 2.25 and 6.25 M GdnHCl. Figures 11A-11J It remains unclear whether the transition observed in DS-Cav1 at lower [GdnHCl] is absent in the trimer DS-Cav1-I53-50A or simply a transition to higher [GdnHCl]. However, it is evident that the native conformation of DS-Cav1 is stabilized through gene fusion with the trimer I53-50A, as reflected in results obtained by measuring D25 binding after heat stress. Data comparing the DS-Cav1-I53-50 nanostructure with the standalone I53-50 nanostructure (DS-Cav1 without fusion) indicate stability maintained upon assembly into an icosahedral nanostructure. Figures 11A-11J This effect likely stems from the extremely high stability of the I53-50A trimer. I53-50A originates from the KDPG aldolase of the hyperthermophilic bacterium *T. maritima*, and only begins to exhibit fluorescence changes at very high (≥5.75 M) concentrations of GdnHCl. Figures 11A-11J ).

[0306] We generated additional constructs to evaluate the number of GS repeat sequences and the need for stable structural domains (such as folded subparts).

[0307] Sequence information

[0308] IPD Name MS (Da) Construct Information RSV_F-10 74005.38 DS-Cav1-8GS-HelExt-50A (SEQ ID NO: 108) RSV_F-11 74293.64 DS-Cav1-12GS-HelExt-50A (SEQ ID NO: 109) RSV_F-12 74551.87 DS-Cav1-16GS-HelExt-50A (SEQ ID NO: 110) RSV_F-13 77212.97 DS-Cav1-PapMV-CP-10GS-HelExt-50A (SEQ ID NO: 111) RSV_F-14 77558.28 DS-Cav1-PapMV-CP-15GS-HelExt-50A (SEQ ID NO: 112) RSV_F-15 77933.62 DS-Cav1-PapMV-CP-20GS-HelExt-50A (SEQ ID NO: 113)

[0309] RSV_F-10 (SEQ ID NO:108)

[0310] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATNRNARRFLGFLLGVGSAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVI EFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY DCKIMTSKTDVSSSVITSLGAIVCCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGSGGSGSGEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0311] >RSV_F-11(SEQ ID NO:109)

[0312] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATNNRARRFLGFLLGVGSAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIE FQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDC KIMTSKTDVSSSVITSLGAIVCGYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGSGGSGSGSGGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0313] >RSV_F-12(SEQ ID NO:110)

[0314] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATNNRARRFLGFLLGVGSAIASGVAVCKLVHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEF QQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGSGGSGSGSGGSGSGGSGGKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0315] >RSV_F-13(SEQ ID NO:111)

[0316] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATNNRARRFLGFLLGVGSAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIE FQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYD CKIMTSKTDVSSSVITSLGAIVCGYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGSGGSGSGSGEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0317] >RSV_F-14(SEQ ID NO:112)

[0318] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTELQLLMQSTPATNNRARRFLGFLLGVGSAIASGVAVCVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRL LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVIT SLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGGSGSGSGGSGSGEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGFPPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0319] >RSV_F-15(SEQ ID NO:113)

[0320] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLL EITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSL GAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGGSGSGSGGSGSGGSSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAI EKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE

[0321] The study focused on expression yield in small-scale transient transfections. Plasmids capable of expressing the relevant constructs were transformed into *E. coli* cells (NEB 5α) and selected on LB+ carbenicillin agar plates. 1 mL cultures were prepared by inoculating bacterial colonies onto TB medium and selecting again with 50 μg / mL carbenicillin. Plasmids were purified from the *E. coli* cultures using the Qiagen Micropreparation Kit according to its protocol. Expi293F... TM ThermoFisher cells were cultured in Expi 293 culture medium supplemented with penicillin (100 u / mL) and streptomycin (100 μg / mL). TMThe medium was cultured in expression medium (ThermoFisher) at 8% CO2, 37°C and 125 rpm shaking.

[0322] One day prior to transfection, cells were seeded at a concentration of 2E6 cells / mL. On the day of transfection, cell viability was determined by counting cells using Countess II (ThermoFisher) with trypan blue. The cell concentration was adjusted to 2.5E6 cells / mL, and cells were seeded in 1 mL volumes into untreated 12-well (Corning) plates. Expifectamine was used according to the manufacturer's instructions. TM Transfect each well with 1 μg of DNA plasmid using ThermoFisher. 18 hours post-transfection, add ThermoFisher's Expifectamine. TM Enhancer components of the transfection kit. Harvest 1 mL of culture 5 days post-transfection and precipitate cells from the supernatant by centrifugation at 1,500 x g at 4°C for 5 minutes. Filter the supernatant through a 0.45 μM filter with a PVDF membrane.

[0323] The filtered supernatant containing the DS-Cav1-I53-50A construct was denatured and boiled at 95°C for 10 min in 2xLaemmli buffer with 2-mercaptoethanol. Sample fractions were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with palilizumab followed by HRP-conjugated anti-human secondary antibody. The blots were imaged using Clarity Western ECL Blotting Substrate (Bio-Rad).

[0324] The filtered supernatant containing the DS-Cav1-I53-50A construct was serially conjugated to NuncMaxiSorp 96-well plates at two-fold dilutions. Pre-fusion conformation-specific antibody D25 was used to detect DS-Cav1-I53-50A, followed by HRP-conjugated anti-human secondary antibody. Protein yield was determined by substrate TMB colorimetric assay, and absorbance was collected at 450 nM.

[0325] Expression yield and binding of the pre-fusion specific mAb D25 (data not shown) indicate that all constructs are well expressed and in their pre-fusion conformation. As is known to those skilled in the art, heterodimerizing domains (e.g., foldants) are typically required for proper expression and folding of pre-fusion F constructs. Our results demonstrate that the I53-50A nanostructure component can support the expression and proper folding of DS-Cav1 without the use of trimerizing domains (such as foldants). The binding of D25 to these constructs indicates that they are antigenically intact and are expected to induce potent immune responses (including neutralizing antibodies) similar to those induced by nanostructures comprising the DS-Cav1-foldant-I53-50 fusion peptide.

[0326] 9.2. Example 2: Cellular Megavirus (CMV)

[0327] Protein-based vaccines for CMV are described, for example, in US Patent Publications US 2016 / 0159864A1 and US 2017 / 0369532A1; International Patent Publication WO 2016 / 092460A3; and in Kirchmeier et al. Expression of enveloped virus-like particles of human cytomegalovirus glycoprotein B antigen induces antibodies with potent and broad-spectrum neutralizing activity. ClinVaccine Immunol. 2014; 21(2):174–80. Homotrimeric complexes of gB, trimeric gH / gL / gO complexes, or pentameric gH / gL / pUL128 / pUL130 / pUL131A complexes are considered three major targets for CMV vaccination.

[0328] The first of these targets, gB, forms a trimer structure with several hydrophobic surfaces. The C-terminus of the extracellular domain of gB is proximal to the transmembrane region and located near the triple axis of the molecule. See Chandramouli et al., Structure of HCMV glycoprotein B in the postfusion conformation bound to aneutralizing human antibody. Nat Commun. 2015 Sep 14; 6:8176. By replacing the linker with the transmembrane region of gB, the CMV gB protein is attached to a nanostructure with an N-terminus, thus having a free N-terminus at or near the triple axis of the nanostructure. The resulting nanostructure displays 20 copies of the gB trimer on its surface and effectively elicits an immune response to CMV gB. As described in International Patent Publication No. WO 2016 / 092460A3, mutations in the gB protein improve the solubility and immunogenicity of nanostructure-based vaccines.

[0329] The second type of these targets, the trimer gH / gL / gO complex, and the third type, the pentamer gH / gL / pUL128 / pUL130 / pUL131A, are formed through the mutual repulsive interaction between the envelope glycoproteins gH / gL and gO or pUL128 / pUL130 / pUL131A. See Ciferri et al., Structural and biochemical studies of HCMV gH / gL / gO and Pentamer reveal mutually exclusive cell entry complexes. Proc. Natl. Acad. Sci. USA 112, 1767–1772 (2015). The gH component is targeted by antibodies that neutralize fibroblast and endothelial / epithelial cell infections. The UL region contains binding sites for antibodies that potently neutralize epithelial and endothelial cell infections.

[0330] The gH component was expressed as a gene fusion with the nanostructured peptide, and gL / gO or gL / pUL128 / pUL130 / pUL131A was co-expressed. The expressed proteins self-assembled into vaccines based on gH / gL / gO or gH / gL / pUL128 / pUL130 / pUL131A nanostructures, respectively. The expression and correct folding of the nanostructures were evaluated by binding to the MSL-109 antibody or its Fab fragment. The correct folding and antigenicity of the pentamer complex were evaluated using the antibody and Fab fragment described in the following literature: Chandramouli et al., Structural basis for potent antibody-mediated neutralization of human cytomegalovirus Sci. Immunol. 2, eaan1457 (2017).

[0331] 9.3. Example 3: Epstein-Barr Virus (EBV)

[0332] Epstein-Barr virus (EBV) represents a major global health problem. Despite its association with infectious mononucleosis and approximately 200,000 cancer cases worldwide each year, there is currently no available vaccine. The primary target of immunity is EBV glycoprotein 350 / 220 (gp350), which binds to B cells via complement receptor 2 (CR2 / CD21). See Kanekiyo et al., Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site. Cell 162(5):1090-1100 (2015). The extracellular domain of gp350 or the D-type of gp350... 123 The fragment was expressed as a gene fusion with a nanostructured polypeptide, the gene fusion being either N-terminal or C-terminal. The resulting gene fusion was expressed, assembled, and formulated into a nanostructure-based vaccine. Antigenicity was determined using monoclonal antibodies 72A1 and 2L10. sequence list <110> Washington Elementary School N. Kim D. Becker L. Stewart <120> Self-assembled nanostructure vaccine <130> ICVX-001 / 02WO 333041-2012 <150> US 62 / 724,721 <151> 2018-08-30 <150> US 62 / 636,757 <151> 2018-02-28 <160> 113 <170> PatentIn version 3.5 <210> 1 <211> 207 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 1 Met Glu Gly Met Asp Pro Leu Ala Val Leu Ala Glu Ser Arg Leu Leu 1 5 10 15 Pro Leu Leu Thr Val Arg Gly Gly Glu Asp Leu Ala Gly Leu Ala Thr 20 25 30 Val Leu Glu Leu Met Gly Val Gly Ala Leu Glu Ile Thr Leu Arg Thr 35 40 45 Glu Lys Gly Leu Glu Ala Leu Lys Ala Leu Arg Lys Ser Gly Leu Leu 50 55 60 Leu Gly Ala Gly Thr Val Arg Ser Pro Lys Glu Ala Glu Ala Ala Leu 65 70 75 80 Glu Ala Gly Ala Ala Phe Leu Val Ser Pro Gly Leu Leu Glu Glu Val 85 90 95 Ala Ala Leu Ala Gln Ala Arg Gly Val Pro Tyr Leu Pro Gly Val Leu 100 105 110 Thr Pro Thr Glu Val Glu Arg Ala Leu Ala Leu Gly Leu Ser Ala Leu 115 120 125 Lys Phe Phe Pro Ala Glu Pro Phe Gln Gly Val Arg Val Leu Arg Ala 130 135 140 Tyr Ala Glu Val Phe Pro Glu Val Arg Phe Leu Pro Thr Gly Gly Ile 145 150 155 160 Lys Glu Glu His Leu Pro His Tyr Ala Ala Leu Pro Asn Leu Leu Ala 165 170 175 Val Gly Gly Ser Trp Leu Leu Gln Gly Asp Leu Ala Ala Val Met Lys 180 185 190 Lys Val Lys Ala Ala Lys Ala Leu Leu Ser Pro Gln Ala Pro Gly 195 200 205 <210> 2 <211> 156 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 2 Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp 1 5 10 15 Met Ala Glu Ala Ala Ile Arg Thr Leu Lys Ala Leu Ser Pro Asn Ile 20 25 30 Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala 35 40 45 Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu 50 55 60 Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile 85 90 95 Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Asp Glu Leu Asp 100 105 110 Ile Leu Ala Leu Val Arg Ala Ile Glu His Ala Ala Asn Val Tyr Tyr 115 120 125 Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu 130 135 140 Arg Gln Gly Arg Glu Asp Ala Gly Pro Ala Arg Glu 145 150 155 <210> 3 <211> 156 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 3 Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp 1 5 10 15 Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile 20 25 30 Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala 35 40 45 Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu 50 55 60 Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile 85 90 95 Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys 100 105 110 Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr 115 120 125 Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu 130 135 140 Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu 145 150 155 <210> 4 <211> 209 <212> PRT <213> artificial sequence <220> <223> Synthesized multi-peptide nanostructures <400> 4 Met Ser Thr Ile Asn Asn Gln Leu Lys Ala Leu Lys Val Ile Pro Val 1 5 10 15 Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu 20 25 30 Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala 35 40 45 Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu 50 55 60 Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys 65 70 75 80 Glu Ala Gly Ala Thr Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr 85 90 95 Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn 100 105 110 Asn Pro Ser Thr Val Glu Ala Ala Leu Glu Met Gly Leu Thr Thr Leu 115 120 125 Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser 130 135 140 Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile 145 150 155 160 Thr Pro Ser Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala 165 170 175 Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Thr Asn Gly Glu 180 185 190 Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn 195 200 205 Pro <210> 5 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 5 Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Thr Asp Val Pro 1 5 10 15 Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser 20 25 30 Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser 50 55 60 Ile Gly Gly Ile Glu Pro Ser Lys Asn Arg Asp His Ser Ala Val Leu 65 70 75 80 Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr 85 90 95 Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 Thr Phe <210> 6 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 6 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 7 <211> 205 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 7 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 8 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 8 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 9 <211> 177 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 9 Met Phe Thr Lys Ser Gly Asp Asp Gly Asn Thr Asn Val Ile Asn Lys 1 5 10 15 Arg Val Gly Lys Asp Ser Pro Leu Val Asn Phe Leu Gly Asp Leu Asp 20 25 30 Glu Leu Asn Ser Phe Ile Gly Phe Ala Ile Ser Lys Ile Pro Trp Glu 35 40 45 Asp Met Lys Lys Asp Leu Glu Arg Val Gln Val Glu Leu Phe Glu Ile 50 55 60 Gly Glu Asp Leu Ser Thr Gln Ser Ser Lys Lys Lys Ile Asp Glu Ser 65 70 75 80 Tyr Val Leu Trp Leu Leu Ala Ala Thr Ala Ile Tyr Arg Ile Glu Ser 85 90 95 Gly Pro Val Lys Leu Phe Val Ile Pro Gly Gly Ser Glu Glu Ala Ser 100 105 110 Val Leu His Val Thr Arg Ser Val Ala Arg Arg Val Glu Arg Asn Ala 115 120 125 Val Lys Tyr Thr Lys Glu Leu Pro Glu Ile Asn Arg Met Ile Ile Val 130 135 140 Tyr Leu Asn Arg Leu Ser Ser Leu Leu Phe Ala Met Ala Leu Val Ala 145 150 155 160 Asn Lys Arg Arg Asn Gln Ser Glu Lys Ile Tyr Glu Ile Gly Lys Ser 165 170 175 Trp <210> 10 <211> 157 <212> PRT <213> artificial sequence <220> <223> Synthesized peptide nanostructure <400> 10 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala 20 25 30 Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His His Glu 115 120 125 Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala 130 135 140 Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 11 <211> 201 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 11 Met Gly His Thr Lys Gly Pro Thr Pro Gln Gln His Asp Gly Ser Ala 1 5 10 15 Leu Arg Ile Gly Ile Val His Ala Arg Trp Asn Lys Thr Ile Ile Met 20 25 30 Pro Leu Leu Ile Gly Thr Ile Ala Lys Leu Leu Glu Cys Gly Val Lys 35 40 45 Ala Ser Asn Ile Val Val Gln Ser Val Pro Gly Ser Trp Glu Leu Pro 50 55 60 Ile Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Leu Gln Thr Pro Ser 65 70 75 80 Ser Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gly Ser Ser Thr Thr 85 90 95 Asp Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Ser Thr Gly Pro Phe 100 105 110 Asp Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gly Glu Thr Met His 115 120 125 Phe Glu Tyr Ile Ala Asp Ser Val Ser His Gly Leu Met Arg Val Gln 130 135 140 Leu Asp Thr Gly Val Pro Val Ile Phe Gly Val Leu Thr Val Leu Thr 145 150 155 160 Asp Asp Gln Ala Lys Ala Arg Ala Gly Val Ile Glu Gly Ser His Asn 165 170 175 His Gly Glu Asp Trp Gly Leu Ala Ala Val Glu Met Gly Val Arg Arg 180 185 190 Arg Asp Trp Ala Ala Gly Lys Thr Glu 195 200 <210> 12 <211> 237 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 12 Met Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly 1 5 10 15 Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val 20 25 30 Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly 35 40 45 Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala 50 55 60 Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu 65 70 75 80 Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Gln Leu Gly 85 90 95 Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu 100 105 110 Lys Thr Val Ala Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His 115 120 125 Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu 130 135 140 Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly 145 150 155 160 Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr 165 170 175 Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg 180 185 190 His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Arg Glu Tyr 195 200 205 Glu Ala Ala Phe Met Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly 210 215 220 Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala 225 230 235 <210> 13 <211> 138 <212> PRT <213> Artificial sequence <220> <223> Synthetically constructed nanostructured polypeptide <400> 13 Met Gly Met Lys Glu Lys Phe Val Leu Ile Ile Thr His Gly Asp Phe 1, 5, 10, 15 Gly Lys Gly Leu Leu Ser Gly Ala Glu Val Ile Ile Gly Lys Gln Glu 20, 25, 30 Asn Val His Thr Val Gly Leu Asn Leu Gly Asp Asn Ile Glu Lys Val 35, 40, 45 Ala Lys Glu Val Met Arg Ile Ile Ile Ala Lys Leu Ala Glu Asp Lys 50, 55, 60 Glu Ile Ile Ile Val Val Asp Leu Phe Gly Gly Ser Pro Phe Asn Ile 65, 70, 75, 80 Ala Leu Glu Met Met Lys Thr Phe Asp Val Lys Val Ile Thr Gly Ile 85, 90, 95 Asn Met Pro Met Leu Val Glu Leu Leu Thr Ser Ile Asn Val Tyr Asp 100, 105, 110 Thr Thr Glu Leu Leu Glu Asn Ile Ser Lys Ile Gly Lys Asp Gly Ile 115 120 125 Lys Val Ile Glu Lys Ser Ser Leu Lys Met 130 135 <210> 14 <211> 154 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 14 Met Lys Tyr Asp Gly Ser Lys Leu Arg Ile Gly Ile Leu His Ala Arg 1 5 10 15 Trp Asn Leu Glu Ile Ile Ala Ala Leu Val Ala Gly Ala Ile Lys Arg 20 25 30 Leu Gln Glu Phe Gly Val Lys Ala Glu Asn Ile Ile Ile Glu Thr Val 35 40 45 Pro Gly Ser Phe Glu Leu Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys 50 55 60 Gln Lys Arg Leu Gly Lys Pro Leu Asp Ala Ile Ile Pro Ile Gly Val 65 70 75 80 Leu Ile Lys Gly Ser Thr Met His Phe Glu Tyr Ile Cys Asp Ser Thr 85 90 95 Thr His Gln Leu Met Lys Leu Asn Phe Glu Leu Gly Ile Pro Val Ile 100 105 110 Phe Gly Val Leu Thr Cys Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala 115 120 125 Gly Leu Ile Glu Gly Lys Met His Asn His Gly Glu Asp Trp Gly Ala 130 135 140 Ala Ala Val Glu Met Ala Thr Lys Phe Asn 145 150 <210> 15 <211> 164 <212> PRT <213> artificial sequence <220> <223> Synthesized peptide nanostructure <400> 15 Met Ala Val Lys Gly Leu Gly Glu Val Asp Gln Lys Tyr Asp Gly Ser 1 5 10 15 Lys Leu Arg Ile Gly Ile Leu His Ala Arg Trp Asn Arg Lys Ile Ile 20 25 30 Leu Ala Leu Val Ala Gly Ala Val Leu Arg Leu Leu Glu Phe Gly Val 35 40 45 Lys Ala Glu Asn Ile Ile Ile Glu Thr Val Pro Gly Ser Phe Glu Leu 50 55 60 Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys Gln Lys Arg Leu Gly Lys 65 70 75 80 Pro Leu Asp Ala Ile Ile Pro Ile Gly Val Leu Ile Lys Gly Ser Thr 85 90 95 Met His Phe Glu Tyr Ile Cys Asp Ser Thr Thr His Gln Leu Met Lys 100 105 110 Leu Asn Phe Glu Leu Gly Ile Pro Val Ile Phe Gly Val Leu Thr Cys 115 120 125 Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala Gly Leu Ile Glu Gly Lys 130 135 140 Met His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala 145 150 155 160 Thr Lys Phe Asn <210> 16 <211> 175 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 16 Met Gly Ala Asn Trp Tyr Leu Asp Asn Glu Ser Ser Arg Leu Ser Phe 1 5 10 15 Thr Ser Thr Lys Asn Ala Asp Ile Ala Glu Val His Arg Phe Leu Val 20 25 30 Leu His Gly Lys Val Asp Pro Lys Gly Leu Ala Glu Val Glu Val Glu 35 40 45 Thr Glu Ser Ile Ser Thr Gly Ile Pro Leu Arg Asp Met Leu Leu Arg 50 55 60 Val Leu Val Phe Gln Val Ser Lys Phe Pro Val Ala Gln Ile Asn Ala 65 70 75 80 Gln Leu Asp Met Arg Pro Ile Asn Asn Leu Ala Pro Gly Ala Gln Leu 85 90 95 Glu Leu Arg Leu Pro Leu Thr Val Ser Leu Arg Gly Lys Ser His Ser 100 105 110 Tyr Asn Ala Glu Leu Leu Ala Thr Arg Leu Asp Glu Arg Arg Phe Gln 115 120 125 Val Val Thr Leu Glu Pro Leu Val Ile His Ala Gln Asp Phe Asp Met 130 135 140 Val Arg Ala Phe Asn Ala Leu Arg Leu Val Ala Gly Leu Ser Ala Val 145 150 155 160 Ser Leu Ser Val Pro Val Gly Ala Val Leu Ile Phe Thr Ala Arg 165 170 175 <210> 17 <211> 208 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 17 Met Thr Asp Tyr Ile Arg Asp Gly Ser Ala Ile Lys Ala Leu Ser Phe 1 5 10 15 Ala Ile Ile Leu Ala Glu Ala Asp Leu Arg His Ile Pro Gln Asp Leu 20 25 30 Gln Arg Leu Ala Val Arg Val Ile His Ala Cys Gly Met Val Asp Val 35 40 45 Ala Asn Asp Leu Ala Phe Ser Glu Gly Ala Gly Lys Ala Gly Arg Asn 50 55 60 Ala Leu Leu Ala Gly Ala Pro Ile Leu Cys Asp Ala Arg Met Val Ala 65 70 75 80 Glu Gly Ile Thr Arg Ser Arg Leu Pro Ala Asp Asn Arg Val Ile Tyr 85 90 95 Thr Leu Ser Asp Pro Ser Val Pro Glu Leu Ala Lys Lys Ile Gly Asn 100 105 110 Thr Arg Ser Ala Ala Ala Leu Asp Leu Trp Leu Pro His Ile Glu Gly 115 120 125 Ser Ile Val Ala Ile Gly Asn Ala Pro Thr Ala Leu Phe Arg Leu Phe 130 135 140 Glu Leu Leu Asp Ala Gly Ala Pro Lys Pro Ala Leu Ile Ile Gly Met 145 150 155 160 Pro Val Gly Phe Val Gly Ala Ala Glu Ser Lys Asp Glu Leu Ala Ala 165 170 175 Asn Ser Arg Gly Val Pro Tyr Val Ile Val Arg Gly Arg Arg Gly Gly 180 185 190 Ser Ala Met Thr Ala Ala Ala Val Asn Ala Leu Ala Ser Glu Arg Glu 195 200 205 <210> 18 <211> 128 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 18 Met Ile Thr Val Phe Gly Leu Lys Ser Lys Leu Ala Pro Arg Arg Glu 1 5 10 15 Lys Leu Ala Glu Val Ile Tyr Ser Ser Leu His Leu Gly Leu Asp Ile 20 25 30 Pro Lys Gly Lys His Ala Ile Arg Phe Leu Cys Leu Glu Lys Glu Asp 35 40 45 Phe Tyr Tyr Pro Phe Asp Arg Ser Asp Asp Tyr Thr Val Ile Glu Ile 50 55 60 Asn Leu Met Ala Gly Arg Ser Glu Glu Thr Lys Met Leu Leu Ile Phe 65 70 75 80 Leu Leu Phe Ile Ala Leu Glu Arg Lys Leu Gly Ile Arg Ala His Asp 85 90 95 Val Glu Ile Thr Ile Lys Glu Gln Pro Ala His Cys Trp Gly Phe Arg 100 105 110 Gly Arg Thr Gly Asp Ser Ala Arg Asp Leu Asp Tyr Asp Ile Tyr Val 115 120 125 <210> 19 <211> 235 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 19 Met Gly Ser Asp Leu Gln Lys Leu Gln Arg Phe Ser Thr Cys Asp Ile 1 5 10 15 Ser Asp Gly Leu Leu Asn Val Tyr Asn Ile Pro Thr Gly Gly Tyr Phe 20 25 30 Pro Asn Leu Thr Ala Ile Ser Pro Pro Gln Asn Ser Ser Ile Val Gly 35 40 45 Thr Ala Tyr Thr Val Leu Phe Ala Pro Ile Asp Asp Pro Arg Pro Ala 50 55 60 Val Asn Tyr Ile Asp Ser Val Pro Pro Asn Ser Ile Leu Val Leu Ala 65 70 75 80 Leu Glu Pro His Leu Gln Ser Gln Phe His Pro Phe Ile Lys Ile Thr 85 90 95 Gln Ala Met Tyr Gly Gly Leu Met Ser Thr Arg Ala Gln Tyr Leu Lys 100 105 110 Ser Asn Gly Thr Val Val Phe Gly Arg Ile Arg Asp Val Asp Glu His 115 120 125 Arg Thr Leu Asn His Pro Val Phe Ala Tyr Gly Val Gly Ser Cys Ala 130 135 140 Pro Lys Ala Val Val Lys Ala Val Gly Thr Asn Val Gln Leu Lys Ile 145 150 155 160 Leu Thr Ser Asp Gly Val Thr Gln Thr Ile Cys Pro Gly Asp Tyr Ile 165 170 175 Ala Gly Asp Asn Asn Gly Ile Val Arg Ile Pro Val Gln Glu Thr Asp 180 185 190 Ile Ser Lys Leu Val Thr Tyr Ile Glu Lys Ser Ile Glu Val Asp Arg 195 200 205 Leu Val Ser Glu Ala Ile Lys Asn Gly Leu Pro Ala Lys Ala Ala Gln 210 215 220 Thr Ala Arg Arg Met Val Leu Lys Asp Tyr Ile 225 230 235 <210> 20 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> Synthetically constructed nanostructured polypeptide <400> 20 Met Ser Gly Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Tyr Glu 1 5 10 15 Leu Lys Gln Ala Ile Ile Ala Phe Leu Lys Met Thr Gly His Glu Pro 20 25 30 Ile Asp Cys Gly Ala Leu Arg Tyr Asp Ala Asp Asp Asp Tyr Pro Ala 35 40 45 Phe Cys Ile Ala Ala Ala Thr Arg Thr Val Ala Asp Pro Gly Ser Leu 50 55 60 Gly Ile Val Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn 65 70 75 80 Lys Val Pro Gly Ala Arg Cys Ala Leu Ala Trp Ser Val Gln Thr Ala 85 90 95 Ala Leu Ala Arg Glu His Asn Asn Ala Gln Leu Ile Gly Ile Gly Gly 100 105 110 Arg Met His Thr Leu Glu Glu Ala Leu Arg Ile Val Lys Ala Phe Val 115 120 125 Thr Thr Pro Trp Ser Lys Ala Gln Arg His Gln Arg Arg Ile Asp Ile 130 135 140 Leu Ala Glu Tyr Glu Arg Thr His Glu Ala Pro Pro Val Pro Gly Ala 145 150 155 160 Pro Ala <210> 21 <211> 157 <212> PRT <213> artificial sequence <220> <223> Synthesized multi-peptide nanostructures <400> 21 Met Gly Asp Asp Ala Arg Ile Ala Ala Ile Gly Asp Val Asp Glu Leu 1 5 10 15 Asn Ser Gln Ile Gly Val Leu Leu Ala Glu Pro Leu Pro Asp Asp Val 20 25 30 Arg Ala Ala Leu Ser Ala Ile Gln His Asp Leu Phe Asp Leu Gly Gly 35 40 45 Glu Leu Cys Ile Pro Gly His Ala Ala Ile Thr Glu Asp His Leu Leu 50 55 60 Arg Leu Ala Leu Trp Leu Val His Tyr Asn Gly Gln Leu Pro Pro Leu 65 70 75 80 Glu Glu Phe Ile Leu Pro Gly Gly Ala Arg Gly Ala Ala Leu Ala His 85 90 95 Val Cys Arg Thr Val Cys Arg Arg Ala Glu Arg Ser Ile Lys Ala Leu 100 105 110 Gly Ala Ser Glu Pro Leu Asn Ile Ala Pro Ala Ala Tyr Val Asn Leu 115 120 125 Leu Ser Asp Leu Leu Phe Val Leu Ala Arg Val Leu Asn Arg Ala Ala 130 135 140 Gly Gly Ala Asp Val Leu Trp Asp Arg Thr Arg Ala His 145 150 155 <210> 22 <211> 157 <212> PRT <213> artificial sequence <220> <223> Synthesized peptide nanostructure <400> 22 Met Ile Leu Ser Ala Glu Gln Ser Phe Thr Leu Arg His Pro His Gly 1 5 10 15 Gln Ala Ala Ala Leu Ala Phe Val Arg Glu Pro Ala Ala Ala Leu Ala 20 25 30 Gly Val Gln Arg Leu Arg Gly Leu Asp Ser Asp Gly Glu Gln Val Trp 35 40 45 Gly Glu Leu Leu Val Arg Val Pro Leu Leu Gly Glu Val Asp Leu Pro 50 55 60 Phe Arg Ser Glu Ile Val Arg Thr Pro Gln Gly Ala Glu Leu Arg Pro 65 70 75 80 Leu Thr Leu Thr Gly Glu Arg Ala Trp Val Ala Val Ser Gly Gln Ala 85 90 95 Thr Ala Ala Glu Gly Gly Glu Met Ala Phe Ala Phe Gln Phe Gln Ala 100 105 110 His Leu Ala Thr Pro Glu Ala Glu Gly Glu Gly Gly Ala Ala Phe Glu 115 120 125 Val Met Val Gln Ala Ala Ala Gly Val Thr Leu Leu Leu Val Ala Met 130 135 140 Ala Leu Pro Gln Gly Leu Ala Ala Gly Leu Pro Pro Ala 145 150 155 <210> twenty three <211> 156 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> twenty three Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp 1 5 10 15 Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile 20 25 30 Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala 35 40 45 Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu 50 55 60 Gly Met Pro Gly Lys Lys Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile 85 90 95 Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys 100 105 110 Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr 115 120 125 Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu 130 135 140 Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu 145 150 155 <210> 24 <211> 209 <212> PRT <213> artificial sequence <220> <223> Synthesized multi-peptide nanostructures <400> 24 Met Asp Asp Ile Asn Asn Gln Leu Lys Arg Leu Lys Val Ile Pro Val 1 5 10 15 Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu 20 25 30 Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala 35 40 45 Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu 50 55 60 Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys 65 70 75 80 Glu Ala Gly Ala Asp Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr 85 90 95 Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn 100 105 110 Asn Pro Ser Thr Val Glu Gln Ala Leu Glu Met Gly Leu Thr Thr Leu 115 120 125 Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser 130 135 140 Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile 145 150 155 160 Thr Pro Asp Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala 165 170 175 Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Arg Asn Gly Glu 180 185 190 Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn 195 200 205 Pro <210> 25 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 25 Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro 1 5 10 15 Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser 20 25 30 Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser 50 55 60 Ile Gly Gly Ile Glu Pro Asp Lys Asn Arg Asp His Ser Ala Val Leu 65 70 75 80 Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr 85 90 95 Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 Thr Phe <210> 26 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 26 Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro 1 5 10 15 Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser 20 25 30 Glu Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser 50 55 60 Ile Gly Gly Ile Glu Pro Asp Lys Asn Glu Asp His Ser Ala Val Leu 65 70 75 80 Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr 85 90 95 Ile His Phe Val Asp Leu Asp Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 Thr Phe <210> 27 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 27 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 28 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 28 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 29 <211> 205 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 29 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 30 <211> 205 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 30 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp 145 150 155 160 Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp 180 185 190 Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu 195 200 205 <210> 31 <211> 205 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 31 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 32 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 32 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 33 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 33 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 34 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 34 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 35 <211> 156 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <220> <221> misc_feature <222> (70)..(70) <223> Xaa is Ala or Lys <400> 35 Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp 1 5 10 15 Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile 20 25 30 Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala 35 40 45 Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu 50 55 60 Gly Met Pro Gly Lys Xaa Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile 85 90 95 Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys 100 105 110 Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr 115 120 125 Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu 130 135 140 Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu 145 150 155 <210> 36 <211> 209 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <220> <221> misc_feature <222> (2)..(2) <223> Xaa is either Ser or Asp. <220> <221> misc_feature <222> (3)..(3) <223> Xaa is Thr or Asp <220> <221> misc_feature <222> (10)..(10) <223> Xaa is either Ala or Arg <220> <221> misc_feature <222> (85)..(85) <223> Xaa is Thr or Asp <220> <221> misc_feature <222> (119)..(119) <223> Xaa is either Ala or Gln <220> <221> misc_feature <222> (163) <223> Xaa is either Ser or Asp. <220> <221> misc_feature <222> (189)..(189) <223> Xaa is Thr or Arg <400> 36 Met Xaa Xaa Ile Asn Asn Gln Leu Lys Xaa Leu Lys Val Ile Pro Val 1 5 10 15 Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu 20 25 30 Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala 35 40 45 Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu 50 55 60 Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys 65 70 75 80 Glu Ala Gly Ala Xaa Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr 85 90 95 Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn 100 105 110 Asn Pro Ser Thr Val Glu Xaa Ala Leu Glu Met Gly Leu Thr Thr Leu 115 120 125 Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser 130 135 140 Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile 145 150 155 160 Thr Pro Xaa Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala 165 170 175 Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Xaa Asn Gly Glu 180 185 190 Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn 195 200 205 Pro <210> 37 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <220> <221> misc_feature <222> (13)..(13) <223> Xaa is Thr or Asp <220> <221> misc_feature <222> (33)..(33) <223> Xaa is Lys or Glu <220> <221> misc_feature <222> (71)..(71) <223> Xaa is either Ser or Asp. <220> <221> misc_feature <222> (74)..(74) <223> Xaa is Arg or Glu <220> <221> misc_feature <222> (101)..(101) <223> Xaa is either Asn or Asp. <220> <221> misc_feature <222> (103)..(103) <223> Xaa is either Asn or Asp. <400> 37 Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Xaa Asp Val Pro 1 5 10 15 Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser 20 25 30 Xaa Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser 50 55 60 Ile Gly Gly Ile Glu Pro Xaa Lys Asn Xaa Asp His Ser Ala Val Leu 65 70 75 80 Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr 85 90 95 Ile His Phe Val Xaa Leu Xaa Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 Thr Phe <210> 38 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <220> <221> misc_feature <222> (9)..(9) <223> Xaa is Tyr or His <220> <221> misc_feature <222> (82)..(82) <223> Xaa is either Asn or Asp. <220> <221> misc_feature <222> (87)..(87) <223> Xaa is either Arg or Asp <220> <221> misc_feature <222> (105)..(105) <223> Xaa is either Ser or Asp. <220> <221> misc_feature <222> (119)..(119) <223> Xaa is Arg or Glu <220> <221> misc_feature <222> (121)..(121) <223> Xaa is Arg or Glu <220> <221> misc_feature <222> (124)..(124) <223> Xaa is either Ala or Asp. <220> <221> misc_feature <222> (126)..(126) <223> Xaa is His or Asp <220> <221> misc_feature <222> (128)..(128) <223> Xaa is Arg or Glu <220> <221> misc_feature <222> (150)..(150) <223> Stone but not Asn <400> 38 Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His No Tyr No Asp Ser No Glu No His No 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 39 <211> 205 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <220> <221> misc_feature <222> (126)..(126) <223> Xaa is Thr or Asp <220> <221> misc_feature <222> (139)..(139) <223> Xaa is Gln or Glu <220> <221> misc_feature <222> (142)..(142) <223> Xaa is Lys or Glu <220> <221> misc_feature <222> (160) <223> Xaa is either Asn or Asp. <220> <221> misc_feature <222> (163) <223> Xaa is either Asn or Asp. <220> <221> misc_feature <222> (166) <223> Xaa is either Glu or Lys <220> <221> misc_feature <222> (169) <223> Xaa is either Lys or Asp <220> <221> misc_feature <222> (179) <223> Xaa is Ser, Lys, or Asp. <220> <221> misc_feature <222> (183)..(183) <223> Xaa is Lys or Glu <220> <221> misc_feature <222> (185)..(185) <223> Xaa is Thr, Asp, or Lys <220> <221> misc_feature <222> (192)..(192) <223> Xaa is either Lys or Asp <220> <221> misc_feature <222> (195)..(195) <223> Xaa is Ala, Glu, or Lys <220> <221> misc_feature <222> (198)..(198) <223> Xaa is either Glu or Lys <220> <221> misc_feature <222> (199)..(199) <223> Xaa is Lys or Glu <400> 39 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa 145 150 155 160 Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa 180 185 190 Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu 195 200 205 <210> 40 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <220> <221> misc_feature <222> (9)..(9) <223> Xaa is Tyr or His <220> <221> misc_feature <222> (38)..(38) <223> Xaa is either Ala or Arg <220> <221> misc_feature <222> (82)..(82) <223> Xaa is either Asn or Asp. <220> <221> misc_feature <222> (87)..(87) <223> Xaa is either Arg or Asp <220> <221> misc_feature <222> (97)..(97) <223> Xaa is an Asp or Asn. <220> <221> misc_feature <222> (105)..(105) <223> Xaa is Ser, Asp, or Asn. <220> <221> misc_feature <222> (119)..(119) <223> Xaa is Arg, Glu, or Asn. <220> <221> misc_feature <222> (121)..(121) <223> Xaa is Arg, Asp, or Glu. <220> <221> misc_feature <222> (122)..(122) <223> Xaa is either Asp or Lys <220> <221> misc_feature <222> (124)..(124) <223> Xaa is either Asp or Lys <220> <221> misc_feature <222> (126)..(126) <223> Xaa is His or Asp <400> 40 Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Xaa Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Xaa Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Xaa Tyr Xaa Xaa Ser Xaa Ala Xaa Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 41 <211> 159 <212> PRT <213> artificial sequence <220> <223> Synthesized peptide nanostructure <400> 41 Met Gly Glu Val Pro Ile Gly Asp Pro Lys Glu Leu Asn Gly Met Glu 1 5 10 15 Ile Ala Ala Val Tyr Leu Gln Pro Ile Glu Met Glu Pro Arg Gly Ile 20 25 30 Asp Leu Ala Ala Ser Leu Ala Asp Ile His Leu Glu Ala Asp Ile His 35 40 45 Ala Leu Lys Asn Asn Pro Asn Gly Phe Pro Glu Gly Phe Trp Met Pro 50 55 60 Tyr Leu Thr Ile Ala Tyr Ala Leu Ala Asn Ala Asp Thr Gly Ala Ile 65 70 75 80 Lys Thr Gly Thr Leu Met Pro Met Val Ala Asp Asp Gly Pro His Tyr 85 90 95 Gly Ala Asn Ile Ala Met Glu Lys Asp Lys Lys Gly Gly Phe Gly Val 100 105 110 Gly Thr Tyr Ala Leu Thr Phe Leu Ile Ser Asn Pro Glu Lys Gln Gly 115 120 125 Phe Gly Arg His Val Asp Glu Glu Thr Gly Val Gly Lys Trp Phe Glu 130 135 140 Pro Phe Val Val Thr Tyr Phe Phe Lys Tyr Thr Gly Thr Pro Lys 145 150 155 <210> 42 <211> 184 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 42 Met Ser Gln Ala Ile Gly Ile Leu Glu Leu Thr Ser Ile Ala Lys Gly 1 5 10 15 Met Glu Leu Gly Asp Ala Met Leu Lys Ser Ala Asn Val Asp Leu Leu 20 25 30 Val Ser Lys Thr Ile Ser Pro Gly Lys Phe Leu Leu Met Leu Gly Gly 35 40 45 Asp Ile Gly Ala Ile Gln Gln Ala Ile Glu Thr Gly Thr Ser Gln Ala 50 55 60 Gly Glu Met Leu Val Asp Ser Leu Val Leu Ala Asn Ile His Pro Ser 65 70 75 80 Val Leu Pro Ala Ile Ser Gly Leu Asn Ser Val Asp Lys Arg Gln Ala 85 90 95 Val Gly Ile Val Glu Thr Trp Ser Val Ala Ala Cys Ile Ser Ala Ala 100 105 110 Asp Leu Ala Val Lys Gly Ser Asn Val Thr Leu Val Arg Val His Met 115 120 125 Ala Phe Gly Ile Gly Gly Lys Cys Tyr Met Val Val Ala Gly Asp Val 130 135 140 Leu Asp Val Ala Ala Ala Val Ala Thr Ala Ser Leu Ala Ala Gly Ala 145 150 155 160 Lys Gly Leu Leu Val Tyr Ala Ser Ile Ile Pro Arg Pro His Glu Ala 165 170 175 Met Trp Arg Gln Met Val Glu Gly 180 <210> 43 <211> 103 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 43 Met Glu Glu Val Val Leu Ile Thr Val Pro Ser Ala Leu Val Ala Val 1 5 10 15 Lys Ile Ala His Ala Leu Val Glu Glu Arg Leu Ala Ala Cys Val Asn 20 25 30 Ile Val Pro Gly Leu Thr Ser Ile Tyr Arg Trp Gln Gly Ser Val Val 35 40 45 Ser Asp His Glu Leu Leu Leu Leu Val Lys Thr Thr Thr His Ala Phe 50 55 60 Pro Lys Leu Lys Glu Arg Val Lys Ala Leu His Pro Tyr Thr Val Pro 65 70 75 80 Glu Ile Val Ala Leu Pro Ile Ala Glu Gly Asn Arg Glu Tyr Leu Asp 85 90 95 Trp Leu Arg Glu Asn Thr Gly 100 <210> 44 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 44 Met Val Arg Gly Ile Arg Gly Ala Ile Thr Val Glu Glu Asp Thr Pro 1 5 10 15 Ala Ala Ile Leu Ala Ala Thr Ile Glu Leu Leu Leu Lys Met Leu Glu 20 25 30 Ala Asn Gly Ile Gln Ser Tyr Glu Glu Leu Ala Ala Val Ile Phe Thr 35 40 45 Val Thr Glu Asp Leu Thr Ser Ala Phe Pro Ala Glu Ala Ala Arg Leu 50 55 60 Ile Gly Met His Arg Val Pro Leu Leu Ser Ala Arg Glu Val Pro Val 65 70 75 80 Pro Gly Ser Leu Pro Arg Val Ile Arg Val Leu Ala Leu Trp Asn Thr 85 90 95 Asp Thr Pro Gln Asp Arg Val Arg His Val Tyr Leu Asn Glu Ala Val 100 105 110 Arg Leu Arg Pro Asp Leu Glu Ser Ala Gln 115 120 <210> 45 <211> 177 <212> PRT <213> artificial sequence <220> <223> Synthesized multi-peptide nanostructures <400> 45 Met Ser Lys Ala Lys Ile Gly Ile Val Thr Val Ser Asp Arg Ala Ser 1 5 10 15 Ala Gly Ile Thr Ala Asp Ile Ser Gly Lys Ala Ile Ile Leu Ala Leu 20 25 30 Asn Leu Tyr Leu Thr Ser Glu Trp Glu Pro Ile Tyr Gln Val Ile Pro 35 40 45 Asp Glu Gln Asp Val Ile Glu Thr Thr Leu Ile Lys Met Ala Asp Glu 50 55 60 Gln Asp Cys Cys Leu Ile Val Thr Thr Gly Gly Thr Gly Pro Ala Lys 65 70 75 80 Arg Asp Val Thr Pro Glu Ala Thr Glu Ala Val Cys Asp Arg Met Met 85 90 95 Pro Gly Phe Gly Glu Leu Met Arg Ala Glu Ser Leu Lys Glu Val Pro 100 105 110 Thr Ala Ile Leu Ser Arg Gln Thr Ala Gly Leu Arg Gly Asp Ser Leu 115 120 125 Ile Val Asn Leu Pro Gly Asp Pro Ala Ser Ile Ser Asp Cys Leu Leu 130 135 140 Ala Val Phe Pro Ala Ile Pro Tyr Cys Ile Asp Leu Met Glu Gly Pro 145 150 155 160 Tyr Leu Glu Cys Asn Glu Ala Met Ile Lys Pro Phe Arg Pro Lys Ala 165 170 175 Lys <210> 46 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 46 Met Val Arg Gly Ile Arg Gly Ala Ile Thr Val Asn Ser Asp Thr Pro 1 5 10 15 Thr Ser Ile Ile Ile Ala Thr Ile Leu Leu Leu Glu Lys Met Leu Glu 20 25 30 Ala Asn Gly Ile Gln Ser Tyr Glu Glu Leu Ala Ala Val Ile Phe Thr 35 40 45 Val Thr Glu Asp Leu Thr Ser Ala Phe Pro Ala Glu Ala Ala Arg Gln 50 55 60 Ile Gly Met His Arg Val Pro Leu Leu Ser Ala Arg Glu Val Pro Val 65 70 75 80 Pro Gly Ser Leu Pro Arg Val Ile Arg Val Leu Ala Leu Trp Asn Thr 85 90 95 Asp Thr Pro Gln Asp Arg Val Arg His Val Tyr Leu Ser Glu Ala Val 100 105 110 Arg Leu Arg Pro Asp Leu Glu Ser Ala Gln 115 120 <210> 47 <211> 172 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 47 Met Arg Ile Thr Thr Lys Val Gly Asp Lys Gly Ser Thr Arg Leu Phe 1 5 10 15 Gly Gly Glu Glu Val Trp Lys Asp Ser Pro Ile Ile Glu Ala Asn Gly 20 25 30 Thr Leu Asp Glu Leu Thr Ser Phe Ile Gly Glu Ala Lys His Tyr Val 35 40 45 Asp Glu Glu Met Lys Gly Ile Leu Glu Glu Ile Gln Asp Asp Ile Tyr 50 55 60 Lys Ile Met Gly Glu Ile Gly Ser Lys Gly Lys Ile Glu Gly Ile Ser 65 70 75 80 Glu Glu Arg Ile Ala Trp Leu Leu Lys Leu Ile Leu Arg Tyr Met Glu 85 90 95 Met Val Asn Leu Lys Ser Phe Val Leu Pro Gly Gly Thr Leu Glu Ser 100 105 110 Ala Lys Leu Asp Val Cys Arg Thr Ile Ala Arg Arg Ala Leu Arg Lys 115 120 125 Val Leu Thr Val Thr Arg Glu Phe Gly Ile Gly Ala Glu Ala Ala Ala 130 135 140 Tyr Leu Leu Pathway Leu Ser Asp Leu Leu Phe Leu Leu Pathway Arg Val Ile 145 150 155 160 Glu Ile Glu Lys Asn Lys Leu Lys Glu Val Arg Ser 165 170 <210> 48 <211> 123 <212> PRT <213> artificial sequence <220> <223> Synthesized peptide nanostructure <400> 48 Met Pro His Leu Val Ile Glu Ala Thr Ala Asn Leu Arg Leu Glu Thr 1 5 10 15 Ser Pro Gly Glu Leu Leu Glu Gln Ala Asn Lys Ala Leu Phe Ala Ser 20 25 30 Gly Gln Phe Gly Glu Ala Asp Ile Lys Ser Arg Phe Val Thr Leu Glu 35 40 45 Ala Tyr Arg Gln Gly Thr Ala Ala Val Glu Arg Ala Tyr Leu His Ala 50 55 60 Cys Leu Ser Ile Leu Asp Gly Arg Asp Ile Ala Thr Arg Thr Leu Leu 65 70 75 80 Gly Ala Ser Leu Cys Ala Val Leu Ala Glu Ala Val Ala Gly Gly Gly 85 90 95 Glu Glu Gly Val Gln Val Ser Val Glu Val Arg Glu Met Glu Arg Leu 100 105 110 Ser Tyr Ala Lys Arg Val Val Ala Arg Gln Arg 115 120 <210> 49 <211> 158 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 49 Met Glu Ser Val Asn Thr Ser Phe Leu Ser Pro Ser Leu Val Thr Ile 1 5 10 15 Arg Asp Phe Asp Asn Gly Gln Phe Ala Val Leu Arg Ile Gly Arg Thr 20 25 30 Gly Phe Pro Ala Asp Lys Gly Asp Ile Asp Leu Cys Leu Asp Lys Met 35 40 45 Ile Gly Val Arg Ala Ala Gln Ile Phe Leu Gly Asp Asp Thr Glu Asp 50 55 60 Gly Phe Lys Gly Pro His Ile Arg Ile Arg Cys Val Asp Ile Asp Asp 65 70 75 80 Lys His Thr Tyr Asn Ala Met Val Tyr Val Asp Leu Ile Val Gly Thr 85 90 95 Gly Ala Ser Glu Val Glu Arg Glu Thr Ala Glu Glu Glu Ala Lys Leu 100 105 110 Ala Leu Arg Val Ala Leu Gln Val Asp Ile Ala Asp Glu His Ser Cys 115 120 125 Val Thr Gln Phe Glu Met Lys Leu Arg Glu Glu Leu Leu Ser Ser Asp 130 135 140 Ser Phe His Pro Asp Lys Asp Glu Tyr Tyr Lys Asp Phe Leu 145 150 155 <210> 50 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 50 Met Pro Val Ile Gln Thr Phe Val Ser Thr Pro Leu Asp His His Lys 1 5 10 15 Arg Leu Leu Leu Ala Ile Ile Tyr Arg Ile Val Thr Arg Val Val Leu 20 25 30 Gly Lys Pro Glu Asp Leu Val Met Met Thr Phe His Asp Ser Thr Pro 35 40 45 Met His Phe Phe Gly Ser Thr Asp Pro Val Ala Cys Val Arg Val Glu 50 55 60 Ala Leu Gly Gly Tyr Gly Pro Ser Glu Pro Glu Lys Val Thr Ser Ile 65 70 75 80 Val Thr Ala Ala Ile Thr Ala Val Cys Gly Ile Val Ala Asp Arg Ile 85 90 95 Phe Val Leu Tyr Phe Ser Pro Leu His Cys Gly Trp Asn Gly Thr Asn 100 105 110 Phe <210> 51 <211> 103 <212> PRT <213> Artificial sequence <220> <223> Synthesized nanostructured peptides <400> 51 Met Glu Glu Val Val Leu Ile Thr Val Pro Ser Ala Leu Val Ala Val 1 5 10 15 Lys Ile Ala His Ala Leu Val Glu Glu Arg Leu Ala Ala Cys Val Asn 20 25 30 Ile Val Pro Gly Leu Thr Ser Ile Tyr Arg Glu Glu Gly Ser Val Val 35 40 45 Ser Asp His Glu Leu Leu Leu Leu Val Lys Thr Thr Thr Asp Ala Phe 50 55 60 Pro Lys Leu Lys Glu Arg Val Lys Glu Leu His Pro Tyr Glu Val Pro 65 70 75 80 Glu Ile Val Ala Leu Pro Ile Ala Glu Gly Asn Arg Glu Tyr Leu Asp 85 90 95 Trp Leu Arg Glu Asn Thr Gly 100 <210> 52 <211> 869 <212> PRT <213> Human Immunodeficiency Virus 1 <400> 52 Met Arg Val Lys Gly Ile Lys Lys Asn Tyr Gln His Trp Trp Arg Gly 1 5 10 15 Gly Ile Met Leu Leu Gly Met Leu Met Ile Cys Ser Ser Ala Glu Lys 20 25 30 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 40 45 Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Gln Asn Pro Glu Met 50 55 60 His Asn Ile Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80 Gln Glu Val Ile Leu Lys Asn Leu Thr Glu Glu Phe Asn Met Trp Lys 85 90 95 Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110 Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125 Asn Cys Thr Asn Ala Glu Ser Leu Asn Cys Thr Ala Thr Asn Gly Thr 130 135 140 Asn Asn Cys Ser Ala Ser Thr Lys Pro Met Glu Glu Met Lys Asn Cys 145 150 155 160 Ser Phe Asn Ile Thr Thr Ser Val Gln Asp Lys Lys Gln Gln Glu Tyr 165 170 175 Ala Leu Phe Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asn Glu Asn 180 185 190 Asp Leu Asn Asn Thr Asn Tyr Thr Ser Tyr Arg Leu Ile Ser Cys Asn 195 200 205 Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile Thr Phe Glu Pro Ile 210 215 220 Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys 225 230 235 240 Asp Lys Arg Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr Val 245 250 255 Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu 260 265 270 Asn Gly Ser Leu Ala Glu Glu Gly Val Val Leu Arg Ser Glu Asn Phe 275 280 285 Thr Asp Asn Ala Lys Asn Ile Ile Val Gln Leu Lys Asp Pro Val Asn 290 295 300 Ile Thr Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Thr Ile 305 310 315 320 Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gly Gln Val Ile Gly Asp Ile 325 330 335 Arg Lys Ala His Cys Asp Leu Asn Gly Thr Glu Trp Asp Asn Ala Leu 340 345 350 Lys Gln Ile Val Glu Glu Leu Arg Lys Gln Tyr Gly Asn Asn Ile Thr 355 360 365 Ile Phe Asn Ser Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser 370 375 380 Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Ala Gln Leu Phe 385 390 395 400 Asn Ser Thr Trp Leu Phe Asn Ser Thr Trp Asn Ser Thr Glu Arg Leu 405 410 415 Gly Asn Asp Thr Glu Arg Thr Asn Asp Thr Ile Thr Leu Pro Cys Lys 420 425 430 Ile Lys Gln Val Ile Asn Met Trp Gln Thr Val Gly Lys Ala Met Tyr 435 440 445 Ala Pro Pro Ile Arg Gly Leu Ile Arg Cys Ser Ser Asn Ile Thr Gly 450 455 460 Leu Ile Leu Thr Arg Asp Gly Ser Gly Asn Thr Thr Gly Asn Glu Thr 465 470 475 480 Phe Arg Pro Gly Gly Gly Asn Met Lys Asp Asn Trp Arg Ser Glu Leu 485 490 495 Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr 500 505 510 Arg Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala Ala Gly Leu 515 520 525 Gly Ala Leu Phe Leu Gly Phe Leu Gly Met Ala Gly Ser Thr Met Gly 530 535 540 Ala Ala Ser Leu Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly 545 550 555 560 Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln 565 570 575 His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg 580 585 590 Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile 595 600 605 Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Thr Val Pro Trp Asn 610 615 620 Ala Ser Trp Ser Asn Lys Ser Leu Asp Asn Ile Trp Glu Asn Met Thr 625 630 635 640 Trp Met Gln Trp Glu Lys Glu Ile Asp Asn Tyr Thr Asp Val Ile Tyr 645 650 655 Lys Leu Leu Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 660 665 670 Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile 675 680 685 Thr Arg Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly 690 695 700 Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser Ile Val Asn Arg 705 710 715 720 Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr Leu Phe Pro Ala 725 730 735 Pro Arg Gly Pro Asp Arg Pro Glu Gly Thr Glu Glu Gly Gly Gly Glu 740 745 750 Arg Gly Arg Asp Ser Ser Asp Arg Ser Ala His Gly Phe Leu Ala Leu 755 760 765 Ile Trp Gly Asp Leu Trp Ser Leu Cys Leu Phe Ser Tyr Arg Arg Leu 770 775 780 Arg Asp Leu Leu Leu Ile Ala Ala Arg Ile Val Glu Leu Leu Gly Arg 785 790 795 800 Arg Gly Trp Glu Val Leu Lys Tyr Trp Trp Ser Leu Leu Gln Tyr Trp 805 810 815 Ser Gln Glu Leu Lys Lys Ser Ala Val Ser Leu Leu Asn Ala Thr Ala 820 825 830 Ile Ala Val Ala Glu Gly Thr Asp Arg Ile Ile Glu Ile Val Gln Arg 835 840 845 Ala Gly Arg Ala Ile Ile His Ile Pro Arg Arg Ile Arg Gln Gly Ala 850 855 860 Glu Arg Ala Leu Leu 865 <210> 53 <211> 492 <212> PRT <213> Human immunodeficiency virus1 <400> 53 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 1 5 10 15 Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Gln Asn Pro Glu Met 20 25 30 His Asn Ile Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 35 40 45 Gln Glu Val Ile Leu Lys Asn Leu Thr Glu Glu Phe Asn Met Trp Lys 50 55 60 Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 65 70 75 80 Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 85 90 95 Asn Cys Thr Asn Ala Glu Ser Leu Asn Cys Thr Ala Thr Asn Gly Thr 100 105 110 Asn Asn Cys Ser Ala Ser Thr Lys Pro Met Glu Glu Met Lys Asn Cys 115 120 125 Ser Phe Asn Ile Thr Thr Ser Val Gln Asp Lys Lys Gln Gln Glu Tyr 130 135 140 Ala Leu Phe Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asn Glu Asn 145 150 155 160 Asp Leu Asn Asn Thr Asn Tyr Thr Ser Tyr Arg Leu Ile Ser Cys Asn 165 170 175 Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile Thr Phe Glu Pro Ile 180 185 190 Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys 195 200 205 Asp Lys Arg Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr Val 210 215 220 Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu 225 230 235 240 Asn Gly Ser Leu Ala Glu Glu Gly Val Val Leu Arg Ser Glu Asn Phe 245 250 255 Thr Asp Asn Ala Lys Asn Ile Ile Val Gln Leu Lys Asp Pro Val Asn 260 265 270 Ile Thr Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Thr Ile 275 280 285 Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gly Gln Val Ile Gly Asp Ile 290 295 300 Arg Lys Ala His Cys Asp Leu Asn Gly Thr Glu Trp Asp Asn Ala Leu 305 310 315 320 Lys Gln Ile Val Glu Glu Leu Arg Lys Gln Tyr Gly Asn Asn Ile Thr 325 330 335 Ile Phe Asn Ser Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser 340 345 350 Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Ala Gln Leu Phe 355 360 365 Asn Ser Thr Trp Leu Phe Asn Ser Thr Trp Asn Ser Thr Glu Arg Leu 370 375 380 Gly Asn Asp Thr Glu Arg Thr Asn Asp Thr Ile Thr Leu Pro Cys Lys 385 390 395 400 Ile Lys Gln Val Ile Asn Met Trp Gln Thr Val Gly Lys Ala Met Tyr 405 410 415 Ala Pro Pro Ile Arg Gly Leu Ile Arg Cys Ser Ser Asn Ile Thr Gly 420 425 430 Leu Ile Leu Thr Arg Asp Gly Ser Gly Asn Thr Thr Gly Asn Glu Thr 435 440 445 Phe Arg Pro Gly Gly Gly Asn Met Lys Asp Asn Trp Arg Ser Glu Leu 450 455 460 Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr 465 470 475 480 Arg Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg 485 490 <210> 54 <211> 191 <212> PRT <213> Human immunodeficiency virus 1 <400> 54 Met Gly Ala Ala Ser Leu Thr Leu Thr Val Gln Ala Arg Gln Leu Leu 1 5 10 15 Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala 20 25 30 Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln 35 40 45 Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu 50 55 60 Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Thr Val Pro 65 70 75 80 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Asp Asn Ile Trp Glu Asn 85 90 95 Met Thr Trp Met Gln Trp Glu Lys Glu Ile Asp Asn Tyr Thr Asp Val 100 105 110 Ile Tyr Lys Leu Leu Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu 115 120 125 Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe 130 135 140 Asp Ile Thr Arg Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met Ile Val 145 150 155 160 Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser Ile Val 165 170 175 Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr Leu 180 185 190 <210> 55 <211> twenty two <212> PRT <213> Human Immunodeficiency Virus 1 <400> 55 Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Arg 1 5 10 15 Trp Leu Trp Tyr Ile Lys 20 <210> 56 <211> 574 <212> PRT <213> Respiratory syncytial virus type A <400> 56 Met Glu Leu Pro Ile Leu Lys Thr Asn Ala Ile Thr Thr Ile Leu Ala 1 5 10 15 Ala Val Thr Leu Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Asn Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Ile 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Val Gly Lys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525 Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Leu Leu Ile Ala Val 530 535 540 Gly Leu Phe Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser 545 550 555 560 Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn 565 570 <210> 57 <211> 566 <212> PRT <213> Influenza A virus <400> 57 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr[[ID=A]] 515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val 530 535 540 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg Ile Cys Ile 565 <210> 58 <211> 584 <212> PRT <21_{3}> Influenza B virus <400> 58 It should be noted that in the translation of the virus name in , it is assumed that "乙型流感病毒" is "Influenza B virus" according to common sense. If there is a more accurate specific translation requirement, it should be adjusted accordingly.Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Lys Thr 50 55 60 Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala 85 90 95 Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile 100 105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Ala Asn Leu Leu Arg Gly 115 120 125 Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu Lys 130 135 140 Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn 145 150 155 160 Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro 165 170 175 Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val Pro 180 185 190 Tyr Ile Cys Ala Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His 195 200 205 Ser Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro 210 215 220 Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser 225 230 235 240 Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln 245 250 255 Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys Thr 260 265 270 Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val Trp 275 280 285 Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile 290 295 300 Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser 305 310 315 320 Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro 325 330 335 Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg 340 345 350 Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala 355 360 365 Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly 370 375 380 Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys 385 390 395 400 Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu 405 410 415 Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp 420 425 430 Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu 435 440 445 Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser 450 455 460 Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu 465 470 475 480 Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn 485 490 495 Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg 500 505 510 Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe 515 520 525 Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp 530 535 540 Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala 545 550 555 560 Val Thr Leu Met Leu Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp 565 570 575 Asn Val Ser Cys Ser Ile Cys Leu 580 <210> 59 <211> 907 <212> PRT <213> Lovebird virus <400> 59 Met Glu Ala Ala Leu Leu Val Cys Gln Tyr Thr Ile Gln Ser Leu Ile 1 5 10 15 His Leu Thr Gly Glu Asp Pro Gly Phe Phe Asn Val Glu Ile Pro Glu 20 25 30 Phe Pro Phe Tyr Pro Thr Cys Asn Val Cys Thr Ala Asp Val Asn Val 35 40 45 Thr Ile Asn Phe Asp Val Gly Gly Lys Lys His Gln Leu Asp Leu Asp 50 55 60 Phe Gly Gln Leu Thr Pro His Thr Lys Ala Val Tyr Gln Pro Arg Gly 65 70 75 80 Ala Phe Gly Gly Ser Glu Asn Ala Thr Asn Leu Phe Leu Leu Glu Leu 85 90 95 Leu Gly Ala Gly Glu Leu Ala Leu Thr Met Arg Ser Lys Lys Leu Pro 100 105 110 Ile Asn Val Thr Thr Gly Glu Glu Gln Gln Val Ser Leu Glu Ser Val 115 120 125 Asp Val Tyr Phe Gln Asp Val Phe Gly Thr Met Trp Cys His His Ala 130 135 140 Glu Met Gln Asn Pro Val Tyr Leu Ile Pro Glu Thr Val Pro Tyr Ile 145 150 155 160 Lys Trp Asp Asn Cys Asn Ser Thr Asn Ile Thr Ala Val Val Arg Ala 165 170 175 Gln Gly Leu Asp Val Thr Leu Pro Leu Ser Leu Pro Thr Ser Ala Gln 180 185 190 Asp Ser Asn Phe Ser Val Lys Thr Glu Met Leu Gly Asn Glu Ile Asp 195 200 205 Ile Glu Cys Ile Met Glu Asp Gly Glu Ile Ser Gln Val Leu Pro Gly 210 215 220 Asp Asn Lys Phe Asn Ile Thr Cys Ser Gly Tyr Glu Ser His Val Pro 225 230 235 240 Ser Gly Gly Ile Leu Thr Ser Thr Ser Pro Val Ala Thr Pro Ile Pro 245 250 255 Gly Thr Gly Tyr Ala Tyr Ser Leu Arg Leu Thr Pro Arg Pro Val Ser 260 265 270 Arg Phe Leu Gly Asn Asn Ser Ile Leu Tyr Val Phe Tyr Ser Gly Asn 275 280 285 Gly Pro Lys Ala Ser Gly Gly Asp Tyr Cys Ile Gln Ser Asn Ile Val 290 295 300 Phe Ser Asp Glu Ile Pro Ala Ser Gln Asp Met Pro Thr Asn Thr Thr 305 310 315 320 Asp Ile Thr Tyr Val Gly Asp Asn Ala Thr Tyr Ser Val Pro Met Val 325 330 335 Thr Ser Glu Asp Ala Asn Ser Pro Asn Val Thr Val Thr Ala Phe Trp 340 345 350 Ala Trp Pro Asn Asn Thr Glu Thr Asp Phe Lys Cys Lys Trp Thr Leu 355 360 365 Thr Ser Gly Thr Pro Ser Gly Cys Glu Asn Ile Ser Gly Ala Phe Ala 370 375 380 Ser Asn Arg Thr Phe Asp Ile Thr Val Ser Gly Leu Gly Thr Ala Pro 385 390 395 400 Lys Thr Leu Ile Ile Thr Arg Thr Ala Thr Asn Ala Thr Thr Thr Thr 405 410 415 His Lys Val Ile Phe Ser Lys Ala Pro Glu Ser Thr Thr Thr Ser Pro 420 425 430 Thr Leu Asn Thr Thr Gly Phe Ala Asp Pro Asn Thr Thr Thr Gly Leu 435 440 445 Pro Ser Ser Thr His Val Pro Thr Asn Leu Thr Ala Pro Ala Ser Thr 450 455 460 Gly Pro Thr Val Ser Thr Ala Asp Val Thr Ser Pro Thr Pro Ala Gly 465 470 475 480 Thr Thr Ser Gly Ala Ser Pro Val Thr Pro Ser Pro Ser Pro Trp Asp 485 490 495 Asn Gly Thr Glu Ser Lys Ala Pro Asp Met Thr Ser Ser Thr Ser Pro 500 505 510 Val Thr Thr Pro Thr Pro Asn Ala Thr Ser Pro Thr Pro Ala Val Thr 515 520 525 Thr Pro Thr Pro Asn Ala Thr Ser Pro Thr Pro Ala Val Thr Thr Pro 530 535 540 Thr Pro Asn Ala Thr Ser Pro Thr Leu Gly Lys Thr Ser Pro Thr Ser 545 550 555 560 Ala Val Thr Thr Pro Thr Pro Asn Ala Thr Ser Pro Thr Leu Gly Lys 565 570 575 Thr Ser Pro Thr Ser Ala Val Thr Thr Pro Thr Pro Asn Ala Thr Ser 580 585 590 Pro Thr Leu Gly Lys Thr Ser Pro Thr Ser Ala Val Thr Thr Pro Thr 595 600 605 Pro Asn Ala Thr Gly Pro Thr Val Gly Glu Thr Ser Pro Gln Ala Asn 610 615 620 Ala Thr Asn His Thr Leu Gly Gly Thr Ser Pro Thr Pro Val Val Thr 625 630 635 640 Ser Gln Pro Lys Asn Ala Thr Ser Ala Val Thr Thr Gly Gln His Asn 645 650 655 Ile Thr Ser Ser Ser Thr Ser Ser Met Ser Leu Arg Pro Ser Ser Asn 660 665 670 Pro Glu Thr Leu Ser Pro Ser Thr Ser Asp Asn Ser Thr Ser His Met 675 680 685 Pro Leu Leu Thr Ser Ala His Pro Thr Gly Gly Glu Asn Ile Thr Gln 690 695 700 Val Thr Pro Ala Ser Ile Ser Thr His His Val Ser Thr Ser Ser Pro 705 710 715 720 Ala Pro Arg Pro Gly Thr Thr Ser Gln Ala Ser Gly Pro Gly Asn Ser 725 730 735 Ser Thr Ser Thr Lys Pro Gly Glu Val Asn Val Thr Lys Gly Thr Pro 740 745 750 Pro Gln Asn Ala Thr Ser Pro Gln Ala Pro Ser Gly Gln Lys Thr Ala 755 760 765 Val Pro Thr Val Thr Ser Thr Gly Gly Lys Ala Asn Ser Thr Thr Gly 770 775 780 Gly Lys His Thr Thr Gly His Gly Ala Arg Thr Ser Thr Glu Pro Thr 785 790 795 800 Thr Asp Tyr Gly Gly Asp Ser Thr Thr Pro Arg Pro Arg Tyr Asn Ala 805 810 815 Thr Thr Tyr Leu Pro Pro Ser Thr Ser Ser Lys Leu Arg Pro Arg Trp 820 825 830 Thr Phe Thr Ser Pro Pro Val Thr Thr Ala Gln Ala Thr Val Pro Val 835 840 845 Pro Pro Thr Ser Gln Pro Arg Phe Ser Asn Leu Ser Met Leu Val Leu 850 855 860 Gln Trp Ala Ser Leu Ala Val Leu Thr Leu Leu Leu Leu Leu Val Met 865 870 875 880 Ala Asp Cys Ala Phe Arg Arg Asn Leu Ser Thr Ser His Thr Tyr Thr 885 890 895 Thr Pro Pro Tyr Asp Asp Ala Glu Thr Tyr Val 900 905 <210> 60 <211> 906 <212> PRT <213> Human cell virus <400> 60 Met Glu Ser Arg Ile Trp Cys Leu Val Val Cys Val Asn Leu Cys Ile 1 5 10 15 Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr Ser His Ala Thr 20 25 30 Ser Ser Thr His Asn Gly Ser His Thr Ser Arg Thr Thr Ser Ala Gln 35 40 45 Thr Arg Ser Val Tyr Ser Gln His Val Thr Ser Ser Glu Ala Val Ser 50 55 60 His Arg Ala Asn Glu Thr Ile Tyr Asn Thr Thr Leu Lys Tyr Gly Asp 65 70 75 80 Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met 85 90 95 Ala Gln Gly Thr Asp Leu Ile Arg Phe Glu Arg Asn Ile Ile Cys Thr 100 105 110 Ser Met Lys Pro Ile Asn Glu Asp Leu Asp Glu Gly Ile Met Val Val 115 120 125 Tyr Lys Arg Asn Ile Val Ala His Thr Phe Lys Val Arg Val Tyr Gln 130 135 140 Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr Ile Tyr Thr Thr Tyr 145 150 155 160 Leu Leu Gly Ser Asn Thr Glu Tyr Val Ala Pro Pro Met Trp Glu Ile 165 170 175 His His Ile Asn Lys Phe Ala Gln Cys Tyr Ser Ser Tyr Ser Arg Val 180 185 190 Ile Gly Gly Thr Val Phe Val Ala Tyr His Arg Asp Ser Tyr Glu Asn 195 200 205 Lys Thr Met Gln Leu Ile Pro Asp Asp Tyr Ser Asn Thr His Ser Thr 210 215 220 Arg Tyr Val Thr Val Lys Asp Gln Trp His Ser Arg Gly Ser Thr Trp 225 230 235 240 Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Leu Thr Ile Thr Thr 245 250 255 Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Ala Thr Ser Thr Gly Asp 260 265 270 Val Val Tyr Ile Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285 Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe Ile Phe Pro Asn Tyr Thr 290 295 300 Ile Val Ser Asp Phe Gly Arg Pro Asn Ala Ala Pro Glu Thr His Arg 305 310 315 320 Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val Ile Ser Trp Asp Ile 325 330 335 Gln Asp Glu Lys Asn Val Thr Cys Gln Leu Thr Phe Trp Glu Ala Ser 340 345 350 Glu Arg Thr Ile Arg Ser Glu Ala Glu Asp Ser Tyr His Phe Ser Ser 355 360 365 Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Gln Glu Val Asn Met 370 375 380 Ser Asp Ser Ala Leu Asp Cys Val Arg Asp Glu Ala Ile Asn Lys Leu 385 390 395 400 Gln Gln Ile Phe Asn Thr Ser Tyr Asn Gln Thr Tyr Glu Lys Tyr Gly 405 410 415 Asn Val Ser Val Phe Glu Thr Ser Gly Gly Leu Val Val Phe Trp Gln 420 425 430 Gly Ile Lys Gln Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445 Ser Ser Leu Asn Ile Thr His Arg Thr Arg Arg Ser Thr Ser Asp Asn 450 455 460 Asn Thr Thr His Leu Ser Ser Met Glu Ser Val His Asn Leu Val Tyr 465 470 475 480 Ala Gln Leu Gln Phe Thr Tyr Asp Thr Leu Arg Gly Tyr Ile Asn Arg 485 490 495 Ala Leu Ala Gln Ile Ala Glu Ala Trp Cys Val Asp Gln Arg Arg Thr 500 505 510 Leu Glu Val Phe Lys Glu Leu Ser Lys Ile Asn Pro Ser Ala Ile Leu 515 520 525 Ser Ala Ile Tyr Asn Lys Pro Ile Ala Ala Arg Phe Met Gly Asp Val 530 535 540 Leu Gly Leu Ala Ser Cys Val Thr Ile Asn Gln Thr Ser Val Lys Val 545 550 555 560 Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys Tyr Ser Arg 565 570 575 Pro Val Val Ile Phe Asn Phe Ala Asn Ser Ser Tyr Val Gln Tyr Gly 580 585 590 Gln Leu Gly Glu Asp Asn Glu Ile Leu Leu Gly Asn His Arg Thr Glu 595 600 605 Glu Cys Gln Leu Pro Ser Leu Lys Ile Phe Ile Ala Gly Asn Ser Ala 610 615 620 Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met Ile Asp Leu Ser Ser 625 630 635 640 Ile Ser Thr Val Asp Ser Met Ile Ala Leu Asp Ile Asp Pro Leu Glu 645 650 655 Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gln Lys Glu Leu Arg 660 665 670 Ser Ser Asn Val Phe Asp Leu Glu Glu Ile Met Arg Glu Phe Asn Ser 675 680 685 Tyr Lys Gln Arg Val Lys Tyr Val Glu Asp Lys Val Val Asp Pro Leu 690 695 700 Pro Pro Tyr Leu Lys Gly Leu Asp Asp Leu Met Ser Gly Leu Gly Ala 705 710 715 720 Ala Gly Lys Ala Val Gly Val Ala Ile Gly Ala Val Gly Gly Ala Val 725 730 735 Ala Ser Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe Gly 740 745 750 Ala Phe Thr Ile Ile Leu Val Ala Ile Ala Val Val Ile Ile Thr Tyr 755 760 765 Leu Ile Tyr Thr Arg Gln Arg Arg Leu Cys Thr Gln Pro Leu Gln Asn 770 775 780 Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val Thr Ser Gly 785 790 795 800 Ser Thr Lys Asp Thr Ser Leu Gln Ala Pro Pro Ser Tyr Glu Glu Ser 805 810 815 Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pro Pro Ser Ser Asp Ala 820 825 830 Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gln Ala Tyr Gln Met Leu 835 840 845 Leu Ala Leu Ala Arg Leu Asp Ala Glu Gln Arg Ala Gln Gln Asn Gly 850 855 860 Thr Asp Ser Leu Asp Gly Gln Thr Gly Thr Gln Asp Lys Gly Gln Lys 865 870 875 880 Pro Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg His 885 890 895 Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900 905 <210> 61 <211> 171 <212> PRT <213> Human cytomegalovirus <400> 61 Met Ser Pro Lys Asp Leu Thr Pro Phe Leu Thr Thr Leu Trp Leu Leu 1 5 10 15 Leu Gly His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45 Cys As n Arg Phe Thr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60 Tyr Ser Pro Glu Lys Thr Ala Glu Ile Arg Gly Ile Val Thr Thr Met 65 70 75 80 Thr His Ser Leu Thr Arg Gln Val Val His Asn Lys Leu Thr Ser Cys 85 90 95 Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg Ile Arg Cys Gly 100 105 110 Lys Val Asn Asp Lys Ala Gln Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125 Pro Tyr Arg Trp Ile Asn Leu Glu Tyr Asp Lys Ile Thr Arg Ile Val 130 135 140 Gly Leu Asp Gln Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160 Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gln 165 170 <210> 62 <211> 214 <212> PRT <213> Human cytomegalovirus <400> 62 Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 1 5 10 15 Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30 Ala Asn Gln Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 40 45 Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60 Pro Arg Ser Pro Leu Gln Phe Ser Gly Phe Gln Arg Val Ser Thr Gly 65 70 75 80 Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 Gln Thr Leu Val Glu Arg Ser Ser Thr Trp Val Lys Lys Val Ile Trp 100 105 110 Tyr Leu Ser Gly Arg Asn Gln Thr Ile Leu Gln Arg Met Pro Arg Thr 115 120 125 Ala Ser Lys Pro Ser Asp Gly Asn Val Gln Ile Ser Val Glu Asp Ala 130 135 140 Lys Ile Phe Gly Ala His Met Val Pro Lys Gln Thr Lys Leu Leu Arg 145 150 155 160 Phe Val Val Asn Asp Gly Thr Arg Tyr Gln Met Cys Val Met Lys Leu 165 170 175 Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gln Val 180 185 190 Arg Leu Thr Phe Thr Glu Ala Asn Asn Gln Thr Tyr Thr Phe Cys Thr 195 200 205 His Pro Asn Leu Ile Val 210 <210> 63 <211> 129 <212> PRT <213> Human cytomegalovirus <400> 63 Met Arg Leu Cys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Val Val 1 5 10 15 Leu Gly Gln Cys Gln Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30 Val Pro His Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gln Thr 35 40 45 Arg Tyr Lys Tyr Val Glu Gln Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60 Tyr Asp Ala Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg Ile 65 70 75 80 Asn Val Thr Glu Val Ser Leu Leu Ile Ser Asp Phe Arg Arg Gln Asn 85 90 95 Arg Arg Gly Gly Thr Asn Lys Arg Thr Thr Phe Asn Ala Ala Gly Ser 100 105 110 Leu Ala Pro His Ala Arg Ser Leu Glu Phe Ser Val Arg Leu Phe Ala 115 120 125 Asn <210> 64 <211> 743 <212> PRT <213> Human cell virus <400> 64 Met Arg Pro Gly Leu Pro Pro Tyr Leu Thr Val Phe Thr Val Tyr Leu 1 5 10 15 Leu Ser His Leu Pro Ser Gln Arg Tyr Gly Ala Asp Ala Ala Ser Glu 20 25 30 Ala Leu Asp Pro His Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg 35 40 45 Pro Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser 50 55 60 Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn 65 70 75 80 Phe Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys 85 90 95 Leu Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu 100 105 110 Thr Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr Ala Leu 115 120 125 Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln Gln Leu Lys 130 135 140 Ala Gln Asp Ser Leu Gly Gln Gln Pro Thr Thr Val Pro Pro Pro Ile 145 150 155 160 Asp Leu Ser Ile Pro His Val Trp Met Pro Pro Gln Thr Thr Pro His 165 170 175 Asp Trp Lys Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe 180 185 190 Asn Gln Thr Cys Ile Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr 195 200 205 Val Thr Pro Cys Leu His Gln Gly Phe Tyr Leu Met Asp Glu Leu Arg 210 215 220 Tyr Val Lys Ile Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser 225 230 235 240 Ile Asp Asp Asp Thr Pro Met Leu Leu Ile Phe Gly His Leu Pro Arg 245 250 255 Val Leu Phe Lys Ala Pro Tyr Gln Arg Asp Asn Phe Ile Leu Arg Gln 260 265 270 Thr Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Ala Gln Leu Asn 275 280 285 Arg His Ser Tyr Leu Lys Asp Ser Asp Phe Leu Asp Ala Ala Leu Asp 290 295 300 Phe Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg 305 310 315 320 Tyr Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met Leu Asp Arg 325 330 335 Arg Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala 340 345 350 Ala Arg Gln Glu Glu Ala Gly Thr Glu Ile Ser Ile Pro Arg Ala Leu 355 360 365 Asp Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met Ile Thr Cys 370 375 380 Leu Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala 385 390 395 400 Val Asp Leu Ala Lys Arg Ala Leu Trp Thr Pro Asp Gln Ile Thr Asp 405 410 415 Ile Thr Ser Leu Val Arg Leu Val Tyr Ile Leu Ser Lys Gln Asn Gln 420 425 430 Gln His Leu Ile Pro Gln Trp Ala Leu Arg Gln Ile Ala Asp Phe Ala 435 440 445 Leu Gln Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala 450 455 460 Arg Gln Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val 465 470 475 480 His Thr Thr Glu Arg Arg Glu Ile Phe Ile Val Glu Thr Gly Leu Cys 485 490 495 Ser Leu Ala Glu Leu Ser His Phe Thr Gln Leu Leu Ala His Pro His 500 505 510 His Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg 515 520 525 Arg Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr 530 535 540 Val Pro Ala Thr Val Pro Ala Ala Leu Ser Ile Leu Ser Thr Met Gln 545 550 555 560 Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly 565 570 575 Glu Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Val Val 580 585 590 Thr Asn Gln Tyr Leu Ile Lys Gly Ile Ser Tyr Pro Val Ser Thr Thr 595 600 605 Val Val Gly Gln Ser Leu Ile Ile Thr Gln Thr Asp Ser Gln Thr Lys 610 615 620 Cys Glu Leu Thr Arg Asn Met His Thr Thr His Ser Ile Thr Ala Ala 625 630 635 640 Leu Asn Ile Ser Leu Glu Asn Cys Ala Phe Cys Gln Ser Ala Leu Leu 645 650 655 Glu Tyr Asp Asp Thr Gln Gly Val Ile Asn Ile Met Tyr Met His Asp 660 665 670 Ser Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val 675 680 685 Ser Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val 690 695 700 Leu Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 720 Met Met Ser Val Tyr Ala Leu Ser Ala Ile Ile Gly Ile Tyr Leu Leu 725 730 735 Tyr Arg Met Leu Lys Thr Cys 740 <210> 65 <211> 278 <212> PRT <213> Human cell virus <400> 65 Met Cys Arg Arg Pro Asp Cys Gly Phe Ser Phe Ser Pro Gly Pro Val 1 5 10 15 Val Leu Leu Trp Cys Cys Leu Leu Leu Pro Ile Val Ser Ser Val Ala 20 25 30 Val Ser Val Ala Pro Thr Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45 Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Gln Gly Asp Lys 50 55 60 Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Arg Arg Asp Gly 65 70 75 80 Pro Leu Ser Gln Leu Ile Arg Tyr Arg Pro Val Thr Pro Glu Ala Ala 85 90 95 Asn Ser Val Leu Leu Asp Asp Ala Phe Leu Asp Thr Leu Ala Leu Leu 100 105 110 Tyr Asn Asn Pro Asp Gln Leu Arg Ala Leu Leu Thr Leu Leu Ser Ser 115 120 125 Asp Thr Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140 Gly Asp Gly Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 150 155 160 Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser Ile Phe Thr Glu 165 170 175 His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Phe Asn Val Val 180 185 190 Val Ala Ile Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205 Pro Val Ser Thr Ala Ala Ala Pro Glu Gly Ile Thr Leu Phe Tyr Gly 210 215 220 Leu Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gln Leu Asp Pro 225 230 235 240 Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255 Leu Lys Gln Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270 Gln Ala Val Asp Ala Arg 275 <210> 66 <211> 272 <212> PRT <213> Borrelia burgdorferi <400> 66 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Val Pro Gly Gly Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Met Ala Thr Val Asp Asn Val Asp Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Ile Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Val Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Leu Glu Val Leu Lys Glu Asp Gly Thr Val Val Ser Arg Lys 100 105 110 Val Thr Ser Lys Asp Lys Ser Thr Thr Glu Ala Lys Phe Asn Glu Lys 115 120 125 Gly Glu Leu Ser Glu Lys Thr Met Thr Arg Ala Asn Gly Thr Thr Leu 130 135 140 Glu Tyr Ser Gln Met Thr Asn Glu Asp Asn Ala Ala Lys Ala Val Glu 145 150 155 160 Thr Leu Lys Asn Gly Ile Lys Phe Glu Gly Asn Leu Ala Ser Gly Lys 165 170 175 Thr Ala Val Glu Ile Lys Glu Gly Thr Val Thr Leu Lys Arg Glu Ile 180 185 190 Asp Lys Asn Gly Lys Val Thr Val Ser Leu Asn Asp Thr Ala Ser Gly 195 200 205 Ser Lys Lys Thr Ala Ser Trp Gln Glu Ser Thr Ser Thr Leu Thr Ile 210 215 220 Ser Ala Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Leu Thr Asn Gly 225 230 235 240 Thr Ile Thr Val Gln Asn Tyr Asp Ser Ala Gly Thr Lys Leu Glu Gly 245 250 255 Ser Ala Ala Glu Ile Lys Lys Leu Asp Glu Leu Lys Asn Ala Leu Arg 260 265 270 <210> 67 <211> 269 <212> PRT <213> Bordetella pertussis <400> 67<^ Met Arg Cys Thr Arg Ala Ile Arg Gln Thr Ala Arg Thr Gly Trp Leu 1 5 10 15 Thr Trp Leu Ala Ile Leu Ala Val Thr Ala Pro Val Thr Ser Pro Ala 20 25 30 Trp Ala Asp Asp Pro Pro Ala Thr Val Tyr Arg Tyr Asp Ser Arg Pro 35 40 45 Pro Glu Asp Val Phe Gln Asn Gly Phe Thr Ala Trp Gly Asn Asn Asp 50 55 60 Asn Val Leu Asp His Leu Thr Gly Arg Ser Cys Gln Val Gly Ser Ser 65 70 75 80 Asn Ser Ala Phe Val Ser Thr Ser Ser Ser Arg Arg Tyr Thr Glu Val 85 90 95 Tyr Leu Glu His Arg Met Gln Glu Ala Val Glu Ala Glu Arg Ala Gly 100 105 110 Arg Gly Thr Gly His Phe Ile Gly Tyr Ile Tyr Glu Val Arg Ala Asp 115 120 125 Asn Asn Phe Tyr Gly Ala Ala Ser Ser Tyr Phe Glu Tyr Val Asp Thr 130 135 140 Tyr Gly Asp Asn Ala Gly Arg Ile Leu Ala Gly Ala Leu Ala Thr Tyr 145 150 155 160 Gln Ser Glu Tyr Leu Ala His Arg Arg Ile Pro Pro Glu Asn Ile Arg 165 170 175 Arg Val Thr Arg Val Tyr His Asn Gly Ile Thr Gly Glu Thr Thr Thr 180 185 190 Thr Glu Tyr Ser Asn Ala Arg Tyr Val Ser Gln Gln Thr Arg Ala Asn 195 200 205 Pro Asn Pro Tyr Thr Ser Arg Arg Ser Val Ala Ser Ile Val Gly Thr 210 215 220 Leu Val Arg Met Ala Pro Val Ile Gly Ala Cys Met Ala Arg Gln Ala 225 230 235 240 Glu Ser Ser Glu Ala Met Ala Ala Trp Ser Glu Arg Ala Gly Glu Ala 245 250 255 Met Val Leu Val Tyr Tyr Glu Ser Ile Ala Tyr Ser Phe 260 265 <210> 68 <211> 495 <212> PRT <213> Dengue fever virus <400> 68 Met Arg Cys Val Gly Ile Gly Asn Arg Asp Phe Val Glu Gly Leu Ser 1 5 10 15 Gly Ala Thr Trp Val Asp Val Val Leu Glu His Gly Ser Cys Val Thr 20 25 30 Thr Met Ala Lys Asp Lys Pro Thr Leu Asp Ile Glu Leu Leu Lys Thr 35 40 45 Glu Val Thr Asn Pro Ala Val Leu Arg Lys Leu Cys Ile Glu Ala Lys 50 55 60 Ile Ser Asn Thr Thr Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala 65 70 75 80 Thr Leu Val Glu Glu Gln Asp Thr Asn Phe Val Cys Arg Arg Thr Phe 85 90 95 Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser 100 105 110 Leu Ile Thr Cys Ala Lys Phe Lys Cys Val Thr Lys Leu Glu Gly Lys 115 120 125 Ile Val Gln Tyr Glu Asn Leu Lys Tyr Ser Val Ile Val Thr Val His 130 135 140 Thr Gly Asp Gln His Gln Val Gly Asn Glu Thr Thr Glu His Gly Thr 145 150 155 160 Thr Ala Thr Ile Thr Pro Gln Ala Pro Thr Ser Glu Ile Gln Leu Thr 165 170 175 Asp Tyr Gly Ala Leu Thr Leu Asp Cys Ser Pro Arg Thr Gly Leu Asp 180 185 190 Phe Asn Glu Met Val Leu Leu Thr Met Glu Lys Lys Ser Trp Leu Val 195 200 205 His Lys Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Thr Ser Gly Ala 210 215 220 Ser Thr Ser Gln Glu Thr Trp Asn Arg Gln Asp Leu Leu Val Thr Phe 225 230 235 240 Lys Thr Ala His Ala Lys Lys Gln Glu Val Val Val Leu Gly Ser Gln 245 250 255 Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Thr 260 265 270 Ser Gly Thr Thr Thr Ile Phe Ala Gly His Leu Lys Cys Arg Leu Lys 275 280 285 Met Asp Lys Leu Thr Leu Lys Gly Met Ser Tyr Val Met Cys Thr Gly 290 295 300 Ser Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln His Gly Thr Val 305 310 315 320 Leu Val Gln Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro 325 330 335 Phe Ser Ser Gln Asp Glu Lys Gly Val Thr Gln Asn Gly Arg Leu Ile 340 345 350 Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys Pro Val Asn Ile Glu 355 360 365 Ala Glu Pro Pro Phe Gly Glu Ser Tyr Ile Val Val Gly Ala Gly Glu 370 375 380 Lys Ala Leu Lys Leu Ser Trp Phe Lys Lys Gly Ser Ser Ile Gly Lys 385 390 395 400 Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala Ile Leu Gly 405 410 415 Asp Thr Ala Trp Asp Phe Gly Ser Ile Gly Gly Val Phe Thr Ser Val 420 425 430 Gly Lys Leu Ile His Gln Ile Phe Gly Thr Ala Tyr Gly Val Leu Phe 435 440 445 Ser Gly Val Ser Trp Thr Met Lys Ile Gly Ile Gly Ile Leu Leu Thr 450 455 460 Trp Leu Gly Leu Asn Ser Arg Ser Thr Ser Leu Ser Met Thr Cys Ile 465 470 475 480 Ala Val Gly Met Val Thr Leu Tyr Leu Gly Val Met Val Gln Ala 485 490 495 <210> 69 <211> 1255 <212> PRT <213> Human SARS coronavirus <400> 69 Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu 1 5 10 15 Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln 20 25 30 His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg 35 40 45 Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser 50 55 60 Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val 65 70 75 80 Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn 85 90 95 Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln 100 105 110 Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys 115 120 125 Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met 130 135 140 Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr 145 150 155 160 Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser 165 170 175 Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly 180 185 190 Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp 195 200 205 Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu 210 215 220 Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro 225 230 235 240 Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr 245 250 255 Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys 275 280 285 Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser 325 330 335 Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser 420 425 430 Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu 435 440 445 Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly 450 455 460 Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp 465 470 475 480 Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val 485 490 495 Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly 500 505 510 Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn 515 520 525 Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg 530 535 540 Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp 545 550 555 560 Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys 565 570 575 Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser 580 585 590 Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr 595 600 605 Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr 610 615 620 Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu 625 630 635 640 His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile 645 650 655 Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys 660 665 670 Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala 675 680 685 Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile 690 695 700 Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys 705 710 715 720 Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu 725 730 735 Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile 740 745 750 Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys 755 760 765 Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe 770 775 780 Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile 785 790 795 800 Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met 805 810 815 Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile 820 825 830 Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr 835 840 845 Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala 850 855 860 Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe 865 870 875 880 Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn 885 890 895 Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala 900 905 910 Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly 915 920 925 Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu 930 935 940 Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn 945 950 955 960 Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp 965 970 975 Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln 980 985 990 Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 995 1000 1005 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp 1010 1015 1020 Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala 1025 1030 1035 Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln 1040 1045 1050 Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys 1055 1060 1065 Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser 1070 1075 1080 Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr 1085 1090 1095 Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly 1100 1105 1110 Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp 1115 1120 1125 Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser 1130 1135 1140 Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val 1145 1150 1155 Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys 1160 1165 1170 Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr 1175 1180 1185 Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile 1190 1195 1200 Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys 1205 1210 1215 Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly 1220 1225 1230 Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys 1235 1240 1245 Gly Val Lys Leu His Tyr Thr 1250 1255 <210> 70 <211> 1353 <212> PRT <213> Middle East Respiratory Syndrome-related Coronavirus <400> 70 Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu 1 5 10 15 Ser Tyr Val Asp Val Gly Pro Asp Ser Ile Lys Ser Ala Cys Ile Glu 20 25 30 Val Asp Ile Gln Gln Thr Phe Phe Asp Lys Thr Trp Pro Arg Pro Ile 35 40 45 Asp Val Ser Lys Ala Asp Gly Ile Ile Tyr Pro Gln Gly Arg Thr Tyr 50 55 60 Ser Asn Ile Thr Ile Thr Tyr Gln Gly Leu Phe Pro Tyr Gln Gly Asp 65 70 75 80 His Gly Asp Met Tyr Val Tyr Ser Ala Gly His Ala Thr Gly Thr Thr 85 90 95 Pro Gln Lys Leu Phe Val Ala Asn Tyr Ser Gln Asp Val Lys Gln Phe 100 105 110 Ala Asn Gly Phe Val Val Arg Ile Gly Ala Ala Ala Asn Ser Thr Gly 115 120 125 Thr Val Ile Ile Ser Pro Ser Thr Ser Ala Thr Ile Arg Lys Ile Tyr 130 135 140 Pro Ala Phe Met Leu Gly Ser Ser Val Gly Asn Phe Ser Asp Gly Lys 145 150 155 160 Met Gly Arg Phe Phe Asn His Thr Leu Val Leu Leu Pro Asp Gly Cys 165 170 175 Gly Thr Leu Leu Arg Ala Phe Tyr Cys Ile Leu Glu Pro Arg Ser Gly 180 185 190 Asn His Cys Pro Ala Gly Asn Ser Tyr Thr Ser Phe Ala Thr Tyr His 195 200 205 Thr Pro Ala Thr Asp Cys Ser Asp Gly Asn Tyr Asn Arg Asn Ala Ser 210 215 220 Leu Asn Ser Phe Lys Glu Tyr Phe Asn Leu Arg Asn Cys Thr Phe Met 225 230 235 240 Tyr Thr Tyr Asn Ile Thr Glu Asp Glu Ile Leu Glu Trp Phe Gly Ile 245 250 255 Thr Gln Thr Ala Gln Gly Val His Leu Phe Ser Ser Arg Tyr Val Asp 260 265 270 Leu Tyr Gly Gly Asn Met Phe Gln Phe Ala Thr Leu Pro Val Tyr Asp 275 280 285 Thr Ile Lys Tyr Tyr Ser Ile Ile Pro His Ser Ile Arg Ser Ile Gln 290 295 300 Ser Asp Arg Lys Ala Trp Ala Ala Phe Tyr Val Tyr Lys Leu Gln Pro 305 310 315 320 Leu Thr Phe Leu Leu Asp Phe Ser Val Asp Gly Tyr Ile Arg Arg Ala 325 330 335 Ile Asp Cys Gly Phe Asn Asp Leu Ser Gln Leu His Cys Ser Tyr Glu 340 345 350 Ser Phe Asp Val Glu Ser Gly Val Tyr Ser Val Ser Ser Phe Glu Ala 355 360 365 Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp 370 375 380 Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys 385 390 395 400 Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser 405 410 415 Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala 420 425 430 Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr 435 440 445 Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile 450 455 460 Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile 465 470 475 480 Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys 485 490 495 Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr 500 505 510 Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser 515 520 525 Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln 530 535 540 Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr 545 550 555 560 Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln 565 570 575 Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn 580 585 590 Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr Ser Leu 595 600 605 Tyr Gly Val Ser Gly Arg Gly Val Phe Gln Asn Cys Thr Ala Val Gly 610 615 620 Val Arg Gln Gln Arg Phe Val Tyr Asp Ala Tyr Gln Asn Leu Val Gly 625 630 635 640 Tyr Tyr Ser Asp Asp Gly Asn Tyr Tyr Cys Leu Arg Ala Cys Val Ser 645 650 655 Val Pro Val Ser Val Ile Tyr Asp Lys Glu Thr Lys Thr His Ala Thr 660 665 670 Leu Phe Gly Ser Val Ala Cys Glu His Ile Ser Ser Thr Met Ser Gln 675 680 685 Tyr Ser Arg Ser Thr Arg Ser Met Leu Lys Arg Arg Asp Ser Thr Tyr 690 695 700 Gly Pro Leu Gln Thr Pro Val Gly Cys Val Leu Gly Leu Val Asn Ser 705 710 715 720 Ser Leu Phe Val Glu Asp Cys Lys Leu Pro Leu Gly Gln Ser Leu Cys 725 730 735 Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val Arg Ser 740 745 750 Val Pro Gly Glu Met Arg Leu Ala Ser Ile Ala Phe Asn His Pro Ile 755 760 765 Gln Val Asp Gln Leu Asn Ser Ser Tyr Phe Lys Leu Ser Ile Pro Thr 770 775 780 Asn Phe Ser Phe Gly Val Thr Gln Glu Tyr Ile Gln Thr Thr Ile Gln 785 790 795 800 Lys Val Thr Val Asp Cys Lys Gln Tyr Val Cys Asn Gly Phe Gln Lys 805 810 815 Cys Glu Gln Leu Leu Arg Glu Tyr Gly Gln Phe Cys Ser Lys Ile Asn 820 825 830 Gln Ala Leu His Gly Ala Asn Leu Arg Gln Asp Asp Ser Val Arg Asn 835 840 845 Leu Phe Ala Ser Val Lys Ser Ser Gln Ser Ser Pro Ile Ile Pro Gly 850 855 860 Phe Gly Gly Asp Phe Asn Leu Thr Leu Leu Glu Pro Val Ser Ile Ser 865 870 875 880 Thr Gly Ser Arg Ser Ala Arg Ser Ala Ile Glu Asp Leu Leu Phe Asp 885 890 895 Lys Val Thr Ile Ala Asp Pro Gly Tyr Met Gln Gly Tyr Asp Asp Cys 900 905 910 Met Gln Gln Gly Pro Ala Ser Ala Arg Asp Leu Ile Cys Ala Gln Tyr 915 920 925 Val Ala Gly Tyr Lys Val Leu Pro Pro Leu Met Asp Val Asn Met Glu 930 935 940 Ala Ala Tyr Thr Ser Ser Leu Leu Gly Ser Ile Ala Gly Val Gly Trp 945 950 955 960 Thr Ala Gly Leu Ser Ser Phe Ala Ala Ile Pro Phe Ala Gln Ser Ile 965 970 975 Phe Tyr Arg Leu Asn Gly Val Gly Ile Thr Gln Gln Val Leu Ser Glu 980 985 990 Asn Gln Lys Leu Ile Ala Asn Lys Phe Asn Gln Ala Leu Gly Ala Met 995 1000 1005 Gln Thr Gly Phe Thr Thr Thr Asn Glu Ala Phe His Lys Val Gln 1010 1015 1020 Asp Ala Val Asn Asn Asn Ala Gln Ala Leu Ser Lys Leu Ala Ser 1025 1030 1035 Glu Leu Ser Asn Thr Phe Gly Ala Ile Ser Ala Ser Ile Gly Asp 1040 1045 1050 Ile Ile Gln Arg Leu Asp Val Leu Glu Gln Asp Ala Gln Ile Asp 1055 1060 1065 Arg Leu Ile Asn Gly Arg Leu Thr Thr Leu Asn Ala Phe Val Ala 1070 1075 1080 Gln Gln Leu Val Arg Ser Glu Ser Ala Ala Leu Ser Ala Gln Leu 1085 1090 1095 Ala Lys Asp Lys Val Asn Glu Cys Val Lys Ala Gln Ser Lys Arg 1100 1105 1110 Ser Gly Phe Cys Gly Gln Gly Thr His Ile Val Ser Phe Val Val 1115 1120 1125 Asn Ala Pro Asn Gly Leu Tyr Phe Met His Val Gly Tyr Tyr Pro 1130 1135 1140 Ser Asn His Ile Glu Val Val Ser Ala Tyr Gly Leu Cys Asp Ala 1145 1150 1155 Ala Asn Pro Thr Asn Cys Ile Ala Pro Val Asn Gly Tyr Phe Ile 1160 1165 1170 Lys Thr Asn Asn Thr Arg Ile Val Asp Glu Trp Ser Tyr Thr Gly 1175 1180 1185 Ser Ser Phe Tyr Ala Pro Glu Pro Ile Thr Ser Leu Asn Thr Lys 1190 1195 1200 Tyr Val Ala Pro Gln Val Thr Tyr Gln Asn Ile Ser Thr Asn Leu 1205 1210 1215 Pro Pro Pro Leu Leu Gly Asn Ser Thr Gly Ile Asp Phe Gln Asp 1220 1225 1230 Glu Leu Asp Glu Phe Phe Lys Asn Val Ser Thr Ser Ile Pro Asn 1235 1240 1245 Phe Gly Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr 1250 1255 1260 Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu 1265 1270 1275 Ser Tyr Ile Asp Leu Lys Glu Leu Gly Asn Tyr Thr Tyr Tyr Asn 1280 1285 1290 Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Val 1295 1300 1305 Ala Leu Ala Leu Cys Val Phe Phe Ile Leu Cys Cys Thr Gly Cys 1310 1315 1320 Gly Thr Asn Cys Met Gly Lys Leu Lys Cys Asn Arg Cys Cys Asp 1325 1330 1335 Arg Tyr Glu Glu Tyr Asp Leu Glu Pro His Lys Val His Val His 1340 1345 1350 <210> 71<00​​​​​​​​​​​​​​​​Ile Pro Leu Gly Val Ile His Asn Ser Thr Leu Gln Val Ser Asp Val 35 40 45 Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln Leu Arg 50 55 60 Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro 65 70 75 80 Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys Val 85 90 95 Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Glu 100 105 110 Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Ala Ala Pro Asp Gly 115 120 125 Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140 Gly Pro Cys Ala Gly Asp Phe Ala Phe His Lys Glu Gly Ala Phe Phe 145 150 155 160 Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Thr Thr Phe 165 170 175 Ala Glu Gly Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys Lys Asp 180 185 190 Phe Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr Glu Asp 195 200 205 Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala Thr Gly 210 215 220 Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val Asp Asn Leu Thr 225 230 235 240 Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro Gln Phe Leu Leu Gln Leu 245 250 255 Asn Glu Thr Ile Tyr Thr Ser Gly Lys Arg Ser Asn Thr Thr Gly Lys 260 265 270 Leu Ile Trp Lys Val Asn Pro Glu Ile Asp Thr Thr Ile Gly Glu Trp 275 280 285 Ala Phe Trp Glu Thr Lys Lys Asn Leu Thr Arg Lys Ile Arg Ser Glu 290 295 300 Glu Leu Ser Phe Thr Val Val Ser Asn Gly Ala Lys Asn Ile Ser Gly 305 310 315 320 Gln Ser Pro Ala Arg Thr Ser Ser Asp Pro Gly Thr Asn Thr Thr Thr 325 330 335 Glu Asp His Lys Ile Met Ala Ser Glu Asn Ser Ser Ala Met Val Gln 340 345 350 Val His Ser Gln Gly Arg Glu Ala Ala Val Ser His Leu Thr Thr Leu 355 360 365 Ala Thr Ile Ser Thr Ser Pro Gln Ser Leu Thr Thr Lys Pro Gly Pro 370 375 380 Asp Asn Ser Thr His Asn Thr Pro Val Tyr Lys Leu Asp Ile Ser Glu 385 390 395 400 Ala Thr Gln Val Glu Gln His His Arg Arg Thr Asp Asn Asp Ser Thr 405 410 415 Ala Ser Asp Thr Pro Ser Ala Thr Thr Ala Ala Gly Pro Pro Lys Ala 420 425 430 Glu Asn Thr Asn Thr Ser Lys Ser Thr Asp Phe Leu Asp Pro Ala Thr 435 440 445 Thr Thr Ser Pro Gln Asn His Ser Glu Thr Ala Gly Asn Asn Asn Thr 450 455 460 His His Gln Asp Thr Gly Glu Glu Ser Ala Ser Ser Gly Lys Leu Gly 465 470 475 480 Leu Ile Thr Asn Thr Ile Ala Gly Val Ala Gly Leu Ile Thr Gly Gly 485 490 495 Arg Arg Thr Arg Arg Glu Ala Ile Val Asn Ala Gln Pro Lys Cys Asn 500 505 510 Pro Asn Leu His Tyr Trp Thr Thr Gln Asp Glu Gly Ala Ala Ile Gly 515 520 525 Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Ile 530 535 540 Glu Gly Leu Met His Asn Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln 545 550 555 560 Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr 565 570 575 Thr Glu Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile Asp Phe 580 585 590 Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys 595 600 605 Cys Ile Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp 610 615 620 Gln Ile Ile His Asp Phe Val Asp Lys Thr Leu Pro Asp Gln Gly Asp 625 630 635 640 Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln Trp Ile Pro Ala Gly Ile 645 650 655 Gly Val Thr Gly Val Ile Ile Ala Val Ile Ala Leu Phe Cys Ile Cys 660 665 670 Lys Phe Val Phe 675<了 <210> 72 <211> 681 <212> PRT <213> Lake Victoria Marburg virus <400> 72 Met Lys Thr Thr Cys Phe Leu Ile Ser Leu Ile Leu Ile Gln Gly Thr 1 5 10 15 Lys Asn Leu Pro Ile Leu Glu Ile Ala Ser Asn Asn Gln Pro Gln Asn )]]20 25 30 Val Asp Ser Val Cys Ser Gly Thr Leu Gln Lys Thr Glu Asp Val His 35 40 45 Leu Met Gly Phe Thr Leu Ser Gly Gln Lys Val Ala Asp Ser Pro Leu 50 55 60 Glu Ala Ser Lys Arg Trp Ala Phe Arg Thr Gly Val Pro Pro Lys Asn 65 70 75 80 Val Glu Tyr Thr Glu Gly Glu Glu Ala Lys Thr Cys Tyr Asn Ile Ser 85 90 95 Val Thr Asp Pro Ser Gly Lys Ser Leu Leu Leu Asp Pro Pro Thr Asn 100 105 110 Ile Arg Asp Tyr Pro Lys Cys Lys Thr Ile His His Ile Gln Gly Gln 115 120 125 It should be noted that there seems to be a misspelling in the tag "<了 ", which should probably be " ". This might cause issues in the correct interpretation of the text.Asn Pro His Ala Gln Gly Ile Ala Leu His Leu Trp Gly Ala Phe Phe 130 135 140 Leu Tyr Asp Arg Ile Ala Ser Thr Thr Met Tyr Arg Gly Lys Val Phe 145 150 155 160 Thr Glu Gly Asn Ile Ala Ala Met Ile Val Asn Lys Thr Val His Lys 165 170 175 Met Ile Phe Ser Arg Gln Gly Gln Gly Tyr Arg His Met Asn Leu Thr 180 185 190 Ser Thr Asn Lys Tyr Trp Thr Ser Ser Asn Gly Thr Gln Thr Asn Asp 195 200 205 Thr Gly Cys Phe Gly Ala Leu Gln Glu Tyr Asn Ser Thr Lys Asn Gln 210 215 220 Thr Cys Ala Pro Ser Lys Ile Pro Pro Pro Leu Pro Thr Ala Arg Pro 225 230 235 240 Glu Ile Lys Leu Thr Ser Thr Pro Thr Asp Ala Thr Lys Leu Asn Thr 245 250 255 Thr Asp Pro Ser Ser Asp Asp Glu Asp Leu Ala Thr Ser Gly Ser Gly 260 265 270 Ser Gly Glu Arg Glu Pro His Thr Thr Ser Asp Ala Val Thr Lys Gln 275 280 285 Gly Leu Ser Ser Thr Met Pro Pro Thr Pro Ser Pro Gln Pro Ser Thr 290 295 300 Pro Gln Gln Gly Gly Asn Asn Thr Asn His Ser Gln Asp Ala Val Thr 305 310 315 320 Glu Leu Asp Lys Asn Asn Thr Thr Ala Gln Pro Ser Met Pro Pro His 325 330 335 Asn Thr Thr Thr Ile Ser Thr Asn Asn Thr Ser Lys His Asn Phe Ser 340 345 350 Thr Leu Ser Ala Pro Leu Gln Asn Thr Thr Asn Asp Asn Thr Gln Ser 355 360 365 Thr Ile Thr Glu Asn Glu Gln Thr Ser Ala Pro Ser Ile Thr Thr Leu 370 375 380 Pro Pro Thr Gly Asn Pro Thr Thr Ala Lys Ser Thr Ser Ser Lys Lys 385 390 395 400 Gly Pro Ala Thr Thr Ala Pro Asn Thr Thr Asn Glu His Phe Thr Ser 405 410 415 Pro Pro Pro Thr Pro Ser Ser Thr Ala Gln His Leu Val Tyr Phe Arg 420 425 430 Arg Lys Arg Ser Ile Leu Trp Arg Glu Gly Asp Met Phe Pro Phe Leu 435 440 445 Asp Gly Leu Ile Asn Ala Pro Ile Asp Phe Asp Pro Val Pro Asn Thr 450 455 460 Lys Thr Ile Phe Asp Glu Ser Ser Ser Ser Gly Ala Ser Ala Glu Glu 465 470 475 480 Asp Gln His Ala Ser Pro Asn Ile Ser Leu Thr Leu Ser Tyr Phe Pro 485 490 495 Asn Ile Asn Glu Asn Thr Ala Tyr Ser Gly Glu Asn Glu Asn Asp Cys 500 505 510 Asp Ala Glu Leu Arg Ile Trp Ser Val Gln Glu Asp Asp Leu Ala Ala 515 520 525 Gly Leu Ser Trp Ile Pro Phe Phe Gly Pro Gly Ile Glu Gly Leu Tyr 530 535 540 Thr Ala Val Leu Ile Lys Asn Gln Asn Asn Leu Val Cys Arg Leu Arg 545 550 555 560 Arg Leu Ala Asn Gln Thr Ala Lys Ser Leu Glu Leu Leu Leu Arg Val 565 570 575 Thr Thr Glu Glu Arg Thr Phe Ser Leu Ile Asn Arg His Ala Ile Asp 580 585 590 Phe Leu Leu Thr Arg Trp Gly Gly Thr Cys Lys Val Leu Gly Pro Asp 595 600 605 Cys Cys Ile Gly Ile Glu Asp Leu Ser Lys Asn Ile Ser Glu Gln Ile 610 615 620 Asp Gln Ile Lys Lys Asp Glu Gln Lys Glu Gly Thr Gly Trp Gly Leu 625 630 635 640 Gly Gly Lys Trp Trp Thr Ser Asp Trp Gly Val Leu Thr Asn Leu Gly 645 650 655 Ile Leu Leu Leu Leu Ser Ile Ala Val Leu Ile Ala Leu Ser Cys Ile 660 665 670 Cys Arg Ile Phe Thr Lys Tyr Ile Gly 675 680 <210> 73 <211> 630 <212> PRT <213> Hantavirus <400> 73 Leu Arg Asn Val Tyr Asp Met Lys Ile Glu Cys Pro His Thr Val Ser 1 5 10 15 Phe Gly Glu Asn Ser Val Ile Gly Tyr Val Glu Leu Pro Pro Val Pro 20 25 30 Leu Ala Asp Thr Ala Gln Met Val Pro Glu Ser Ser Cys Asn Met Asp 35 40 45 Asn His Gln Ser Leu Asn Thr Ile Thr Lys Tyr Thr Gln Val Ser Trp 50 55 60 Arg Gly Lys Ala Asp Gln Ser Gln Ser Ser Gln Asn Ser Phe Glu Thr 65 70 75 80 Val Ser Thr Glu Val Asp Leu Lys Gly Thr Cys Val Leu Lys His Lys 85 90 95 Met Val Glu Glu Ser Tyr Arg Ser Arg Lys Ser Val Thr Cys Tyr Asp 100 105 110 Leu Ser Cys Asn Ser Thr Tyr Cys Lys Pro Thr Leu Tyr Met Ile Val 115 120 125 Pro Ile His Ala Cys Asn Met Met Lys Ser Cys Leu Ile Ala Leu Gly 130 135 140 Pro Tyr Arg Val Gln Val Val Tyr Glu Arg Ser Tyr Cys Met Thr Gly 145 150 155 160 Val Leu Ile Glu Gly Lys Cys Phe Val Pro Asp Gln Ser Val Val Ser 165 170 175 Ile Ile Lys His Gly Ile Phe Asp Ile Ala Ser Val His Ile Val Cys 180 185 190 Phe Phe Val Ala Val Lys Gly Asn Thr Tyr Lys Ile Phe Glu Gln Val 195 200 205 Lys Lys Ser Phe Glu Ser Thr Cys Asn Asp Thr Glu Asn Lys Val Gln 210 215 220 Gly Tyr Tyr Ile Cys Ile Val Gly Gly Asn Ser Ala Pro Ile Tyr Val 225 230 235 240 Pro Thr Leu Asp Asp Phe Arg Ser Met Glu Ala Phe Thr Gly Ile Phe 245 250 255 Arg Ser Pro His Gly Glu Asp His Asp Leu Ala Gly Glu Glu Ile Ala 260 265 270 Ser Tyr Ser Ile Val Gly Pro Ala Asn Ala Lys Val Pro His Ser Ala 275 280 285 Ser Ser Asp Thr Leu Ser Leu Ile Ala Tyr Ser Gly Ile Pro Ser Tyr 290 295 300 Ser Ser Leu Ser Ile Leu Thr Ser Ser Thr Glu Ala Lys His Val Phe 305 310 315 320 Ser Pro Gly Leu Phe Pro Lys Leu Asn His Thr Asn Cys Asp Lys Ser 325 330 335 Ala Ile Pro Leu Ile Trp Thr Gly Met Ile Asp Leu Pro Gly Tyr Tyr 340 345 350 Glu Ala Val His Pro Cys Thr Val Phe Cys Val Leu Ser Gly Pro Gly 355 360 365 Ala Ser Cys Glu Ala Phe Ser Glu Gly Gly Ile Phe Asn Ile Thr Ser 370 375 380 Pro Met Cys Leu Val Ser Lys Gln Asn Arg Phe Arg Leu Thr Glu Gln 385 390 395 400 Gln Val Asn Phe Val Cys Gln Arg Val Asp Met Asp Ile Val Val Tyr 405 410 415 Cys Asn Gly Gln Arg Lys Val Ile Leu Thr Lys Thr Leu Val Ile Gly 420 425 430 Gln Cys Ile Tyr Thr Ile Thr Ser Leu Phe Ser Leu Leu Pro Gly Val 435 440 445 Ala His Ser Ile Ala Val Glu Leu Cys Val Pro Gly Phe His Gly Trp 450 455 460 Ala Thr Ala Ala Leu Leu Val Thr Phe Cys Phe Gly Trp Val Leu Ile 465 470 475 480 Pro Ala Ile Thr Phe Ile Ile Leu Thr Val Leu Lys Phe Ile Ala Asn 485 490 495 Ile Phe His Thr Ser Asn Gln Glu Asn Arg Leu Lys Ser Val Leu Arg 500 505 510 Lys Ile Lys Glu Glu Phe Glu Lys Thr Lys Gly Ser Met Val Cys Asp 515 520 525 Val Cys Lys Tyr Glu Cys Glu Thr Tyr Lys Glu Leu Lys Ala His Gly 530 535 540 Val Ser Cys Pro Gln Ser Gln Cys Pro Tyr Cys Phe Thr His Cys Glu 545 550 555 560 Pro Thr Glu Ala Ala Phe Gln Ala His Tyr Lys Val Cys Gln Val Thr 565 570 575 His Arg Phe Arg Asp Asp Leu Lys Lys Thr Val Thr Pro Gln Asn Phe 580 585 590 Thr Pro Gly Cys Tyr Arg Thr Leu Asn Leu Phe Arg Tyr Lys Ser Arg 595 600 605 Cys Tyr Ile Phe Thr Met Trp Ile Phe Leu Leu Val Leu Glu Ser Ile 610 615 620 Leu Trp Ala Ala Ser Ala 625 630 <210> 74 <211> 487 <212> PRT <213> Hantavirus <400> 74 Ser Glu Thr Pro Leu Thr Pro Val Trp Asn Asp Asn Ala His Gly Val 1 5 10 15 Gly Ser Val Pro Met His Thr Asp Leu Glu Leu Asp Phe Ser Leu Thr 20 25 30 Ser Ser Ser Lys Tyr Thr Tyr Arg Arg Lys Leu Thr Asn Pro Leu Glu 35 40 45 Glu Ala Gln Ser Ile Asp Leu His Ile Glu Ile Glu Glu Gln Thr Ile 50 55 60 Gly Val Asp Val His Ala Leu Gly His Trp Phe Asp Gly Arg Leu Asn 65 70 75 80 Leu Lys Thr Ser Phe His Cys Tyr Gly Ala Cys Thr Lys Tyr Glu Tyr 85 90 95 Pro Trp His Thr Ala Lys Cys His Tyr Glu Arg Asp Tyr Gln Tyr Glu 100 105 110 Thr Ser Trp Gly Cys Asn Pro Ser Asp Cys Pro Gly Val Gly Thr Gly 115 120 125 Cys Thr Ala Cys Gly Leu Tyr Leu Asp Gln Leu Lys Pro Val Gly Ser 130 135 140 Ala Tyr Lys Ile Ile Thr Ile Arg Tyr Ser Arg Arg Val Cys Val Gln 145 150 155 160 Phe Gly Glu Glu Asn Leu Cys Lys Ile Ile Asp Met Asn Asp Cys Phe 165 170 175 Val Ser Arg His Val Lys Val Cys Ile Ile Gly Thr Val Ser Lys Phe 180 185 190 Ser Gln Gly Asp Thr Leu Leu Phe Phe Gly Pro Leu Glu Gly Gly Gly 195 200 205 Leu Ile Phe Lys His Trp Cys Thr Ser Thr Cys Gln Phe Gly Asp Pro 210 215 220 Gly Asp Ile Met Ser Pro Arg Asp Lys Gly Phe Leu Cys Pro Glu Phe 225 230 235 240 Pro Gly Ser Phe Arg Lys Lys Cys Asn Phe Ala Thr Thr Pro Ile Cys 245 250 255 Glu Tyr Asp Gly Asn Met Val Ser Gly Tyr Lys Lys Val Met Ala Thr 260 265 270 Ile Asp Ser Phe Gln Ser Phe Asn Thr Ser Thr Met His Phe Thr Asp 275 280 285 Glu Arg Ile Glu Trp Lys Asp Pro Asp Gly Met Leu Arg Asp His Ile 290 295 300 Asn Ile Leu Val Thr Lys Asp Ile Asp Phe Asp Asn Leu Gly Glu Asn 305 310 315 320 Pro Cys Lys Ile Gly Leu Gln Thr Ser Ser Ile Glu Gly Ala Trp Gly 325 330 335 Ser Gly Val Gly Phe Thr Leu Thr Cys Leu Val Ser Leu Thr Glu Cys 340 345 350 Pro Thr Phe Leu Thr Ser Ile Lys Ala Cys Asp Lys Ala Ile Cys Tyr 355 360 365 Gly Ala Glu Ser Val Thr Leu Thr Arg Gly Gln Asn Thr Val Lys Val 370 375 380 Ser Gly Lys Gly Gly His Ser Gly Ser Thr Phe Arg Cys Cys His Gly 385 390 395 400 Glu Asp Cys Ser Gln Ile Gly Leu His Ala Ala Ala Pro His Leu Asp 405 410 415 Lys Val Asn Gly Ile Ser Glu Ile Glu Asn Ser Lys Val Tyr Asp Asp 420 425 430 Gly Ala Pro Gln Cys Gly Ile Lys Cys Trp Phe Val Lys Ser Gly Glu 435 440 445 Trp Ile Ser Gly Ile Phe Ser Gly Asn Trp Ile Val Leu Ile Val Leu 450 455 460 Cys Val Phe Leu Leu Phe Ser Leu Val Leu Leu Ser Ile Leu Cys Pro 465 470 475 480 Val Arg Lys His Lys Lys Ser 485 <210> 75 <211> 226 <212> PRT <213> Hepatitis B <400> 75 Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Lys Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asn Ser Trp Trp Thr Ser Leu Ser Phe Leu Gly Gly Asn Thr Val Cys 35 40 45 Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly 100 105 110 Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Lys Thr Pro Ala 115 120 125 Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys 145 150 155 160 Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Ile 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile 195 200 205 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 <210> 76<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 85 90 95 Lys Ile Ile Gly Asp Glu Val Gly Leu Arg Thr Pro Gln Arg Phe Thr 100 105 110 Asp Leu Val Lys Phe Ile Ser Asp Lys Ile Lys Phe Leu Asn Pro Asp 115 120 125 Arg Glu Tyr Asp Phe Arg Asp Leu Thr Trp Cys Ile Asn Pro Pro Glu 130 135 140 Arg Ile Lys Leu Asp Tyr Asp Gln Tyr Cys Ala Asp Val Ala Ala Glu 145 150 155 160 Glu Leu Met Asn Ala Leu Val Asn Ser Thr Leu Leu Glu Thr Arg Thr 165 170 175 Thr Asn Gln Phe Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr 180 185 190 Thr Ile Arg Gly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu 195 200 205 Tyr Leu Gly Arg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser 210 215 220 Gln Gly Met Tyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser 225 230 235 240 Ser Lys Arg Ser Glu Leu Ser Gln Leu Ser Met Tyr Arg Val Phe Glu 245 250 255 Val Gly Val Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met 260 265 270 Thr Asn Tyr Leu Glu Gln Pro Val Ser Asn Asp Leu Ser Asn Cys Met 275 280 285 Val Ala Leu Gly Glu Leu Lys Leu Ala Ala Leu Cys His Gly Glu Asp 290 295 300 Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser Phe Gln 305 310 315 320 Leu Val Lys Leu Gly Val Trp Lys Ser Pro Thr Asp Met Gln Ser Trp 325 330 335 Val Pro Leu Ser Thr Asp Asp Pro Val Ile Asp Arg Leu Tyr Leu Ser 340 345 350 Ser His Arg Gly Val Ile Ala Asp Asn Gln Ala Lys Trp Ala Val Pro 355 360 365 Thr Thr Arg Thr Asp Asp Lys Leu Arg Met Glu Thr Cys Phe Gln Gln 370 375 380 Ala Cys Lys Gly Lys Ile Gln Ala Leu Cys Glu Asn Pro Glu Trp Ala 385 390 395 400 Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val Leu Ser Val Asp 405 410 415 Leu Ser Leu Thr Val Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly 420 425 430 Pro Leu Ile Thr His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His 435 440 445 Asn Asn Val Tyr Trp Leu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu 450 455 460 Gly Val Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro 465 470 475 480 Tyr Leu Phe Asn Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala 485 490 495 Pro Thr Tyr Leu Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser 500 505 510 Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr 515 520 525 Tyr Asp Thr Ser Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser 530 535 540 Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys 545 550 555 560 Gly Val Pro Ile Glu Leu Gln Val Glu Cys Phe Thr Trp Asp Gln Lys 565 570 575 Leu Trp Cys Arg His Phe Cys Val Leu Ala Asp Ser Glu Ser Gly Gly 580 585 590 His Ile Thr His Ser Gly Met Glu Gly Met Gly Val Ser Cys Thr Val 595 600 605 Thr Arg Glu Asp Gly Thr Asn Arg Arg 610 615 <210> 77 <211> 550 <212> PRT <213> Measles virus <400> 77 Met Gly Leu Lys Val Asn Val Ser Ala Ile Phe Met Ala Val Leu Leu 1 5 10 15 Thr Leu Gln Thr Pro Thr Gly Gln Ile His Trp Gly Asn Leu Ser Lys 20 25 30 Ile Gly Val Val Gly Ile Gly Ser Ala Ser Tyr Lys Val Met Thr Arg 35 40 45 Ser Ser His Gln Ser Leu Val Ile Lys Leu Met Pro Asn Ile Thr Leu 50 55 60 Leu Asn Asn Cys Thr Arg Val Glu Ile Ala Glu Tyr Arg Arg Leu Leu 65 70 75 80 Arg Thr Val Leu Glu Pro Ile Arg Asp Ala Leu Asn Ala Met Thr Gln 85 90 95 Asn Ile Arg Pro Val Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg 100 105 110 Phe Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 115 120 125 Ala Gln Ile Thr Ala Gly Ile Ala Leu His Gln Ser Met Leu Asn Ser 130 135 140 Gln Ala Ile Asp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gln Ala 145 150 155 160 Ile Glu Ala Ile Arg Gln Ala Gly Gln Glu Met Ile Leu Ala Val Gln 165 170 175 Gly Val Gln Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser Met Asn Gln 180 185 190 Leu Ser Cys Asp Leu Ile Gly Gln Lys Leu Gly Leu Lys Leu Leu Arg 195 200 205 Tyr Tyr Thr Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro 210 215 220 Ile Ser Ala Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly 225 230 235 240 Asp Ile Asn Lys Val Leu Glu Lys Leu Gly Tyr Ser Gly Gly Asp Leu 245 250 255 Leu Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val 260 265 270 Asp Thr Glu Ser Tyr Phe Ile Val Leu Ser Ile Ala Tyr Pro Thr Leu 275 280 285 Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Ser Tyr 290 295 300 Asn Ile Gly Ser Gln Glu Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala 305 310 315 320 Thr Gln Gly Tyr Leu Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe 325 330 335 Met Pro Glu Gly Thr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser 340 345 350 Pro Leu Leu Gln Glu Cys Leu Arg Gly Ser Thr Lys Ser Cys Ala Arg 355 360 365 Thr Leu Val Ser Gly Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly 370 375 380 Asn Leu Ile Ala Asn Cys Ala Ser Ile Leu Cys Lys Cys Tyr Thr Thr 385 390 395 400 Gly Thr Ile Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr Ile Ala 405 410 415 Ala Asp His Cys Pro Val Val Glu Val Asn Gly Val Thr Ile Gln Val 420 425 430 Gly Ser Arg Arg Tyr Pro Asp Ala Val Tyr Leu His Arg Ile Asp Leu 435 440 445 Gly Pro Pro Ile Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly 450 455 460 Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu Ser Ser 465 470 475 480 Asp Gln Ile Leu Arg Ser Met Lys Gly Leu Ser Ser Thr Ser Ile Val 485 490 495 Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly Ile Pro Ala 500 505 510 Leu Ile Cys Cys Cys Arg Gly Arg Cys Asn Lys Lys Gly Glu Gln Val 515 520 525 Gly Met Ser Arg Pro Gly Leu Lys Pro Asp Leu Thr Gly Thr Ser Lys 530 535 540 Ser Tyr Val Arg Ser Leu 545 550 <210> 78 <211> 500 <212> PRT <213> Zika virus <400> 78 Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser 1 5 10 15 Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr 20 25 30 Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr 35 40 45 Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser 50 55 60 Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala 65 70 75 80 Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu 85 90 95 Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser 100 105 110 Leu Val Thr Cys Ala Lys Phe Thr Cys Ser Lys Lys Met Thr Gly Lys 115 120 125 Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His 130 135 140 Gly Ser Gln His Ser Gly Met Ile Gly Tyr Glu Thr Asp Glu Asp Arg 145 150 155 160 Ala Lys Val Glu Val Thr Pro Asn Ser Pro Arg Ala Glu Ala Thr Leu 165 170 175 Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro Arg Thr Gly Leu 180 185 190 Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn Lys His Trp Leu 195 200 205 Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro Trp His Ala Gly 210 215 220 Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu Ala Leu Val Glu 225 230 235 240 Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val Val Val Leu Gly Ser 245 250 255 Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu 260 265 270 Met Asp Gly Ala Lys Gly Arg Leu Phe Ser Gly His Leu Lys Cys Arg 275 280 285 Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys 290 295 300 Thr Ala Ala Phe Thr Phe Thr Lys Val Pro Ala Glu Thr Leu His Gly 305 310 315 320 Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys 325 330 335 Ile Pro Val Gln Met Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly 340 345 350 Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser 355 360 365 Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val 370 375 380 Ile Gly Val Gly Asp Lys Lys Ile Thr His His Trp His Arg Ser Gly 385 390 395 400 Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg Gly Ala Lys Arg 405 410 415 Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly 420 425 430 Val Phe Asn Ser Leu Gly Lys Gly Ile His Gln Ile Phe Gly Ala Ala 435 440 445 Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser Gln Ile Leu Ile 450 455 460 Gly Thr Leu Leu Val Trp Leu Gly Leu Asn Thr Lys Asn Gly Ser Ile 465 470 475 480 Ser Leu Thr Cys Leu Ala Leu Gly Gly Val Met Ile Phe Leu Ser Thr 485 490 495 Ala Val Ser Ala 500 <210> 79 <211> 378 <212> PRT <213> Plasmodium vivax <400> 79 Met Lys Asn Phe Ile Leu Leu Ala Val Ser Ser Ile Leu Leu Val Asp 1 5 10 15 Leu Phe Pro Thr His Cys Gly His Asn Val Asp Leu Ser Lys Ala Ile 20 25 30 Asn Leu Asn Gly Val Asn Phe Asn Asn Val Asp Ala Ser Ser Leu Gly 35 40 45 Ala Ala His Val Gly Gln Ser Ala Ser Arg Gly Arg Gly Leu Gly Glu 50 55 60 Asn Pro Asp Asp Glu Glu Gly Asp Ala Lys Lys Lys Lys Asp Gly Lys 65 70 75 80 Lys Ala Glu Pro Lys Asn Pro Arg Glu Asn Lys Leu Lys Gln Pro Gly 85 90 95 Asp Arg Ala Asp Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro 100 105 110 Ala Gly Asp Arg Ala Asp Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly 115 120 125 Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro Ala Gly Asp Arg Ala 130 135 140 Asp Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro Ala Gly Asp 145 150 155 160 Arg Ala Asp Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala 165 170 175 Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln 180 185 190 Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Asp 195 200 205 Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg 210 215 220 Ala Asp Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly 225 230 235 240 Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro 245 250 255 Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asn Gly Ala Gly Gly 260 265 270 Gln Ala Ala Gly Gly Asn Ala Gly Gly Gly Gln Gly Gln Asn Asn Glu 275 280 285 Gly Ala Asn Ala Pro Asn Glu Lys Ser Val Lys Glu Tyr Leu Asp Lys 290 295 300 Val Arg Ala Thr Val Gly Thr Glu Trp Thr Pro Cys Ser Val Thr Cys 305 310 315 320 Gly Val Gly Val Arg Val Arg Arg Arg Val Asn Ala Ala Asn Lys Lys 325 330 335 Pro Glu Asp Leu Thr Leu Asn Asp Leu Glu Thr Asp Val Cys Thr Met 340 345 350 Asp Lys Cys Ala Gly Ile Phe Asn Val Val Ser Asn Ser Leu Gly Leu 355 360 365 Val Ile Leu Leu Val Leu Ala Leu Phe Asn 370 375 <210> 80 <211> 546 <212> PRT <213> Nipah virus <400> 80 Met Val Val Ile Leu Asp Lys Arg Cys Tyr Cys Asn Leu Leu Ile Leu 1 5 10 15 Ile Leu Met Ile Ser Glu Cys Ser Val Gly Ile Leu His Tyr Glu Lys 20 25 30 Leu Ser Lys Ile Gly Leu Val Lys Gly Val Thr Arg Lys Tyr Lys Ile 35 40 45 Lys Ser Asn Pro Leu Thr Lys Asp Ile Val Ile Lys Met Ile Pro Asn 50 55 60 Val Ser Asn Met Ser Gln Cys Thr Gly Ser Val Met Glu Asn Tyr Lys 65 70 75 80 Thr Arg Leu Asn Gly Ile Leu Thr Pro Ile Lys Gly Ala Leu Glu Ile 85 90 95 Tyr Lys Asn Asn Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly 100 105 110 Val Ile Met Ala Gly Val Ala Ile Gly Ile Ala Thr Ala Ala Gln Ile 115 120 125 Thr Ala Gly Val Ala Leu Tyr Glu Ala Met Lys Asn Ala Asp Asn Ile 130 135 140 Asn Lys Leu Lys Ser Ser Ile Glu Ser Thr Asn Glu Ala Val Val Lys 145 150 155 160 Leu Gln Glu Thr Ala Glu Lys Thr Val Tyr Val Leu Thr Ala Leu Gln 165 170 175 Asp Tyr Ile Asn Thr Asn Leu Val Pro Thr Ile Asp Lys Ile Ser Cys 180 185 190 Lys Gln Thr Glu Leu Ser Leu Asp Leu Ala Leu Ser Lys Tyr Leu Ser 195 200 205 Asp Leu Leu Phe Val Phe Gly Pro Asn Leu Gln Asp Pro Val Ser Asn 210 215 220 Ser Met Thr Ile Gln Ala Ile Ser Gln Ala Phe Gly Gly Asn Tyr Glu 225 230 235 240 Thr Leu Leu Arg Thr Leu Gly Tyr Ala Thr Glu Asp Phe Asp Asp Leu 245 250 255 Leu Glu Ser Asp Ser Ile Thr Gly Gln Ile Ile Tyr Val Asp Leu Ser 260 265 270 Ser Tyr Tyr Ile Ile Val Arg Val Tyr Phe Pro Ile Leu Thr Glu Ile 275 280 285 Gln Gln Ala Tyr Ile Gln Glu Leu Leu Pro Val Ser Phe Asn Asn Asp 290 295 300 Asn Ser Glu Trp Ile Ser Ile Val Pro Asn Phe Ile Leu Val Arg Asn 305 310 315 320 Thr Leu Ile Ser Asn Ile Glu Ile Gly Phe Cys Leu Ile Thr Lys Arg 325 330 335 Ser Val Ile Cys Asn Gln Asp Tyr Ala Thr Pro Met Thr Asn Asn Met 340 345 350 Arg Glu Cys Leu Thr Gly Ser Thr Glu Lys Cys Pro Arg Glu Leu Val 355 360 365 Val Ser Ser His Val Pro Arg Phe Ala Leu Ser Asn Gly Val Leu Phe 370 375 380 Ala Asn Cys Ile Ser Val Thr Cys Gln Cys Gln Thr Thr Gly Arg Ala 385 390 395 400 Ile Ser Gln Ser Gly Glu Gln Thr Leu Leu Met Ile Asp Asn Thr Thr 405 410 415 Cys Pro Thr Ala Val Leu Gly Asn Val Ile Ile Ser Leu Gly Lys Tyr 420 425 430 Leu Gly Ser Val Asn Tyr Asn Ser Glu Gly Ile Ala Ile Gly Pro Pro 435 440 445 Val Phe Thr Asp Lys Val Asp Ile Ser Ser Gln Ile Ser Ser Met Asn 450 455 460 Gln Ser Leu Gln Gln Ser Lys Asp Tyr Ile Lys Glu Ala Gln Arg Leu 465 470 475 480 Leu Asp Thr Val Asn Pro Ser Leu Ile Ser Met Leu Ser Met Ile Ile 485 490 495 Leu Tyr Val Leu Ser Ile Ala Ser Leu Cys Ile Gly Leu Ile Thr Phe 500 505 510 Ile Ser Phe Ile Ile Val Glu Lys Lys Arg Asn Thr Tyr Ser Arg Leu 515 520 525 Glu Asp Arg Arg Val Arg Pro Thr Ser Ser Gly Asp Leu Tyr Tyr Ile 530 535 540 Gly Thr 545 <210> 81 <211> 602 <212> PRT <213> Nippa virus <400> 81 Met Pro Ala Glu Asn Lys Lys Val Arg Phe Glu Asn Thr Thr Ser Asp 1 5 10 15 Lys Gly Lys Ile Pro Ser Lys Val Ile Lys Ser Tyr Tyr Gly Thr Met 20 25 30 Asp Ile Lys Lys Ile Asn Glu Gly Leu Leu Asp Ser Lys Ile Leu Ser 35 40 45 Ala Phe Asn Thr Val Ile Ala Leu Leu Gly Ser Ile Val Ile Ile Val 50 55 60 Met Asn Ile Met Ile Ile Gln Asn Tyr Thr Arg Ser Thr Asp Asn Gln 65 70 75 80 Ala Val Ile Lys Asp Ala Leu Gln Gly Ile Gln Gln Gln Ile Lys Gly 85 90 95 Leu Ala Asp Lys Ile Gly Thr Glu Ile Gly Pro Lys Val Ser Leu Ile 100 105 110 Asp Thr Ser Ser Thr Ile Thr Ile Pro Ala Asn Ile Gly Leu Leu Gly 115 120 125 Ser Lys Ile Ser Gln Ser Thr Ala Ser Ile Asn Glu Asn Val Asn Glu 130 135 140 Lys Cys Lys Phe Thr Leu Pro Pro Leu Lys Ile His Glu Cys Asn Ile 145 150 155 160 Ser Cys Pro Asn Pro Leu Pro Phe Arg Glu Tyr Arg Pro Gln Thr Glu 165 170 175 Gly Val Ser Asn Leu Val Gly Leu Pro Asn Asn Ile Cys Leu Gln Lys 180 185 190 Thr Ser Asn Gln Ile Leu Lys Pro Lys Leu Ile Ser Tyr Thr Leu Pro 195 200 205 Val Val Gly Gln Ser Gly Thr Cys Ile Thr Asp Pro Leu Leu Ala Met 210 215 220 Asp Glu Gly Tyr Phe Ala Tyr Ser His Leu Glu Arg Ile Gly Ser Cys 225 230 235 240 Ser Arg Gly Val Ser Lys Gln Arg Ile Ile Gly Val Gly Glu Val Leu 245 250 255 Asp Arg Gly Asp Glu Val Pro Ser Leu Phe Met Thr Asn Val Trp Thr 260 265 270 Pro Pro Asn Pro Asn Thr Val Tyr His Cys Ser Ala Val Tyr Asn Asn 275 280 285 Glu Phe Tyr Tyr Val Leu Cys Ala Val Ser Thr Val Gly Asp Pro Ile 290 295 300 Leu Asn Ser Thr Tyr Trp Ser Gly Ser Leu Met Met Thr Arg Leu Ala 305 310 315 320 Val Lys Pro Lys Ser Asn Gly Gly Gly Tyr Asn Gln His Gln Leu Ala 325 330 335 Leu Arg Ser Ile Glu Lys Gly Arg Tyr Asp Lys Val Met Pro Tyr Gly 340 345 350 Pro Ser Gly Ile Lys Gln Gly Asp Thr Leu Tyr Phe Pro Ala Val Gly 355 360 365 Phe Leu Val Arg Thr Glu Phe Lys Tyr Asn Asp Ser Asn Cys Pro Ile 370 375 380 Thr Lys Cys Gln Tyr Ser Lys Pro Glu Asn Cys Arg Leu Ser Met Gly 385 390 395 400 Ile Arg Pro Asn Ser His Tyr Ile Leu Arg Ser Gly Leu Leu Lys Tyr 405 410 415 Asn Leu Ser Asp Gly Glu Asn Pro Lys Val Val Phe Ile Glu Ile Ser 420 425 430 Asp Gln Arg Leu Ser Ile Gly Ser Pro Ser Lys Ile Tyr Asp Ser Leu 435 440 445 Gly Gln Pro Val Phe Tyr Gln Ala Ser Phe Ser Trp Asp Thr Met Ile 450 455 460 Lys Phe Gly Asp Val Leu Thr Val Asn Pro Leu Val Val Asn Trp Arg 465 470 475 480 Asn Asn Thr Val Ile Ser Arg Pro Gly Gln Ser Gln Cys Pro Arg Phe 485 490 495 Asn Thr Cys Pro Glu Ile Cys Trp Glu Gly Val Tyr Asn Asp Ala Phe 500 505 510 Leu Ile Asp Arg Ile Asn Trp Ile Ser Ala Gly Val Phe Leu Asp Ser 515 520 525 Asn Gln Thr Ala Glu Asn Pro Val Phe Thr Val Phe Lys Asp Asn Glu 530 535 540 Ile Leu Tyr Arg Ala Gln Leu Ala Ser Glu Asp Thr Asn Ala Gln Lys 545 550 555 560 Thr Ile Thr Asn Cys Phe Leu Leu Lys Asn Lys Ile Trp Cys Ile Ser 565 570 575 Leu Val Glu Ile Tyr Asp Thr Gly Asp Asn Val Ile Arg Pro Lys Leu 580 585 590 Phe Ala Val Lys Ile Pro Glu Gln Cys Thr 595 600 <210> 82 <211> 775 <212> PRT <213> Rotavirus A <400> 82 Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Ser Val 1 5 10 15 Asp Leu His Asp Glu Ile Glu Gln Ile Gly Ser Glu Lys Thr Gln Asn 20 25 30 Val Thr Ile Asn Pro Ser Pro Phe Ala Gln Thr Arg Tyr Ala Pro Val 35 40 45 Asn Trp Gly His Gly Glu Ile Asn Asp Ser Thr Thr Val Glu Pro Ile 50 55 60 Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Thr Pro Pro Asn Asp Tyr 65 70 75 80 Trp Ile Leu Ile Asn Ser Asn Thr Asn Gly Val Val Tyr Glu Ser Thr 85 90 95 Asn Asn Ser Asp Phe Trp Thr Ala Val Val Ala Ile Glu Pro His Val 100 105 110 Asn Pro Val Asp Arg Gln Tyr Thr Ile Phe Gly Glu Ser Lys Gln Phe 115 120 125 Asn Val Ser Asn Asp Ser Asn Lys Trp Lys Phe Leu Glu Met Phe Arg 130 135 140 Ser Ser Ser Gln Asn Glu Phe Tyr Asn Arg Arg Thr Leu Thr Ser Asp 145 150 155 160 Thr Arg Phe Val Gly Ile Leu Lys Tyr Gly Gly Arg Val Trp Thr Phe 165 170 175 His Gly Glu Thr Pro Arg Ala Thr Thr Asp Ser Ser Ser Thr Ala Asn 180 185 190 Leu Asn Asn Ile Ser Ile Thr Ile His Ser Glu Phe Tyr Ile Ile Pro 195 200 205 Arg Ser Gln Glu Ser Lys Cys Asn Glu Tyr Ile Asn Asn Gly Leu Pro 210 215 220 Pro Ile Gln Asn Thr Arg Asn Val Val Pro Leu Pro Leu Ser Ser Arg 225 230 235 240 Ser Ile Gln Tyr Lys Arg Ala Gln Val Asn Glu Asp Ile Ile Val Ser 245 250 255 Lys Thr Ser Leu Trp Lys Glu Met Gln Tyr Asn Arg Asp Ile Ile Ile 260 265 270 Arg Phe Lys Phe Gly Asn Ser Ile Val Lys Met Gly Gly Leu Gly Tyr 275 280 285 Lys Trp Ser Glu Ile Ser Tyr Lys Ala Ala Asn Tyr Gln Tyr Asn Tyr 290 295 300 Leu Arg Asp Gly Glu Gln Val Thr Ala His Thr Thr Cys Ser Val Asn 305 310 315 320 Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp Phe 325 330 335 Gly Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Tyr Val Tyr Val 340 345 350 Asp Tyr Trp Asp Asp Ser Lys Ala Phe Arg Asn Met Val Tyr Val Arg 355 360 365 Ser Leu Ala Ala Asn Leu Asn Ser Val Lys Cys Thr Gly Gly Ser Tyr 370 375 380 Asn Phe Ser Ile Pro Val Gly Ala Trp Pro Val Met Asn Gly Gly Ala 385 390 395 400 Val Ser Leu His Phe Ala Gly Val Thr Leu Ser Thr Gln Phe Thr Asp 405 410 415 Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Ser Leu Thr Val Asp 420 425 430 Glu Pro Pro Phe Ser Ile Leu Arg Thr Arg Thr Val Asn Leu Tyr Gly 435 440 445 Leu Pro Ala Ala Asn Pro Asn Asn Gly Asn Glu Tyr Tyr Glu Ile Ser 450 455 460 Gly Arg Phe Ser Leu Ile Tyr Leu Val Pro Thr Asn Asp Asp Tyr Gln 465 470 475 480 Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg Gln 485 490 495 Leu Thr Asp Leu Arg Glu Glu Phe Asn Ser Leu Ser Gln Glu Ile Ala 500 505 510 Met Ala Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe Ser 515 520 525 Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Leu Thr Lys Ser Met Ala 530 535 540 Thr Ser Val Met Lys Lys Phe Arg Lys Ser Lys Leu Ala Thr Ser Ile 545 550 555 560 Ser Glu Met Thr Asn Ser Leu Ser Asp Ala Ala Ser Ser Ala Ser Arg 565 570 575 Asn Val Ser Ile Arg Ser Asn Leu Ser Ala Ile Ser Asn Trp Thr Asn 580 585 590 Val Ser Asn Asp Val Ser Asn Val Thr Asn Ser Leu Asn Asp Ile Ser 595 600 605 Thr Gln Thr Ser Thr Ile Ser Lys Lys Phe Arg Leu Lys Glu Met Ile 610 615 620 Thr Gln Thr Glu Gly Met Ser Phe Asp Asp Ile Ser Ala Ala Val Leu 625 630 635 640 Lys Thr Lys Ile Asp Met Ser Thr Gln Ile Gly Lys Asn Thr Leu Pro 645 650 655 Asp Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Lys Arg Ser Tyr 660 665 670 Arg Ile Leu Lys Asp Asp Glu Val Met Glu Ile Asn Thr Glu Gly Lys 675 680 685 Phe Phe Ala Tyr Lys Ile Asn Thr Phe Asp Glu Val Pro Phe Asp Val 690 695 700 Asn Lys Phe Ala Glu Leu Val Thr Asp Ser Pro Val Ile Ser Ala Ile 705 710 715 720 Ile Asp Phe Lys Thr Leu Lys Asn Leu Asn Asp Asn Tyr Gly Ile Thr 725 730 735 Arg Thr Glu Ala Leu Asn Leu Ile Lys Ser Asn Pro Asn Met Leu Arg 740 745 750 Asn Phe Ile Asn Gln Asn Asn Pro Ile Ile Arg Asn Arg Ile Glu Gln 755 760 765 Leu Ile Leu Gln Cys Lys Leu 770 775 <210> 83 <211> 230 <212> PRT <213> Rotavirus A <400> 83 Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Ser Val 1 5 10 15 Asp Leu His Asp Glu Ile Glu Gln Ile Gly Ser Glu Lys Thr Gln Asn 20 25 30 Val Thr Ile Asn Pro Ser Pro Phe Ala Gln Thr Arg Tyr Ala Pro Val 35 40 45 Asn Trp Gly His Gly Glu Ile Asn Asp Ser Thr Thr Val Glu Pro Ile 50 55 60 Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Thr Pro Pro Asn Asp Tyr 65 70 75 80 Trp Ile Leu Ile Asn Ser Asn Thr Asn Gly Val Val Tyr Glu Ser Thr 85 90 95 Asn Asn Ser Asp Phe Trp Thr Ala Val Val Ala Ile Glu Pro His Val 100 105 110 Asn Pro Val Asp Arg Gln Tyr Thr Ile Phe Gly Glu Ser Lys Gln Phe 115 120 125 Asn Val Ser Asn Asp Ser Asn Lys Trp Lys Phe Leu Glu Met Phe Arg 130 135 140 Ser Ser Ser Gln Asn Glu Phe Tyr Asn Arg Arg Thr Leu Thr Ser Asp 145 150 155 160 Thr Arg Phe Val Gly Ile Leu Lys Tyr Gly Gly Arg Val Trp Thr Phe 165 170 175 His Gly Glu Thr Pro Arg Ala Thr Thr Asp Ser Ser Ser Thr Ala Asn 180 185 190 Leu Asn Asn Ile Ser Ile Thr Ile His Ser Glu Phe Tyr Ile Ile Pro 195 200 205 Arg Ser Gln Glu Ser Lys Cys Asn Glu Tyr Ile Asn Asn Gly Leu Pro 210 215 220 Pro Ile Gln Asn Thr Arg 225 230 <210> 84 <211> 539 <212> PRT <213> Human metapneumovirus <400> 84 Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ser Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile 115 120 ...

Claims

1. A nanostructure, said nanostructure comprising: A plurality of first polypeptides and a plurality of antigens, wherein the antigens are attached to the first polypeptides in sequence from the N-terminus to the C-terminus, thereby forming a conjugate of the first polypeptides and the antigens; as well as A plurality of second polypeptides, wherein the plurality of second polypeptides are attached to the plurality of first polypeptides; The amino acid sequence of the conjugate is shown in SEQ ID NO: 95, and the amino acid sequence of the second polypeptide is shown in SEQ ID NO:

34. The nanostructure has icosahedral symmetry and comprises 20 trimer subunits formed by the plurality of first polypeptides and 12 pentamer subunits formed by the plurality of second polypeptides.

2. A vaccine comprising the nanostructure of claim 1, wherein the vaccine is capable of eliciting a neutralizing antibody response against respiratory syncytial virus (RSV).

3. The vaccine of claim 2, wherein the neutralizing antibody response is protective against RSV infection.

4. The vaccine of claim 2, wherein the neutralizing antibody response is broadly neutralizing against different strains of RSV.

5. Use of the vaccine according to claim 2 in the preparation of a kit for inducing immunity against RSV in a subject.

6. The use according to claim 5, wherein the kit further comprises an adjuvant.

7. The use according to claim 5, wherein the kit further comprises a second vaccine, and wherein the second vaccine is selected from the group consisting of: protein-based vaccines, live vaccines, whole-cell vaccines, DNA vaccines, or RNA vaccines.

8. The use according to claim 7, wherein the protein-based vaccine is a nanoparticle-based vaccine.

9. The use according to claim 7, wherein the live vaccine is a live attenuated vaccine.

10. The use according to claim 7, wherein the vaccine according to claim 2 is a primary immunization vaccine, and the second vaccine is a booster vaccine.

11. The use according to claim 7, wherein the second vaccine is a primary immunization vaccine, and the vaccine according to claim 2 is a booster vaccine.

12. The use according to claim 5, wherein the kit induces directed affinity maturation.

13. The use according to claim 5, wherein the kit results in a broadly neutralizing immune response.

14. A pharmaceutical composition comprising the vaccine according to claim 2.

15. A method for preparing the nanostructure according to claim 1 by co-expression, the method comprising: The conjugates of the plurality of first polypeptides and the plurality of antigens are expressed in the first recombinant expression system. The plurality of second polypeptides were expressed in a second recombinant expression system. This results in the nanostructure, and The nanostructure was purified.

16. A method for preparing the nanostructure according to claim 1 by co-expression, the method comprising: The plurality of first polypeptides and conjugates of the plurality of antigens, as well as the plurality of second polypeptides, are expressed in a single recombinant expression system. This results in the nanostructure, and The nanostructure was purified.

17. The method of claim 16, wherein the conjugates of the plurality of first polypeptides and the plurality of antigens, as well as the plurality of second polypeptides, are encoded by a single open reading frame.

18. The method of claim 17, wherein the single open reading frame encodes a self-cleaving peptide.

Images