Antigenic ebola virus polypeptides

Abstract

The present disclosure relates to fusion proteins comprising one or more EBV polypeptides and ferritin, and their use in eliciting antibodies against EBV.

Description

Cross-reference to related applications

[0001] This application claims the benefit of European Patent Application No. 24305361.8, filed on March 8, 2024, the entire contents of which are incorporated herein by reference. CRADA Statement

[0002] This invention was created in the course of a collaborative research and development agreement with the National Institutes of Health, U.S. Department of Health and Human Services. The U.S. government holds certain rights to this invention. sequence list

[0003] This application contains a sequence list that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy created on February 26, 2025, is named “01121-0054-00PCT.xml” and has a size of 85,801 bytes. introduction

[0004] Even with numerous successes in vaccinology, new breakthroughs are still needed to protect humans from many life-threatening infectious diseases. Many currently licensed vaccines rely on technologies from a decade ago to produce attenuated live or inactivated pathogens, which have inherent safety concerns and, in many cases, only stimulate short-lived, weak immune responses requiring multiple doses. While advances in genetic and biochemical engineering have made it possible to develop therapeutics for challenging disease targets, their application in vaccinology is not yet fully realized. Recombinant protein technology now allows for the design of optimal antigens. Furthermore, nanoparticles have increasingly demonstrated potential for optimal antigen presentation and targeted drug delivery. Nanoparticles with multiple attached antigens have shown increased binding affinity through the multivalent display of their molecular cargo, as well as the ability to cross biological barriers more efficiently due to their nanoscale size. Helicobacter pylori (H. pylori) ferritin nanoparticles fused with the influenza virus hemagglutinin (HA) protein have been shown to improve antigen stability and increase immunogenicity in mouse influenza models (see Kanekiyo et al., Nature 499:102-106 (2013)). When used with adjuvants, this fusion protein self-assembles into octahedral nanoparticles exhibiting eight trimer HA spikes to deliver robust immune responses in a variety of preclinical models.

[0005] Epstein-Barr virus (EBV) infects approximately 95% of the adult population worldwide and is known to be associated with two types of B-cell lymphoma (Burkit lymphoma and Hodgkin lymphoma). The virus can also infect epithelial cells and is associated with nasopharyngeal carcinoma. Furthermore, EBV causes most cases of infectious mononucleosis in developed countries, primarily affecting children and young adults. Infectious mononucleosis can lead to a long recovery period of up to one month. Currently, there is no approved vaccine on the market, therefore a preventative vaccine is urgently needed. Summary of the Invention

[0006] The purpose of this disclosure is to provide compositions, kits, methods, and uses that can provide one or more of the advantages discussed above, or at least provide useful options to the public. Therefore, the following examples are disclosed herein.

[0007] Example 1 is a fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gH polypeptide and / or (ii) an EBV gL polypeptide; and (b) Ferritin of SEQ ID NO: 1, comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof.

[0008] Example 2 is a fusion protein as described in Example 1, wherein the EBV polypeptide further comprises the EBV gp42 polypeptide.

[0009] Example 3 is a fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gp42 polypeptide and (ii) an EBV gH polypeptide and / or an EBV gL polypeptide; and (b) Ferritin of SEQ ID NO: 1, comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof.

[0010] Example 4 is a fusion protein as described in any one of Examples 1-3, wherein the ferritin contains glutamine at residue N102 corresponding to SEQ ID NO: 1 and glutamine at residue N145 corresponding to SEQ ID NO: 1, optionally wherein the ferritin further contains glutamine at residue N18 corresponding to SEQ ID NO: 1 and serine at residue C30 corresponding to SEQ ID NO: 1.

[0011] Example 5 is a fusion protein as described in any one of Examples 1-4, wherein a bullfrog ferritin extension sequence is attached to the N-terminus of the ferritin, and wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO: 2.

[0012] Example 6 is a fusion protein as described in any one of Examples 1-5, wherein the EBV polypeptide comprises: (i) EBVgp42 polypeptide, (ii) EBV gH polypeptide, and (iii) EBV gL polypeptide.

[0013] Example 7 is a fusion protein as described in Example 6, wherein the EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide, and the ferritin are arranged in order from the N-terminus to the C-terminus.

[0014] Example 8 is a fusion protein as described in any one of Examples 1-7, which further includes a linker between each EBV peptide.

[0015] Example 9 is a fusion protein as described in Example 8, wherein the linker between the EBV gL polypeptide and the EBV gH polypeptide is 44 to 48 amino acids, and the linker between the EBV gH polypeptide and the EBV gp42 polypeptide is 32 amino acids.

[0016] Example 10 is a fusion protein as described in Example 9, wherein the linker between the EBV gL polypeptide and the EBV gH polypeptide is 46 amino acids, and the linker between the EBV gH polypeptide and the EBV gp42 polypeptide is 32 amino acids.

[0017] Example 11 is a fusion protein as described in Example 8, wherein the linker separates the EBV gL polypeptide and the EBV gH polypeptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 3; and / or wherein the linker separates the EBV gH polypeptide and the EBV gp42 polypeptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 4.

[0018] Example 12 is a fusion protein as described in any one of Examples 1-11, further comprising an additional linker separating the ferritin and the EBV gp42 polypeptide.

[0019] Example 13 is a fusion protein as described in Example 12, wherein the additional linker has a length of 88 amino acids.

[0020] Example 14 is a fusion protein as described in Example 13, wherein the additional linker consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 5.

[0021] Example 15 is a fusion protein as described in any one of Examples 1-14, wherein the ferritin contains the amino acid sequence of SEQ ID NO: 23.

[0022] Example 16 is a fusion protein as described in any one of Examples 1-15, wherein the EBV gp42 polypeptide comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with residues 856-1038 of any one of SEQ ID NO: 9-11.

[0023] Example 17 is a fusion protein as described in Example 16, wherein the EBV gH polypeptide comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with residues 162-823 of any one of SEQ ID NO: 9-11; optionally, wherein the EBV gH polypeptide comprises leucine at residue 189 corresponding to any one of SEQ ID NO: 9-11; glutamic acid at residue 368 corresponding to any one of SEQ ID NO: 9-11; and glutamic acid at residue 429 corresponding to any one of SEQ ID NO: 9-11.

[0024] Example 18 is a fusion protein as described in Example 16 or 17, wherein the EBV gL polypeptide comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with residues 1-115 of any one of SEQ ID NO: 9-11; optionally, wherein the EBV gL polypeptide comprises alanine at residue 36 corresponding to any one of SEQ ID NO: 9-11; glutamine at residue 47 corresponding to any one of SEQ ID NO: 9-11; and glutamine at residue 105 corresponding to any one of SEQ ID NO: 9-11.

[0025] Example 19 is a fusion protein as described in any one of Examples 1-18, comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the following: (a) amino acid residues 1-1133 of any one of SEQ ID NO: 9-11, (b) amino acid residues 1-1148 of any one of SEQ ID NO: 36, 38, or 40, or (c) amino acid residues 1-1156 of any one of SEQ ID NO: 37, 39, or 41.

[0026] Example 20 is a fusion protein as described in any one of Examples 1-19, optionally comprising a mammalian leader sequence located at the N-terminus of the fusion protein, optionally wherein the fusion protein comprises EBV gL polypeptide, EBV gH polypeptide, EBV gp42 polypeptide and ferritin, wherein the EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide and the ferritin are arranged in N-terminus to C-terminus within the EBV polypeptide, and the mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide.

[0027] Example 21 is a fusion protein as described in Example 20, having a mammalian leader sequence, wherein the mammalian leader sequence is selected from SEQ ID NO: 12 or SEQ ID NO: 13.

[0028] Example 22 is a fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising an EBV gL polypeptide and an EBV gH polypeptide, wherein the EBV gL polypeptide comprises the sequence of residues 1-115 of SEQ ID NO: 11, and the EBV gH polypeptide comprises the sequence of residues 162-823 of SEQ ID NO: 11. (b) A ferritin polypeptide of SEQ ID NO: 1, or a ferritin polypeptide having SEQ ID NO: 1 and at least one mutation selected from one or more of the following: glutamine at a residue corresponding to position N18 of SEQ ID NO: 1; serine at a residue corresponding to position C30 of SEQ ID NO: 1; glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; and glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; and (c) A bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO: 2, and optionally a mammalian leader sequence located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; The EBV gL polypeptide, the EBV gH polypeptide, and the ferritin are arranged sequentially from the N-terminus to the C-terminus within the EBV polypeptide, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide.

[0029] Example 23 is a fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising an EBV gL polypeptide, an EBV gH polypeptide and an EBV gp42 polypeptide, wherein the EBV gL polypeptide comprises the amino acid sequence of residues 1-115 of SEQ ID NO: 11, the EBV gH polypeptide comprises the amino acid sequence of residues 162-823 of SEQ ID NO: 11, and the EBV gp42 polypeptide comprises the amino acid sequence of residues 856-1038 of SEQ ID NO: 11; (b) Ferritin containing SEQ ID NO: 24; and (c) Optionally located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; The EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide, and the ferritin are arranged in the EBV polypeptide in the order from the N-terminus to the C-terminus, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide.

[0030] Example 24 is a fusion protein as described in Example 22 or 23, wherein the linker separates the EBV gL polypeptide and the EBV gH polypeptide, and contains the amino acid sequence of SEQ ID NO: 3.

[0031] Example 25 is a fusion protein as described in any one of Examples 23-24, wherein the linker separates the EBV gH polypeptide and the EBV gp42 polypeptide and contains the amino acid sequence of SEQ ID NO: 4.

[0032] Example 26 is a fusion protein as described in any one of Examples 23-25, wherein the linker separates the EBV gp42 polypeptide from the ferritin and contains the amino acid sequence of SEQ ID NO: 5.

[0033] Example 27 is a fusion protein as described in any one of Examples 22-26, wherein the ferritin consists of the amino acid sequence of SEQ ID NO: 24.

[0034] Example 28 is a fusion protein comprising the amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41.

[0035] Example 29 is a fusion protein consisting of an amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41.

[0036] Example 30 is a fusion protein comprising (i) an EBV gp220 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO: 14, and (ii) ferritin comprising an amino acid sequence of any one of SEQ ID NO: 23, 24, 42, and 43.

[0037] Example 31 is a fusion protein as described in Example 30, wherein the amino acid sequence of the fusion protein consists of SEQ ID NO: 25 or 35.

[0038] Example 32 is a ferritin nanoparticle containing a fusion protein as described in any one of Examples 1-29.

[0039] Example 33 is a composition or kit comprising a first ferritin nanoparticle as described in Example 32, and optionally a second ferritin nanoparticle comprising a second fusion protein containing an EBV gp220 peptide and ferritin.

[0040] Example 34 is a composition or kit as described in Example 33, wherein the EBV gp220 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO: 14.

[0041] Example 35 is a composition or kit as described in Examples 33 or 34, wherein the ferritin in the second fusion protein contains a serine or cysteine ​​residue at position 110 of SEQ ID NO: 1; optionally, wherein the ferritin contains an amino acid sequence having at least 80% identity with residues 435-600 of SEQ ID NO: 14.

[0042] Example 36 is a composition or kit as described in Example 33, wherein the second fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, optionally wherein position C544 of SEQ ID NO: 14 is substituted with serine.

[0043] Example 37 is a composition or kit comprising: (a) a first ferritin nanoparticle comprising a fusion protein consisting of an amino acid sequence of any one of SEQ ID NO: 9-11; or (b) a second ferritin nanoparticle comprising a second fusion protein consisting of an amino acid sequence of SEQ ID NO: 14; or (b) a second ferritin nanoparticle comprising a second fusion protein consisting of an amino acid sequence of SEQ ID NO: 14, wherein position C544 of SEQ ID NO: 14 is replaced with serine.

[0044] Example 38 is a composition or kit comprising a first ferritin nanoparticle and a second ferritin nanoparticle, the first ferritin nanoparticle comprising a fusion protein consisting of the amino acid sequence of SEQ ID NO: 11, and the second ferritin nanoparticle comprising a second fusion protein consisting of the amino acid sequence of SEQ ID NO: 14.

[0045] Example 39 is a pharmaceutical composition comprising a fusion protein as described in any one of Examples 1-31, or ferritin nanoparticles as described in Example 32, or a composition as described in any one of Examples 33-38; and a pharmaceutically acceptable carrier.

[0046] Example 40 is a pharmaceutical composition as described in Example 39, wherein the pharmaceutically acceptable carrier comprises an adjuvant.

[0047] Example 41 is a bivalent vaccine comprising the composition as described in any one of Examples 33-38, wherein the fusion protein and the second fusion protein are present in a 1:1 weight ratio, or wherein the fusion protein and the second fusion protein are present in a 1:1 molar ratio.

[0048] Example 42 is a fusion protein as described in any one of Examples 1-31, a composition as described in any one of Examples 33-38, ferritin particles as described in Example 32, a pharmaceutical composition as described in Example 39 or Example 40, or a bivalent vaccine as described in Example 41, for use in a method of inducing an immune response to EBV in a subject, protecting a subject from EBV infection, or preventing EBV-related diseases in a subject.

[0049] Example 43 is a method for inducing an immune response to EBV in a subject, protecting the subject from EBV infection, or preventing EBV-related diseases in a subject, the method comprising administering to the subject a fusion protein as described in any one of Examples 1-31, a composition as described in any one of Examples 33-38, ferritin particles as described in Example 32, a pharmaceutical composition as described in Example 39 or Example 40, or a bivalent vaccine as described in Example 41.

[0050] Example 44 is a nucleic acid that encodes a fusion protein as described in any one of Examples 1-31.

[0051] Example 45 is an expression vector comprising the nucleic acid as described in Example 44; and optionally further comprising the nucleic acid encoding a second fusion protein of the composition as described in any one of Examples 32-37.

[0052] Example 46 is a host cell containing the nucleic acid as described in Example 44 or the expression vector as described in Example 45.

[0053] Example 47 is a method for preparing a fusion protein as described in any one of Examples 1 to 31, the method comprising: culturing a host cell as described in Example 46 under conditions in which the fusion protein is expressed in the host cell, and recovering the fusion protein from the cultured host cell culture.

[0054] Example 48 is the method described in Example 47, wherein the host cells are Chinese hamster ovary cells.

[0055] Example 49 is the method as described in Example 47 or Example 48, wherein the host cells are dihydrofolate reductase (DHFR) deficient; and the expression vector further comprises a polynucleotide sequence encoding DHFR.

[0056] Example 50 is the method as described in Example 49, the method comprising culturing these host cells in the presence of a DHFR selector; optionally wherein the DHFR selector comprises methotrexate.

[0057] Example 51 is the method as described in any one of Examples 47-50, wherein the expression vector comprises a polynucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41.

[0058] Example 52 is the method as described in Example 51, wherein the expression vector comprises a polynucleotide sequence of any one of SEQ ID NO: 19-22.

[0059] Example 53 is a fusion protein that can be obtained or produced by the method described in any one of Examples 47-52.

[0060] Example 54 is a ferritin consisting of the amino acid sequence of SEQ ID NO: 24.

[0061] Further objects and advantages will be set forth in the description which follows, and / or will be apparent from the description, or may be learned by practice. These objects and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0062] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and not limiting of the claims.

[0063] Certain embodiments are illustrated in conjunction with the accompanying drawings, which are incorporated in and form part of this specification, and together with the description, serve to explain the principles described herein. Attached Figure Description

[0064] Figure 1A -B shows the clonal titer of EBV ferritin gp220 and the viable packaged cell density (VCD) on day 5 of batch culture. Clones were cultured at 1 x 10⁻⁶ cells / year. 6 Inoculate at a density of vc / mL in 10 mL batch cultures in a rotary tube. The batch culture medium is chemically defined (CD)CHO / 30% (v / v) Efficient Feed B, supplemented with 4 mM L-glutamine. On day 5 (or... Figure 1A The culture medium harvest (shown as six clones on day 6, as indicated by the white column) was collected and analyzed using Vi-Cell XR for viable cell density determination. Individual harvest samples were clarified by centrifugation, and the titers of the clarified harvests were determined using the Octet method (Octet Red 96). The black column shows the titers of the corresponding pre-sorting cells on day 5 of batch culture. Figure 1A The clone sets A and H are shown; Figure 1B The B and I clone sets are displayed.

[0065] Figure 2The fed-batch clone screening productivity results are shown. Fed-batch clone screening was run using an Ambr® 15-mL system (Sartorius Stedim) with in-cell basal and fed media. For each clone, cells were seeded into an Ambr reactor to achieve 1 x 102 cells in a total working volume of 14.8 mL. 6 The target seeding density is [number] viable cells / mL. Daily culture feeding begins on day 2. Starting on day 2, cultures are sampled for daily viable cell density and viability percentage measurements (Vi-Cell XR). Cultures are sampled on days 5, 7, and 12 for titer analysis; these samples are clarified by centrifugation and frozen at -80°C for storage. After the run is complete (day 12), the clarified harvest is thawed and titers are determined using the Octet method (Octet Red 96). For each clone, the mean specific productivity (up to day 7) is determined by dividing the change in titer by the change in integrated viable cell concentration (IVCC) between day 0 and day 7. Culture titers are displayed as columns (black - day 5; white - day 7; stripes - day 12), and mean specific productivity is displayed as a single point for each clone.

[0066] Figure 3A -C shows the antigenic epitope confirmed by octet analysis of the binding of C18 gL / gH / gp42 nanoparticles (SEQ ID NO: 11) to the antibody. Figure 3A -C show the use of Ammo1 antibody ( Figure 3A ), E1D1 antibody ( Figure 3B ) and 1D8 antibody ( Figure 3C Binding curves of C18 gL / gH / gp42 nanoparticles. Each antibody binds to different epitopes on the C18 antigen.

[0067] Figures 4A-4B The results showed that on days 0 and 21, 1 µg of C18 gL / gH / gp42 ferritin nanoparticles were used. Figure 4A (SEQ ID NO: 11) or gp350D123 ( Figure 4B ELISA of serum from mice immunized with ferritin nanoparticles (SEQ ID NO: 14). ELISA plates were coated with His-labeled antigen monomers. Controls were pre-collected blood samples from mice before immunization. Each group contained 5 mice.

[0068] Figure 5A -B shows the electron microscopy negative staining analysis of the nanoparticles evaluated as in Example 5. Figure 5A The gL / gH / gp42 C18 ferritin nanoparticles (SEQ ID NO: 11) are shown. Figure 5B The gp350D123 ferritin nanoparticles (SEQ ID NO: 14) are shown. Uniform particle formation with very few aggregates is visible. Magnification: 73,000x. Scale bar: 200 nm. Detailed Implementation

[0069] Fusion proteins comprising one or more EBV peptides and ferritin of the present invention are provided, which may be antigenic when administered alone, with an adjuvant as a separate molecule, and / or as part of nanoparticles (e.g., ferritin particles). Such fusion proteins and compositions comprising such fusion proteins can be used to elicit an antibody response against Epstein-Barr virus (EBV). The fusion proteins may comprise an EBV peptide (e.g., gL, gH, gL / gH, gp220, or gp42 peptides, or combinations thereof) and a ferritin polymerizing domain. In some embodiments, the ferritin comprises or consists of the amino acid of any one of SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the ferritin comprises or consists of the amino acid of SEQ ID NO: 23. In some embodiments, the ferritin comprises or consists of the amino acid of SEQ ID NO: 24. In some embodiments, the ferritin comprises or consists of the amino acid of SEQ ID NO: 42. In some embodiments, the ferritin comprises or consists of the amino acid of SEQ ID NO: 43. The nucleic acid encoding the fusion protein described herein is also provided. A. Definition

[0070] As used herein, “EBV polypeptide” refers to a polypeptide containing all or part of the amino acid sequence encoded by EBV. Similarly, gL, gH, gp42, and gp220 polypeptides refer to polypeptides containing all or part of the gL, gH, gp42, and gp220 amino acid sequences encoded by EBV, respectively. A polypeptide having, for example, at least 80% identity with an EBV-encoded polypeptide will necessarily contain a portion of the EBV-encoded polypeptide. The terms “gL polypeptide,” “gH polypeptide,” “gp42 polypeptide,” and “gp220 polypeptide” are used interchangeably with “EBV gL polypeptide,” “EBV gH polypeptide,” “EBV gp42 polypeptide,” and “EBV gp220 polypeptide,” respectively. Immunization with EBV polypeptides, which are part or all of the antigenic polypeptide, can confer protection against EBV infection. Unless the context otherwise requires, any polypeptide containing an EBV polypeptide disclosed herein may contain all or part of a plurality of sequences encoded by EBV (e.g., all or part of gL and gH of EBV, or all or part of gL, gH and gp42 of EBV).

[0071] As used herein, “monomer” or “monomer construct” refers to a construct expressed as a single-chain protein. A monomer may contain gL and gH of EBV expressed in a single chain, or gL, gH, and gp42 of EBV expressed in a single chain.

[0072] As used herein, "ferritin" or "ferritin protein" refers to a protein having a detectable sequence identity with Helicobacter pylori ferritin (e.g., SEQ ID NO: 1, 23, 24, 42, or 43). In certain embodiments, ferritin comprises or consists of the amino acid sequence of SEQ ID NO: 23 or 24. In other specific embodiments, ferritin comprises or consists of the amino acid sequence of SEQ ID NO: 42 or 43.

[0073] As used in this article, "wild-type ferritin" refers to ferritin whose sequence contains naturally occurring sequences.

[0074] As used herein, a "ferritin monomer" refers to a single ferritin molecule that is not assembled with other ferritin molecules (or, where applicable, a single ferritin heavy or light chain). A "ferritin multimer" comprises multiple associated ferritin monomers. "Ferritin" includes monomeric ferritin and polymeric ferritin.

[0075] As used herein, “ferritin particles” refers to ferritin that self-assembles into a spherical form. Ferritin particles are sometimes referred to as “ferritin nanoparticles” or simply “nanoparticles.” In some embodiments, ferritin particles contain 24 ferritin monomers (or, where applicable, a total of 24 heavy and light chains).

[0076] As used herein, “heterogeneous ferritin” refers to ferritin comprising Helicobacter pylori ferritin with an N-terminal extension of bullfrog ferritin. An exemplary sequence used as an N-terminal extension of bullfrog ferritin is shown as SEQ ID NO: 2. In heterogeneous ferritin, the N-terminal extension of bullfrog ferritin may be fused with Helicobacter pylori ferritin (see SEQ ID NO: 24). As used herein, “bullfrog ferritin extension sequence” is a polypeptide comprising the sequence of SEQ ID NO: 2. Heterogeneous ferritin is sometimes also referred to as “bfpFerr” or “bfp ferritin”. Any construct comprising the bullfrog ferritin extension sequence may be provided without that sequence. Exemplary bullfrog ferritin extension sequences are provided in Table 2. With the bullfrog ferritin extension sequences shown in Table 2, the same construct may be prepared without that sequence.

[0077] As used herein, "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of the N (asparagine) residue. This is achieved through N-glycosylation, forming an N-glycan. This glycan can be a polysaccharide.

[0078] As used in this article, "glycosylation" refers to the addition of sugar units to a protein.

[0079] As used herein, an “immune response” refers to the response of cells of the immune system (e.g., B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells) to a stimulus (e.g., an antigen or vaccine). An immune response can include any cells of the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses. As used herein, a “protective immune response” refers to an immune response that protects a subject from infection (e.g., prevents infection or the occurrence of infection-related disease). Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activity of lymphocytes (e.g., B cells or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc. An “antibody response” is an immune response that produces antibodies.

[0080] As used herein, “antigen” refers to a factor that elicits an immune response; and / or a factor that binds to T cell receptors (e.g., when presented by MHC molecules) or to antibodies (e.g., produced by B cells) when exposed to or administered to an organism. In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Alternatively or additionally, in some embodiments, an antigen elicits a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism species. In some embodiments, an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of members of the target organism species. In some embodiments, the antigen binds to antibodies and / or T-cell receptors and may or may not induce a specific physiological response in an organism. In some embodiments, for example, the antigen may bind to antibodies and / or T-cell receptors in vitro, regardless of whether such interaction occurs in vivo. In some embodiments, the antigen reacts with products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The antigen may include antigenic ferritins comprising ferritins (e.g., containing one or more mutations) and non-ferritin polypeptides as described herein.

[0081] As used herein, “adjuvant” refers to a substance or agent that enhances an immune response against an antigen. Adjuvants may include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions of antigen solutions emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Sometimes, killed mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) may also be used as adjuvants (e.g., see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants may also include biomolecules such as Toll-like receptor (TLR) agonists and co-stimulatory molecules. The adjuvant can be administered as a single molecule in the composition or covalently bound (conjugated) to ferritin or fusion proteins (e.g., antigenic ferritin peptides) disclosed herein.

[0082] As used herein, a “fusion protein” means a protein comprising two or more of the polypeptides disclosed herein (e.g., two or more of gL, gH, gp220, gp42 polypeptides, ferritin, or combinations thereof). Fusion proteins can be monomeric constructs (i.e., constructs expressed as single-chain proteins) or multimeric forms. As disclosed herein, fusion proteins may contain additional components, such as, but not limited to, one or more linkers separating two or more of the polypeptides. Fusion proteins may comprise or consist of the following: “antigenic polypeptides,” “antigenic EBV polypeptides,” or “antigenic ferritin polypeptides” as disclosed herein.

[0083] As used in this article, "antigenic EBV polypeptide" refers to a polypeptide containing all or part of an EBV amino acid sequence of sufficient length to make the molecule antigenic relative to EBV. Antigenicity can be characterized by an EBV sequence that is part of a construct that further contains a heterologous sequence, such as ferritin. That is, if the EBV sequence is part of a construct that further contains a heterologous sequence, then it is sufficient for the construct to serve as an antigen for generating anti-EBV antibodies, regardless of whether an EBV sequence without a heterologous sequence could do so.

[0084] The terms "antigenic ferritin peptide" and "antigenic ferritin" are used interchangeably herein and refer to a peptide comprising ferritin and a nonferritin peptide (e.g., an EBV peptide) of sufficient length to make the molecule antigenic relative to the nonferritin peptide. Antigenicity can be a characteristic of the nonferritin sequence as part of a larger construct. That is, it is sufficient that the construct can act as an antigen against the nonferritin peptide, regardless of whether the nonferritin peptide, which does not contain ferritin, can do so. In some embodiments, the nonferritin peptide is an EBV peptide, in which case the antigenic ferritin peptide is also an "antigenic EBV peptide".

[0085] As used herein, "antigenic polypeptide" refers to a polypeptide that is either or both of antigenic ferritin polypeptide and antigenic EBV polypeptide. In some embodiments, the ferritin in the antigenic ferritin polypeptide comprises or is composed of any one of the following amino acids: SEQ ID NO: 23, 24, 42, and 43.

[0086] As used herein, “surface-exposed” amino acids refer to amino acid residues in proteins (e.g., ferritin) that, where applicable, are accessible to solvent molecules when the protein is polymerized and in its native three-dimensional conformation. Thus, for example, in the case of ferritin forming 24-mers, surface-exposed amino acid residues are those whose side chains are accessible to solvents when ferritin assembles into 24-mers (e.g., as ferritin polymers or ferritin particles).

[0087] As used herein, “subject” means any member of the animal kingdom. In some embodiments, “subject” means a human. In some embodiments, “subject” means a non-human animal. In some embodiments, a subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, a non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In some embodiments of the invention, a subject is an adult, a adolescent, or an infant. The terms “individual” or “patient” are used and are intended to be used interchangeably with human subjects.

[0088] As used herein, the terms "vaccination" or "administration of a vaccine" refer to the administration of a composition designed to, for example, produce an immune response to a pathogenic factor. Vaccination may be administered before, during, and / or after exposure to a pathogenic factor and / or the appearance of one or more symptoms, and in some embodiments, shortly before, during, and / or after exposure to the pathogenic factor. In some embodiments, vaccination comprises multiple administrations of a vaccine composition at appropriate intervals.

[0089] This disclosure describes nucleic acid sequences and amino acid sequences that share a certain degree of identity with a given nucleic acid sequence or amino acid sequence (reference sequence).

[0090] Sequence identity between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. Sequence identity between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0091] The terms "identity %", "sameness %", or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed across the entire length of the sequences to be compared. The comparison of two sequences is typically performed by comparing the sequences with respect to segments or "comparison windows" after the best alignment to identify local regions of the respective sequences. The best alignment for comparison can be performed manually or with the aid of the local homology algorithm described in Smith and Waterman, 1981, Ads App. Math. [Advances in Applied Mathematics] 2, 482; the local homology algorithm described in Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48, 443; the similarity search algorithm described in Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88, 2444; or with the aid of computer programs using the algorithms described (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

[0092] The identity percentage is obtained by determining the number of identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0093] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (in some embodiments, consecutive nucleotides). In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0094] Nucleic acid sequences or amino acid sequences having a specific degree of identity with a given nucleic acid sequence or amino acid sequence may possess at least one functional property of the given sequence, and in some cases, are functionally equivalent to the given sequence. In some embodiments, nucleic acid sequences or amino acid sequences having a specific degree of identity with a given nucleic acid sequence or amino acid sequence are functionally equivalent to the given sequence.

[0095] As used herein, the term “kit” refers to a packaged set of related components, such as one or more compounds or compositions, and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants.

[0096] As used herein, “host cell” (or “host cell population”) means any cell or population of cells of any organism intended for the production of a recombinant protein encoded by an expression vector or for the propagation of an expression vector introduced into a host cell. “Recombinant cell” refers to a host cell containing a heterologous expression vector, which may or may not be integrated into the host cell chromosome. In some embodiments, the host cell has been transfected with an expression vector containing a polynucleotide sequence encoding the fusion protein disclosed herein and is capable of producing the fusion protein as disclosed herein. Therefore, host cells that can be used in this disclosure may include progeny of the original cell if such cells have been transformed with nucleic acids. A variety of suitable host cells (such as those disclosed herein) are useful herein.

[0097] The term "vector" or "expression vector" refers to a receptor nucleic acid molecule modified to contain or incorporate a provided nucleic acid sequence (e.g., a nucleic acid sequence encoding the fusion protein disclosed herein). One exemplary type of vector used herein is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another exemplary type of vector used herein is a viral vector (e.g., an adeno-associated virus vector), into which additional DNA segments can be ligated into a viral genome.

[0098] As used herein, when nucleic acids have been introduced into cells, the cells (e.g., a population of host cells) have been “transfected” with such nucleic acids, for example, with an expression vector containing a polynucleotide sequence encoding the fusion protein disclosed herein. Cells can be transfected with one or more (e.g., one, two, three, or four) expression vectors or other nucleic acids by any method known in the art (including, but not limited to, electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex), for example. As used herein, the term “transfection” encompasses any means of introducing one or more (e.g., one, two, three, or four) nucleic acids into the cell. B. Fusion protein containing EBV peptide and ferritin

[0099] In some embodiments, a fusion protein is provided comprising an EBV peptide and ferritin, optionally wherein the ferritin comprises or is composed of the sequence of any one of SEQ ID NO: 23, 24, 42, and 43, and optionally includes a bullfrog ferritin extension sequence (e.g., the bullfrog ferritin extension sequence of SEQ ID NO: 2) at the N-terminus of the ferritin. The EBV peptide may be any of the EBV peptides described herein, such as gL, gH, gL / gH, gL / gH / gp42, gp220, gp42 peptides, or combinations thereof. In such embodiments, the fusion protein may be a monomeric construct (i.e., a construct expressed as a single-chain protein). The monomeric construct may comprise any one or more of the peptides disclosed herein (e.g., gL, gH, gL / gH, gL / gH / gp42, gp220, gp42 peptides) and the ferritin of the present invention expressed in a single chain. As disclosed elsewhere herein, fusion proteins may contain additional components, such as, but not limited to, one or more linkers that separate two or more of the polypeptides disclosed herein. In some embodiments, the fusion proteins disclosed herein comprise or consist of: an antigenic polypeptide, an antigenic EBV polypeptide, and / or an antigenic ferritin polypeptide.

[0100] In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 1-1133 of any one of SEQ ID NO: 9-11, and the ferritin of the present invention. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 9-11, 14, 25, or 35-41, wherein the ferritin comprises or is composed of amino acids of any one of SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, wherein the ferritin comprises or consists of the amino acids of SEQ ID NO: 23 or 24. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10, wherein the ferritin comprises or consists of the amino acids of any one of SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, wherein the ferritin comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 25, wherein the ferritin comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36, and the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43.In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37, and the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 38, and the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 39, and the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 40, and the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 41, and the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14 or 35, wherein the ferritin comprises or consists of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises an amino acid sequence in any one of SEQ ID NO: 9-11, 25, or 35.In certain embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14 or 35, optionally wherein the ferritin is the ferritin of SEQ ID NO: 14 (where residue C544 is substituted with serine) or the ferritin of SEQ ID NO: 35 (where residue C574 is substituted with serine). Further fusion proteins and their components are disclosed in International Application PCT / US2019 / 025419, which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the ferritin is Helicobacter pylori ferritin (see SEQ ID NO: 1, 23, 24, 42 or 43 for exemplary Helicobacter pylori ferritin sequences).

[0102] In some embodiments, ferritin nanoparticles are provided comprising a fusion protein as disclosed herein, the fusion protein comprising an EBV peptide and ferritin, wherein the ferritin comprises or consists of amino acids of SEQ ID NO: 23 or 24. In some embodiments, ferritin nanoparticles are provided comprising a fusion protein as disclosed herein, the fusion protein comprising an EBV peptide and ferritin, wherein the ferritin comprises or consists of amino acids of SEQ ID NO: 42 or 43.

[0103] In some embodiments, a bullfrog ferritin extension sequence is attached to the N-terminus of ferritin. In some embodiments, the bullfrog ferritin extension sequence attached to the N-terminus of ferritin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2. In some embodiments, the bullfrog ferritin extension sequence attached to the N-terminus of ferritin comprises the amino acid sequence of SEQ ID NO: 2.

[0104] In a particular embodiment, the fusion protein comprises an EBV polypeptide comprising: (i) an EBV gH polypeptide and / or (ii) an EBV gL polypeptide; and ferritin comprising the amino acid sequence of SEQ ID NO: 1; and further comprising at least one of: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; and / or (iii) a bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the fusion protein comprises (a) an Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gH polypeptide and / or (ii) an EBV gL polypeptide; and (b) ferritin of SEQ ID NO: 1, the ferritin comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof. In some such embodiments, the fusion protein further comprises an EBV gp42 polypeptide. In a particular embodiment, the fusion protein comprises (a) an Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gp42 polypeptide and (ii) an EBV gH polypeptide and / or an EBV gL polypeptide; and (b) ferritin of SEQ ID NO: 1, the ferritin comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; and (iii) a bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO: 2.

[0105] In some embodiments, ferritin comprises glutamine at a residue at position N102 of SEQ ID NO: 1 and glutamine at a residue at position N145 of SEQ ID NO: 1, optionally wherein ferritin further comprises glutamine at a residue at position N18 of SEQ ID NO: 1 and serine at a residue at position C30 of SEQ ID NO: 1.

[0106] In other specific embodiments, ferritin contains glutamine at the residue corresponding to position N102 of SEQ ID NO: 1, glutamine at the residue corresponding to position N145 of SEQ ID NO: 1, and glutamine at the residue corresponding to position N18 of SEQ ID NO: 1.

[0107] In some embodiments, the fusion protein comprises (i) an EBV gp42 polypeptide, (ii) an EBV gH polypeptide, and (iii) an EBV gL polypeptide, optionally wherein the EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide, and ferritin are arranged in an N-terminal to C-terminal sequence within the fusion protein. In some such embodiments, the fusion protein comprises one or more or each of linkers between the EBV gL polypeptide and the EBV gH polypeptide, between the EBV gH polypeptide and the EBV gp42 polypeptide, and between the EBV gp42 polypeptide and ferritin. In some embodiments, the linkers have a length of at least 15 amino acids, as disclosed elsewhere herein. In such embodiments, each linker may be independently selected from any linker disclosed herein, such as a linker having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID Nos: 3-8 or 26-34. In a particular embodiment, the linker between the EBV gL peptide and the EBV gH peptide is 44 to 48 amino acids long, and the linker between the EBV gH peptide and the EBV gp42 peptide is 32 amino acids long. In another particular embodiment, the linker between the EBV gL peptide and the EBV gH peptide is 46 amino acids long, and the linker between the EBV gH peptide and the EBV gp42 peptide is 32 amino acids long. In some embodiments, the fusion protein lacks one or more of the linkers (e.g., one or more of the linkers between the EBV gL peptide and the EBV gH peptide, between the EBV gH peptide and the EBV gp42 peptide, and / or between the EBV gp42 peptide and ferritin). In a particular embodiment, the linker separates the EBV gL peptide and the EBV gH peptide and contains an amino acid sequence having at least 80% identity with SEQ ID NO: 3; and / or the linker separates the EBV gH peptide and the EBV gp42 peptide and contains an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In certain embodiments, the linker separates the EBV gL peptide and the EBV gH peptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 3; and / or the linker separates the EBV gH peptide and the EBV gp42 peptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In some embodiments, the linker separates the EBV gp42 peptide and ferritin and contains an amino acid sequence having at least 80% identity with SEQ ID NO: 5. In some embodiments, the linker separating the EBV gp42 peptide and ferritin contains the amino acid sequence of SEQ ID NO: 5.In some embodiments, the linker separating the EBV gp42 peptide and ferritin consists of the amino acid sequence of SEQ ID NO: 5.

[0108] In a particular embodiment, the fusion protein comprises the amino acid sequence of any one of SEQ ID Nos: 9-11, 14, 25, or 35-41. In other particular embodiments, the fusion protein consists of the amino acid sequence of any one of SEQ ID Nos: 9-11, 14, 25, or 35-41.

[0109] In some embodiments, the fusion protein comprises a mammalian leader sequence located at the N-terminus of the fusion protein, optionally wherein the fusion protein comprises the EBV gL peptide, EBV gH peptide, EBV gp42 peptide, and ferritin of the present invention, wherein the EBV gL peptide, the EBV gH peptide, the EBV gp42 peptide, and the ferritin are arranged in N-terminus to C-terminus within the EBV peptide, and the mammalian leader sequence is located at the N-terminus of the EBV gL peptide. Any of the disclosed constructs comprising a mammalian leader sequence may be provided without the sequence. Exemplary mammalian leader sequences are provided in Table 2. With the mammalian leader sequences shown in Table 2, the same constructs may be prepared without the sequence.

[0110] In some embodiments, the composition comprises (a) a first fusion protein comprising the EBV gL polypeptide, EBV gH polypeptide, and / or EBV gp42 polypeptide of the present invention and ferritin, and (b) a second fusion protein comprising the EBV gp220 polypeptide and optionally metronidin. In some embodiments, the first fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 1-1133 of any one of SEQ ID NO: 9-11 and the ferritin of the present invention. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 9-11 or 36-41, wherein the ferritin comprises or is composed of amino acids of any one of SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the EBVgp220 polypeptide of the second fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO: 14, 25, or 35, and optionally metronidazole. In some embodiments, the gp220 polypeptide further comprises a mammalian leader sequence linked to the N-terminus of the gp220 polypeptide, optionally wherein the leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. In a particular embodiment, the second fusion protein further comprises ferritin. In a particular embodiment, ferritin comprises a serine or cysteine ​​residue corresponding to position 110 of SEQ ID NO: 1; optionally wherein ferritin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 435-600 of SEQ ID NO: 14. In certain embodiments, the second fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, 25, or 35. In some embodiments, the ferritin in the second fusion protein comprises a serine or cysteine ​​residue corresponding to position 110 of SEQ ID NO: 1. In some such embodiments, the ferritin comprises an amino acid sequence having at least 80% identity with residues 435-600 of SEQ ID NO: 14.In a particular embodiment, the composition comprises a first fusion protein consisting of the amino acid sequence of SEQ ID NO: 11 and a second fusion protein consisting of the amino acid sequence of SEQ ID NO: 14.

[0111] In some embodiments containing a second fusion protein, the first fusion protein and the second fusion protein are present in a weight ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or wherein the first fusion protein and the second fusion protein are present in a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0112] In a particular embodiment, the fusion protein comprises an EBV polypeptide comprising an EBV gL polypeptide and an EBV gH polypeptide, wherein the EBV gL polypeptide comprises the sequence of residues 1-115 of SEQ ID NO: 11, and the EBV gH polypeptide comprises the sequence of residues 162-823 of SEQ ID NO: 11; a ferritin polypeptide of SEQ ID NO: 1, or a ferritin polypeptide having SEQ ID NO: 1 and at least one mutation, the at least one mutation being selected from any one or more of the following: glutamine at a residue corresponding to position N18 of SEQ ID NO: 1; serine at a residue corresponding to position C30 of SEQ ID NO: 1; glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; and optionally a bullfrog ferritin extension sequence linked to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the sequence of SEQ ID NO: The fusion protein contains the amino acid sequence of SEQ ID NO: 2, and optionally a mammalian leader sequence located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or 13. In such embodiments, the EBV gL peptide, EBV gH peptide, and ferritin are arranged in N-terminal to C-terminal sequence within the fusion protein, and optionally the mammalian leader sequence is located at the N-terminus of the EBV gL peptide. In some such embodiments, a linker separates the EBV gL peptide and the EBV gH peptide and comprises the amino acid sequence of SEQ ID NO: 3. Some such embodiments further comprise a second fusion protein comprising the EBV gp220 peptide and ferritin, and the second fusion protein comprises the amino acid sequence of SEQ ID NO: 14 or 35, optionally wherein the ferritin comprises or consists of the amino acid sequence of any one of SEQ ID NO: 23, 24, 42, and 43. In certain embodiments, the second fusion protein comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 35. In some embodiments where the second fusion protein comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14 or 35, the second fusion protein further comprises a serine residue at residue C544 of SEQ ID NO: 14 or at residue C574 of SEQ ID NO: 35.

[0113] In other specific embodiments, the fusion protein comprises an EBV polypeptide comprising an EBV gL polypeptide, an EBV gH polypeptide, and an EBV gp42 polypeptide, wherein the EBV gL polypeptide comprises amino acid sequences of residues 1-115 of SEQ ID NO: 11, the EBV gH polypeptide comprises amino acid sequences of residues 162-823 of SEQ ID NO: 11, and the EBV gp42 polypeptide comprises amino acid sequences of residues 856-1038 of SEQ ID NO: 11; ferritin comprising SEQ ID NO: 24; and optionally a mammalian leader sequence located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises amino acid sequences of SEQ ID NO: 12 or 13. In such embodiments, the EBV gL polypeptide, EBV gH polypeptide, EBV gp42 polypeptide, and ferritin are arranged in N-terminus to C-terminus within the fusion protein, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide. In some such embodiments, the linker separates the EBV gL peptide and the EBV gH peptide and contains the amino acid sequence of SEQ ID NO: 3. In some such embodiments, the linker separates the EBV gH peptide and the EBV gp42 peptide and contains the amino acid sequence of SEQ ID NO: 4. Some such embodiments further contain a second fusion protein comprising the EBV gp220 peptide and ferritin, and the second fusion protein contains the amino acid sequence of SEQ ID NO: 14, 25, or 35, optionally wherein the ferritin comprises or consists of the amino acid sequence of any one of SEQ ID NO: 23, 24, 42, and 43. 1. Ferritin mutation

[0114] This disclosure provides the Helicobacter pylori ferritin sequence of SEQ ID NO: 1, comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) combinations thereof. In some embodiments, the ferritin of this disclosure comprises a mammalian leader sequence attached to the N-terminus of the ferritin. In some such embodiments, the ferritin of this disclosure comprises a bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, the bullfrog ferritin extension sequence optionally comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ferritin comprises glutamine at a residue corresponding to position N102 of SEQ ID NO: 1 and glutamine at a residue corresponding to position N145 of SEQ ID NO: 1. In some such embodiments, the ferritin further comprises glutamine at a residue corresponding to position N18 of SEQ ID NO: 1 and serine at a residue corresponding to position C30 of SEQ ID NO: 1. In some embodiments, ferritin comprises or is composed of any one of the following amino acids: SEQ ID NO: 23, 23, 42 or 43. 2. Removal of internal cysteine

[0115] In some embodiments, the ferritin comprises a mutation in which an internal cysteine ​​residue is replaced with a non-cysteine ​​amino acid. Removal of the native internal cysteine ​​residue ensures that only one unpaired cysteine ​​residue is present in each ferritin monomer and avoids undesirable reactions such as disulfide formation. In some embodiments, C30 of the Helicobacter pylori ferritin of SEQ ID NO: 1 is replaced with a non-cysteine ​​amino acid. In some embodiments, C30 of the Helicobacter pylori ferritin of SEQ ID NO: 1 is replaced with serine (C30S), although any non-cysteine ​​residue, such as alanine, glycine, threonine, or asparagine, can be used. Similar amino acids can be found in non-Helicobacter pylori ferritin by pairwise or structural alignment. Therefore, in some embodiments, the internal cysteine ​​replaced with a non-cysteine ​​is the amino acid residue that aligns to C30 of the Helicobacter pylori ferritin of SEQ ID NO: 1. An exemplary ferritin sequence showing the C30S mutation is shown in SEQ ID NO: 23. In some embodiments, when there is more than one internal cysteine ​​in the ferritin, two or more (e.g., each) internal cysteines are replaced by non-cysteine ​​amino acids (such as serine or amino acids selected from serine, alanine, glycine, threonine or asparagine). 3. Glycosylation

[0116] Human-compatible glycosylation can contribute to the safety and efficacy of recombinant pharmaceutical products. Regulatory approval may depend on demonstrating appropriate glycosylation as a critical quality attribute (see Zhang et al., Drug Discovery Today 21(5):740-765 (2016)). N-glycans can be generated by glycosylation of asparagine side chains and can differ structurally between humans and other organisms, such as bacteria and yeast. Therefore, it may be desirable to reduce or eliminate non-human glycosylation and / or N-glycan formation in ferritin according to this disclosure. In some embodiments, controlling the glycosylation of ferritin improves the efficacy and / or safety of the composition, particularly when used for human vaccination.

[0117] In some embodiments, ferritin is mutated to suppress the formation of N-glycans. In some embodiments, the mutated ferritin has reduced glycosylation compared to its corresponding wild-type ferritin.

[0118] In some embodiments, ferritin comprises a mutation replacing surface-exposed asparagine with a non-asparagine amino acid. In some embodiments, the surface-exposed asparagine is N18 of the Helicobacter pylori ferritin of SEQ ID NO: 1 or a position corresponding to position 18 of the Helicobacter pylori ferritin of SEQ ID NO: 1, as determined by pairwise or structural alignment. In some embodiments, mutating such asparagine (e.g., N18 of the Helicobacter pylori ferritin) reduces the glycosylation of ferritin. In some embodiments, the mutation replaces asparagine with glutamine. In some embodiments, ferritin is Helicobacter pylori ferritin containing the N18Q mutation. Thus, in some embodiments, ferritin comprises glutamine at a residue corresponding to position 18 of SEQ ID NO: 1.

[0119] Mammals exposed to glycosylated proteins produced in bacteria or yeast can generate an immune response to these proteins because the glycosylation pattern of a given protein in bacteria or yeast may differ from that of the same protein in mammals. Therefore, some therapeutic glycosylated proteins may not be suitable for production in bacteria or yeast.

[0120] In some embodiments, reduced ferritin glycosylation through amino acid mutations promotes protein production in bacteria or yeast. In some embodiments, reduced ferritin glycosylation decreases the likelihood of adverse reactions in mammals following administration of mutant ferritin expressed in bacteria or yeast. In some embodiments, mutant ferritin produced in bacteria or yeast exhibits lower reactivity in human subjects due to reduced glycosylation. In some embodiments, the incidence of hypersensitivity reactions in human subjects is lower after treatment with mutant ferritin with reduced glycosylation compared to wild-type ferritin.

[0121] In some embodiments, compositions containing mutant ferritin with reduced glycosylation degrade more slowly in subjects compared to compositions containing wild-type ferritin or compositions containing corresponding ferritin with wild-type glycosylation. In some embodiments, compositions containing mutant ferritin with reduced glycosylation have a lower clearance rate in subjects compared to compositions containing wild-type ferritin or compositions containing corresponding ferritin with wild-type glycosylation. In some embodiments, compositions containing mutant ferritin with reduced glycosylation have a longer serum half-life compared to compositions containing wild-type ferritin or compositions containing corresponding ferritin with wild-type glycosylation. 4. Mutant Combinations

[0122] In some embodiments, ferritin comprises more than one type of mutation described herein. In some embodiments, ferritin comprises one or more mutations independently selected from: mutations that reduce glycosylation, mutations that remove internal cysteine ​​residues, and mutations that generate surface-exposed cysteine ​​residues. In some embodiments, ferritin comprises mutations that reduce glycosylation, mutations that remove internal cysteine ​​residues, and mutations that generate surface-exposed cysteine ​​residues. In a particular embodiment, ferritin comprises serine at position 110 corresponding to the amino acid of SEQ ID NO: 1. In some embodiments, ferritin comprises or consists of the amino acid of SEQ ID NO: 23. In some embodiments, ferritin comprises or consists of the amino acid of SEQ ID NO: 24. In some embodiments, ferritin comprises or consists of the amino acid of SEQ ID NO: 42. In some embodiments, ferritin comprises or consists of the amino acid of SEQ ID NO: 43.

[0123] Unless otherwise stated, the location of ferritin mutations in this paragraph refers to the location corresponding to the location in SEQ ID NO: 1. A location “corresponds” to a given location if the location in the amino acid sequence is aligned to a given location in SEQ ID NO: 1 using a standard sequence alignment algorithm (e.g., the Smith-Waltman algorithm with default parameters). In some embodiments, ferritin contains N18Q and C30S mutations. In some embodiments, ferritin contains N18Q, C30S, and E11C mutations. In some embodiments, ferritin contains N18Q, C30S, and S71C mutations. In some embodiments, ferritin contains N18Q, C30S, and A74C mutations. In some embodiments, ferritin contains N18Q, C30S, and K79C mutations. In some embodiments, ferritin contains N18Q, C30S, and S99C mutations. In some embodiments, ferritin contains N18Q, C30S, and S110C mutations. In some embodiments, ferritin does not contain the S110C mutation. In some embodiments, ferritin comprises N18Q mutation, C30S mutation, N102Q mutation, and N145Q mutation. In some embodiments, ferritin comprises a mutation corresponding to any of the aforementioned mutation sets, wherein the corresponding mutation changes N to Q or C to S at a position determined by comparison of the ferritin amino acid sequence with the Helicobacter pylori ferritin amino acid sequence (SEQ ID NO: 1, 23, 24, 42, or 43). Exemplary ferritins comprising more than one type of mutation (e.g., N18Q mutation, C30S mutation, N102Q mutation, and N145Q mutation) are provided in SEQ ID NO: 23, 24, 42, and 43.

[0124] In some specific embodiments, ferritin comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some specific embodiments, ferritin comprises or consists of the amino acid sequence of residues 1134-1299 of any one of SEQ ID Nos: 9-11. In some embodiments, ferritin comprises glutamine at residue 1151 corresponding to position 1151 of SEQ ID Nos: 9-11; serine at residue 1163 corresponding to position 1163 of any one of SEQ ID Nos: 9-11; glutamine at residue 1235 corresponding to position 1235 of any one of SEQ ID Nos: 9-11; and glutamine at residue 1278 corresponding to position 1278 of any one of SEQ ID Nos: 9-11. C. A fusion protein containing gL and gH peptides and ferritin.

[0125] EBV possesses three glycoproteins (glycoprotein B (gB), gH, and gL), which form a core membrane fusion mechanism to allow viral penetration into cells. gL and gH have been previously described, for example, in Matsuura et al., Proc Natl AcadSci US A. [Proceedings of the National Academy of Sciences] 2010 Dec 28; 107(52):22641-6. Monomers and trimers of gL and gH used as vaccines have been described, for example, in Cui et al., Vaccine. [Vaccines] 2016 Jul 25; 34(34):4050-5. gH and gL proteins associate to form a heterodimeric complex, which is considered essential for efficient membrane fusion and binding to epithelial cell receptors (required for viral entry).

[0126] The fusion protein disclosed herein may comprise EBV gL and EBV gH, as well as ferritin disclosed herein (e.g., ferritin of any one of SEQ ID NO: 23, 24, 42, and 43). In some embodiments, the fusion protein is formed into nanoparticles (e.g., ferritin particles) through polymerization of ferritin, for example. In some embodiments, the fusion protein according to this disclosure comprises an EBV gL peptide and an EBV gH peptide, and a linker having a length of at least 15 amino acids that separates the EBV gL peptide and the EBV gH peptide. It has been found that relatively long linkers can provide benefits, such as improved expression and / or immunogenicity. In some embodiments, the linker separates the EBV gL peptide and the EBV gH peptide and has a length of at least 15 amino acids (e.g., 15-100 amino acids, such as 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-20 amino acids). Such linkers can have lengths of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, 44 to 48 amino acids, 44 amino acids, 46 amino acids, 48 ​​amino acids, or 40 to 50 amino acids. In some embodiments, the linker separating the EBV gL peptide and the EBV gH peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID No: 3-8 and 29-30. In a particular embodiment, the linker separating the EBV gL peptide and the EBV gH peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3.

[0127] In some embodiments, the EBV gH and / or gL peptides are full-length gH and / or gL (see GenBank accessions CEQ35765.1 and YP_001129472.1 for exemplary full-length sequences, respectively). In some embodiments, the EBV gH and / or gL peptides are fragments of gH and / or gL. In some embodiments, the gL peptide is a gL(D7) construct with a 7-amino acid deletion at the C-terminus of gL. In some embodiments, the gH peptide contains a mutation at C137, such as the C137A mutation. In some embodiments, the C137 mutation removes a native, unpaired cysteine ​​residue to avoid nonspecific conjugation. In some embodiments, the gH peptide contains a mutation that removes a cysteine ​​residue corresponding to cysteine ​​137 of SEQ ID NO: 17, such as the C137A mutation. In some embodiments, the C137 mutation removes a native, unpaired cysteine ​​residue to avoid nonspecific conjugation.

[0128] In some embodiments, the EBV gL polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 16. In some embodiments, the EBV gH polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 17. In some embodiments, the EBV gL polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 1-115 of any one of SEQ ID NO: 9-11. In some embodiments, the EBV gH polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 162-823 of any one of SEQ ID NO: 9-11.

[0129] In some embodiments, the fusion protein may contain the gL polypeptide, gH polypeptide, and ferritin of the present invention in an N-terminal to C-terminal sequence. In other words, in some embodiments, the EBV gL polypeptide, EBV gH polypeptide, and ferritin of the present invention are arranged as single chains in an N-terminal to C-terminal sequence within the fusion protein. In some embodiments, the ferritin comprises or is composed of the amino acids of SEQ ID NO: 23 or 24. In some embodiments, the ferritin comprises or is composed of the amino acids of SEQ ID NO: 42 or 43.

[0130] In some embodiments, a mammalian leader sequence (also referred to as a signal sequence) is appended to the N-terminus of an EBV polypeptide, such as a gH or gL polypeptide, for example, at the N-terminus of the EBV polypeptide (e.g., at the N-terminus of a fusion protein containing the EBV polypeptide). In some embodiments, when expressed in mammalian cells, the mammalian leader sequence results in the secretion of the disclosed fusion protein. In some embodiments, the mammalian leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 12 or 13.

[0131] The natural EBV gH and / or gL sequences are shown in GenBank accession number NC_009334.1 (human herpesvirus 4, complete genome, dated March 26, 2010). For some constructs disclosed herein, amino acids 23-137 of the gL amino acid sequence in NC_009334.1 are used as the gL peptide, and the natural signal peptide (amino acids 1-22 of the NCBI sequence) is replaced with the IgGκ leader sequence. For some constructs, amino acids 19-678 of the gH amino acid sequence in NC_009334.1 are used as the gH peptide. In some embodiments, gL and gH are linked via a linker as shown in the sequence listing herein. In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID Nos: 3-8. In a particular embodiment, the connector separates the EBV gL peptide and the EBV gH peptide and has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3.

[0132] In some embodiments, the EBV gH polypeptide comprises an amino acid sequence having at least 80% identity with residues 162-823 of any one of SEQ ID Nos: 9-11; optionally, the EBV gH polypeptide comprises leucine at residue 189 corresponding to any one of SEQ ID Nos: 9-11; glutamic acid at residue 368 corresponding to any one of SEQ ID Nos: 9-11; and glutamic acid at residue 429 corresponding to any one of SEQ ID Nos: 9-11. In some embodiments, the EBV gL polypeptide comprises an amino acid sequence having at least 80% identity with residues 1-115 of any one of SEQ ID Nos: 9-11; optionally, the EBV gL polypeptide comprises alanine at residue 36 corresponding to any one of SEQ ID Nos: 9-11; glutamine at residue 47 corresponding to any one of SEQ ID Nos: 9-11; and glutamine at residue 105 corresponding to any one of SEQ ID Nos: 9-11.

[0133] In some embodiments, the gL and gH peptides are expressed as single-chain monomers. In some embodiments, the monomer composition comprises or consists of the sequence shown in the sequence listing and referred to as "monomer" in the specification. A single chain containing gL and gH peptides may be referred to as "gL / gH," which may be used interchangeably with "gH_gL," "gL_gH," or "gL / gH."

[0134] The gL / gH polypeptide can be combined with any of the ferritins discussed herein. For example, in some embodiments, the fusion protein comprises the monomeric or trimeric gL / gH polypeptide (+ / - gp42 and / or gp220) of the present invention and ferritin, optionally wherein the ferritin comprises or is composed of any one of the amino acids of SEQ ID NO: 23, 24, 42 and 43.

[0135] Additionally, in some embodiments, any fusion protein comprising the EBV gL / gH peptide of the present invention and ferritin may be present in a composition comprising another peptide disclosed herein. D. A fusion protein containing gp220 polypeptide and ferritin.

[0136] In some embodiments, the fusion protein disclosed herein may comprise the gp220 peptide and the ferritin disclosed herein (e.g., ferritin of residues 435-600 of SEQ ID NO: 23, 24, 42, and 43 or SEQ ID NO: 14). gp220-hybrid bullfrog / Helicobacter pylori ferritin nanoparticles have previously been described in Kanekiyo Cell. [Cell] 2015 Aug 27; 162(5):1090-100.

[0137] In some embodiments, the gp220 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 18. In some embodiments, the EBV gp220 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO: 14.

[0138] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is appended to the N-terminus of the gp220 polypeptide. In some embodiments, when expressed in mammalian cells, the mammalian leader sequence leads to the secretion of the protein. In some embodiments, the mammalian leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 12 or 13.

[0139] The gp220 polypeptide can be combined with any of the ferritins discussed herein. For example, in some embodiments, the composition comprises a single-chain fusion protein containing the gp220 polypeptide and optionally ferritin, and a single-chain fusion protein containing gL / gH and / or gp42 and ferritin, wherein the ferritin comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the fusion protein comprises the gp220 polypeptide disclosed herein and ferritin, and the ferritin comprises a serine or cysteine ​​(e.g., serine) at residue 110 of SEQ ID NO: 1; optionally, wherein the ferritin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 435-600 of SEQ ID NO: 14, or wherein the ferritin comprises or is composed of any one of SEQ ID NO: 23, 24, 42, and 43.

[0140] Additionally, in some embodiments, any fusion protein comprising the gp220 peptide and ferritin may be present in a composition comprising another fusion protein and / or another peptide disclosed herein.

[0141] In some embodiments, the fusion protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 14, optionally further comprising a mammalian leader sequence (such as the mammalian leader sequence of SEQ ID NO: 12 or 13), and optionally lacking a bullfrog ferritin extension sequence (e.g., the bullfrog ferritin extension sequence of SEQ ID NO: 2) linked to the N-terminus of ferritin. In some embodiments, the fusion protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 35, optionally lacking a bullfrog ferritin extension sequence (e.g., the bullfrog ferritin extension sequence of SEQ ID NO: 2) linked to the N-terminus of ferritin. In some embodiments, the fusion protein comprising the gp220 polypeptide and ferritin comprises ferritin, wherein at least one or all of the surface-exposed cysteine ​​residues are replaced by different amino acids (e.g., serine). In some embodiments, the fusion protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 25, 14, or 35, and the ferritin of the fusion protein comprises a serine residue corresponding to position 110 of SEQ ID NO: 1 (i.e., a serine residue at position C544 of SEQ ID NO: 14 or at position C574 of SEQ ID NO: 35). In one embodiment, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 25. E. A fusion protein containing gp42 peptide and ferritin

[0142] In some embodiments, the fusion protein disclosed herein may comprise a gp42 polypeptide and a ferritin disclosed herein (e.g., ferritin of any one of SEQ ID NO: 23, 24, 42, and 43). An exemplary gp42 sequence is provided as SEQ ID NO: 15. Additional exemplary gp42 sequences suitable for inclusion in fusions (e.g., fusions with gL and gH polypeptides) are provided as residues 856-1038 of any one of SEQ ID No: 9-11.

[0143] In some embodiments, the gp42 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 15. In some embodiments, the gp42 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 856-1038 of any one of SEQ ID Nos: 9-11.

[0144] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is appended to the N-terminus of the gp42 polypeptide. In some embodiments, when expressed in mammalian cells, the mammalian leader sequence leads to the secretion of the protein. In some embodiments, the mammalian leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 12 or 13.

[0145] In some embodiments, the fusion protein comprising gH and / or gL peptides further comprises the gp42 peptide. Any fusion protein comprising the above-described gH and / or gL peptides may further comprise the gp42 peptide. In some embodiments, the gp42 peptide is located at the C-terminus of one or more gH and / or gL peptides, as illustrated in SEQ ID Nos: 9-11 and 36-41. In some embodiments, the gp42 peptide is located at the N-terminus of ferritin, also illustrated in SEQ ID Nos: 9-11 and 36-41. Thus, for example, the fusion protein may comprise the gL peptide, gH peptide, gp42 peptide, and ferritin of the present invention in N-terminal to C-terminal order. In other words, in some embodiments, the EBV gL peptide, EBV gH peptide, EBV gp42 peptide, and ferritin of the present invention are arranged in N-terminal to C-terminal order within the fusion protein. In some embodiments, ferritin comprises or is composed of any one of the amino acids in SEQ ID NOs: 23, 24, 42, and 43.

[0146] Linkers (such as those described herein) can separate the gp42 peptide from one or more EBV peptides and / or ferritin located at its N-terminus and / or C-terminus. In some embodiments, the linker separates each EBV peptide in the fusion protein (e.g., gL peptide, gH peptide, and gp42 peptide), and additional linkers may be present between ferritin and the EBV peptide (e.g., gp42 peptide).

[0147] In some embodiments, the linker separates the EBV gH peptide and the EBV gp42 peptide and has a length of at least 15 amino acids (e.g., 15-100 amino acids, such as 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-20 amino acids). Such linkers may have lengths of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, 32 amino acids, or 40 to 50 amino acids. In some embodiments, the linker separating the EBV gH peptide and the EBV gp42 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID Nos: 3-8. In a particular embodiment, the linker separating the EBV gH peptide and the EBV gp42 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 4.

[0148] In some embodiments, the linker separates the EBV gp42 peptide and ferritin. Such linkers may have a length of at least 15 amino acids (e.g., 15-100 amino acids, such as 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-20 amino acids), or a length of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, or 40 to 50 amino acids, 70 to 90 amino acids, 80 to 90 amino acids, or 88 amino acids). In some embodiments, such linkers comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO: 3-8. In a particular embodiment, the additional adapter comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 5.

[0149] The gp42 peptide can be combined with any of the ferritins discussed herein to form a fusion protein. For example, in some embodiments, the fusion protein comprises the gp42 peptide (+ / - gL / gH and / or gp220) and ferritin. In some embodiments, the ferritin comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43.

[0150] In some embodiments, the fusion protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO: 9-11, optionally further comprising a mammalian leader sequence (such as the mammalian leader sequence of SEQ ID NO: 12 or 13, as shown in SEQ ID NO: 36-41), and optionally lacking the bullfrog ferritin extension sequence (the bullfrog ferritin extension sequence of SEQ ID NO: 2) linked to the N-terminus of ferritin. In some embodiments, ferritin comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43.

[0151] Additionally, in some embodiments, any fusion protein comprising the gp42 peptide and ferritin of the present invention may be present in a composition comprising another fusion protein and / or peptide disclosed herein. F. Connector

[0152] In some embodiments, the fusion protein in portion B or C includes a linker between the gL and gH peptides. In some embodiments, the fusion protein in portion B or E includes a linker between the gH and gp42 peptides. In some embodiments, the fusion protein in portions B, C, D, or E includes a linker between the EBV peptide of the present invention and ferritin. The present invention provides a relatively long linker between the gL and gH sequences that can provide increased immunogenicity. In some embodiments, the linker is a peptide linker that can promote the expression of the fusion protein (e.g., from a single open reading frame). In some embodiments, the linker is a glycine-serine linker. Exemplary linkers are provided in Table 2.

[0153] In some embodiments, the linker is at least 15 amino acids long. In some embodiments, the linker is at least 25 amino acids long. In some embodiments, the linker is at least 30 amino acids long. In some embodiments, the linker is at least 35 amino acids long. In some embodiments, the linker is at least 40 amino acids long. In some embodiments, the linker is less than or equal to 60 amino acids long. In some embodiments, the linker is less than or equal to 50 amino acids long. In some embodiments, the linker is about 16, 28, 40, 46, or 47 amino acids long. In some embodiments, the linker is flexible. In some embodiments, the linker contains a cysteine ​​residue, for example, for use as a conjugation site, such as an adjuvant; an exemplary linker containing a cysteine ​​residue is provided as SEQ ID NO: 29 (SGGGSGSASSGASASGSSCSGSGSGSSSASSGASSGGASGGGSGGSG). In some embodiments, the linker contains a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 29, and further contains a cysteine ​​residue corresponding to the cysteine ​​residue in SEQ ID NO: 29. In some embodiments, the linker comprises at least 25 amino acids (e.g., 25 to 60 amino acids), wherein cysteine ​​is located in the range from the 8th amino acid from the N-terminus to the 8th amino acid from the C-terminus, or within 10 amino acids of the central residue or bond of the linker.

[0154] In some embodiments, the linker comprises glycine (G) and / or serine (S) amino acids. In some embodiments, the linker comprises or consists of the following: glycine (G), serine (S), asparagine (N), and / or alanine (A) amino acids, and optionally cysteine ​​as discussed above. In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with GGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 30). In some embodiments, the linker comprises GGGGSGGGGGGGGGSG (SEQ ID NO: 31), GGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 32), SEQ ID NO: 30, or GS. In some embodiments, the linker comprises FR1 (GGSGSASAEAAAKEAAAKAGGSGGSG; SEQ ID NO: 33) or FR2 (GGSGSASAEAAAKEAAAKEAAAKASGGSGGSG; SEQ ID NO: 34). In some embodiments, the linker comprises or consists of SEQ ID NO: 3, 6, or 8. In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 6. In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 8.

[0155] In some embodiments, the linker separates the EBV gL peptide and the EBV gH peptide and has a length of at least 15 amino acids (e.g., 15-100 amino acids, such as 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-20 amino acids). Such linkers can have lengths of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, 44 to 48 amino acids, 44 amino acids, 46 amino acids, 48 ​​amino acids, or 40 to 50 amino acids. In some embodiments, the adapter separating the EBV gL peptide and the EBV gH peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID Nos: 3-8 and 29-30. In a particular embodiment, the adapter separating the EBV gL peptide and the EBV gH peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3. In a particular embodiment, the adapter separating the EBV gL peptide and the EBV gH peptide comprises or consists of the following: SEQ ID NO: 3.

[0156] In some embodiments, the linker separates the EBV gH peptide and the EBV gp42 peptide and has a length of at least 15 amino acids (e.g., 15-100 amino acids, such as 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-20 amino acids). Such linkers may have lengths of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, 32 amino acids, or 40 to 50 amino acids. In some embodiments, the linker separating the EBV gH peptide and the EBV gp42 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID Nos: 3-8. In a specific embodiment, the connector separating the EBV gH peptide and the EBV gp42 peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 4. In a specific embodiment, the connector separating the EBV gH peptide and the EBV gp42 peptide comprises or consists of the following: SEQ ID NO: 4.

[0157] In some embodiments, the linker separates the EBV gp42 polypeptide and ferritin. Such linkers may have a length of at least 15 amino acids (e.g., 15-100 amino acids, such as 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-20 amino acids), or a length of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, or 40 to 50 amino acids, 70 to 90 amino acids, 80 to 90 amino acids, or 88 amino acids). In some embodiments, such linkers comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO: 3-8. In a specific embodiment, the linker separating the EBV gp42 peptide and ferritin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 5. In a specific embodiment, the linker separating the EBV gp42 peptide and ferritin comprises or consists of the following: SEQ ID NO: 5.

[0158] In some embodiments, the fusion protein comprising gL, gH, and gp42 of the present invention and ferritin comprises linkers between gL and gH, between gH and gp42, and between gp42 and ferritin. In one embodiment, the linker separating the EBV gL peptide and the EBV gH peptide comprises or consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the linker separating the EBV gL peptide and the EBV gH peptide has 40 to 50 amino acids. In one embodiment, the linker separating the EBV gL peptide and the EBV gH peptide has 44, 46, or 47 amino acids. In one embodiment, the linker separating the EBV gH peptide and the EBV gp42 peptide comprises or consists of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the linker separating the EBV gH peptide and the EBV gp42 peptide has 30 to 40 amino acids. In one embodiment, the linker separating the EBV gH peptide and the EBV gp42 peptide has 32 amino acids. In one embodiment, the linker separating the EBV gp42 peptide and ferritin comprises or consists of the amino acid sequence of SEQ ID NO: 5. In one embodiment, the linker separating the EBV gp42 peptide and ferritin has 80 to 90 amino acids. In one embodiment, the linker separating the EBV gp42 peptide and ferritin has 88 amino acids.

[0159] In a specific embodiment, the linker between the EBV gL peptide and the EBV gH peptide is 44 to 48 amino acids, and the linker between the EBV gH peptide and the EBV gp42 peptide is 32 amino acids. In another specific embodiment, the linker between the EBV gL peptide and the EBV gH peptide is 46 amino acids, and the linker between the EBV gH peptide and the EBV gp42 peptide is 32 amino acids. In yet another specific embodiment, the linker separates the EBV gL peptide and the EBV gH peptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 3; and / or the linker separates the EBV gH peptide and the EBV gp42 peptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 4.

[0160] In some specific embodiments, the fusion protein further comprises an additional linker separating the ferritin and the EBV gp42 polypeptide. In some specific embodiments, the additional linker is 88 amino acids in length. In some specific embodiments, the additional linker consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 5. Mutations in G.gL, gH, gp42, linker, and / or ferritin sequences can eliminate potential oxidation, deamidation, or isoflavone formation sites.

[0161] In some embodiments, the fusion protein of portions B, C, or E contains one or more mutations to eliminate potential oxidation, deamidation, or isoflavone formation sites, such as the exemplary mutations listed in Table 1 below.

[0162] For example, in some embodiments, the gL sequence contains one or more mutations to eliminate potential succinimide / isoaspartic acid or deamidation sites. For example, the gL sequence may contain a G to A mutation at position 36 corresponding to SEQ ID NO: 16; an N to Q mutation at position 47 corresponding to SEQ ID NO: 16; and / or an N to Q mutation at position 105 corresponding to SEQ ID NO: 16. In some embodiments, the gL sequence contains each of the following: a G to A mutation at position 36 corresponding to SEQ ID NO: 16; an N to Q mutation at position 47 corresponding to SEQ ID NO: 16; and an N to Q mutation at position 105 corresponding to SEQ ID NO: 16. A position “corresponds” to a given position in SEQ ID NO: 16 if the position in the amino acid sequence is aligned to a given position in SEQ ID NO: 16 according to a standard sequence alignment algorithm (e.g., the Smith-Waltman algorithm using default parameters).

[0163] In some embodiments, the linker contains one or more mutations to eliminate potential deamidation sites, such as N to G mutations.

[0164] In some embodiments, the gH sequence contains one or more mutations to eliminate potential succinimide / isoaspartic acid or oxidation sites. For example, the gH sequence may contain one or more of the following: M to L mutations at positions 28, 240, or 568 corresponding to SEQ ID NO: 17; D to E mutations at positions 207 or 268 corresponding to SEQ ID NO: 17; M to I mutations at positions 338 or 478 corresponding to SEQ ID NO: 17; or N to Q mutations at position 492 corresponding to SEQ ID NO: 17. In some embodiments, the gH sequence contains each of the following: M to L mutations at position 28 corresponding to SEQ ID NO: 17 and D to E mutations at positions 207 and 268 corresponding to SEQ ID NO: 17.

[0165] In some embodiments, the gp42 sequence contains one or more mutations to eliminate potential deamidation sites. For example, the gp42 sequence may contain one or more of the following: an N-to-Q mutation at position 131 or 162 corresponding to SEQ ID NO: 15; or an N-to-S mutation at position 160 corresponding to SEQ ID NO: 15. In some embodiments, the gp42 sequence contains each of the following: an N-to-Q mutation at positions 131 and 162 corresponding to SEQ ID NO: 15 and an N-to-S mutation at position 160 corresponding to SEQ ID NO: 15. In some embodiments, the gp42 sequence does not contain any of these mutations.

[0166] In some embodiments, the ferritin sequence contains one or more mutations to eliminate potential deamidation, oxidation, or isoflavone formation sites. For example, the ferritin sequence may contain M to I mutations at position 17 corresponding to SEQ ID NO: 1; M to L mutations at position 26 corresponding to SEQ ID NO: 1; G to A mutations at position 37 corresponding to SEQ ID NO: 1; or N to Q mutations at positions 102 or 145 corresponding to SEQ ID NO: 1.

[0167] Exemplary mutations are shown in Table 1 below.

[0168] H. Exemplary compositions, kits, nucleic acids, uses, and methods

[0169] In some embodiments, the present invention provides a method for immunizing or inoculating a subject against EBV infection. The present invention further provides a method for inducing an immune response against EBV in a subject. In some embodiments, the method of the present invention includes administering to the subject an effective amount of the pharmaceutical composition described herein. In some embodiments, the method of the present invention includes administering to the subject an effective amount of the fusion protein or nanoparticles described herein.

[0170] In some embodiments, a composition comprising one or more of the fusion proteins described herein and a pharmaceutically acceptable mediator, adjuvant, or excipient is provided.

[0171] In some embodiments, the fusion protein, nanoparticles, or compositions described herein are administered to a subject (e.g., a human or any of the subjects discussed below) to immunize against infection caused by EBV. In some embodiments, the fusion protein or nanoparticles described herein are administered to a subject (e.g., a human) to generate a protective immune response against future EBV infection. In some embodiments, a fusion protein is administered. In some embodiments, a fusion protein comprising an EBV peptide and ferritin is administered, wherein the ferritin may have one or more mutations described herein. In some embodiments, a fusion protein or nanoparticle is administered having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NOs: 9-11, 14, 25, or 36-41. In some embodiments, a fusion protein or nanoparticle having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 9-11 or 36-41, and a fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, 25, or 35. In some embodiments, the ferritin of the fusion protein or nanoparticle comprises or consists of the amino acid of SEQ ID NO: 23. In some embodiments, the ferritin of the fusion protein or nanoparticle comprises or consists of the amino acid of SEQ ID NO: 24. In some embodiments, the ferritin of the fusion protein or nanoparticle comprises or consists of the amino acid of SEQ ID NO: 42. In some embodiments, the ferritin of the fusion protein or nanoparticle comprises or consists of the amino acid of SEQ ID NO: 43. In some embodiments, a fusion protein or nanoparticle comprising any one of SEQ ID NO: 9-11 or 36-41 is applied. In some embodiments, a fusion protein or nanoparticle comprising any one of SEQ ID NO: 9-11 or 36-41 and a fusion protein comprising SEQ ID NO: 14, 25 or 35 are applied.

[0172] In some embodiments, a protective immune response reduces the incidence of hospitalization. In some embodiments, a protective immune response reduces the incidence of EBV infection, mononucleosis, complications of mononucleosis (e.g., hepatitis, encephalitis, severe hemolytic anemia, or splenomegaly), nasopharyngeal carcinoma, gastric cancer, or B-cell lymphoma (e.g., Burkitt lymphoma or Hodgkin lymphoma), and multiple sclerosis.

[0173] In some embodiments, the composition comprises a fusion protein. In some embodiments, the composition comprises a fusion protein comprising the gH peptide and ferritin of the present invention. In some embodiments, the composition comprises a fusion protein comprising the gL peptide and ferritin of the present invention. In some embodiments, the composition comprises a fusion protein comprising the gp42 peptide and ferritin of the present invention. In some embodiments, the composition comprises a fusion protein comprising the gp220 peptide and optionally metronidazole.

[0174] In some embodiments, the composition comprises more than one fusion protein. In some embodiments, the composition comprises one or more fusion proteins comprising more than one EBV peptide of the present invention and at least one ferritin. In some embodiments, the EBV vaccine comprises nanoparticles containing a gp220 peptide and optionally a metronidazole, and nanoparticles containing gH and gL peptides of the present invention, and optionally a gp42 peptide and ferritin, respectively.

[0175] In some embodiments, any one or more of the fusion proteins, nanoparticles, or compositions described herein are provided for use in immune responses against EBV-induced infections. In some embodiments, any one or more of the fusion proteins, nanoparticles, or compositions described herein (e.g., in sections B, C, D, or E) are provided for use in generating a protective immune response against future EBV infection.

[0176] In some embodiments, the compositions disclosed herein comprise first ferritin nanoparticles and optionally second ferritin nanoparticles, the first ferritin nanoparticles comprising a fusion protein disclosed herein (e.g., a fusion protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 9-11, 14, 25, or 35-41), and the second ferritin nanoparticles comprising a second fusion protein containing an EBV gp220 peptide and ferritin (e.g., a fusion protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 14, 25, or 35). In some embodiments, the EBV gp220 polypeptide of the composition comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO: 14. In some embodiments, the ferritin of the second fusion protein comprises a serine or cysteine ​​residue corresponding to position 110 of SEQ ID NO: 1; optionally, the ferritin comprises an amino acid sequence having at least 80% sequence identity with residues 435-600 of SEQ ID NO: 14. In some embodiments, the second fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, optionally wherein position C544 of SEQ ID NO: 14 is substituted with a serine.

[0177] In some embodiments, the composition disclosed herein comprises: (a) a first ferritin nanoparticle comprising a fusion protein consisting of an amino acid sequence of any one of SEQ ID NO: 9-11; or (b) a second ferritin nanoparticle comprising a second fusion protein consisting of an amino acid sequence of SEQ ID NO: 14; or (c) a second ferritin nanoparticle comprising a second fusion protein consisting of an amino acid sequence of SEQ ID NO: 14, wherein position C544 of SEQ ID NO: 14 is replaced with serine.

[0178] In a particular embodiment, the composition disclosed herein comprises: (a) a first ferritin nanoparticle comprising a fusion protein consisting of the amino acid sequence of SEQ ID NO: 11; and (b) a second ferritin nanoparticle comprising a second fusion protein consisting of the amino acid sequence of SEQ ID NO: 14. 1. Subjects

[0179] In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans.

[0180] In some embodiments, the subject is an adult (aged 18 or older). In some embodiments, the subject is a child or adolescent (aged 18 or younger). In some embodiments, the subject is an older adult (aged 60 or older). In some embodiments, the subject is a non-older adult (aged 18 or older and aged 60 or younger).

[0181] In some embodiments, the composition is suitably formulated for the intended route of administration. Examples of suitable routes of administration include intramuscular, transdermal, subcutaneous, intranasal, oral, or transdermal administration.

[0182] In some embodiments, the composition is administered to the subject more than once. In some embodiments, enhanced administration improves the immune response.

[0183] In some embodiments, any one or more of the fusion proteins or compositions described herein are used in mammals such as primates (e.g., non-human primates such as monkeys (e.g., macaques, such as rhesus monkeys or cynomolgus monkeys) or apes), rodents (e.g., mice or rats) or domesticated mammals (e.g., dogs, rabbits, cats, horses, sheep, cattle, goats, camels or donkeys). 2. Adjuvant

[0184] Adjuvants can be administered to subjects in conjunction with the fusion proteins and / or nanoparticles described herein, wherein administration of such combinations can generate higher titers of antibodies against one or more fusion proteins in subjects compared to administration of one or more fusion proteins without adjuvants. Adjuvants can promote earlier, more effective, or more durable immune responses to one or more fusion proteins.

[0185] In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition comprises more than one adjuvant. In some embodiments, the composition does not comprise an adjuvant.

[0186] In some embodiments, the adjuvant comprises aluminum. In some embodiments, the adjuvant is aluminum phosphate. In some embodiments, the adjuvant is alum (Alyhydrogel '85 2%; Brenntag - catalog number 21645-51-2).

[0187] In some embodiments, the adjuvant is an organic adjuvant. In some embodiments, the adjuvant is an oil-based adjuvant. In some embodiments, the adjuvant comprises an oil-in-water nanoemulsion.

[0188] In some embodiments, the adjuvant comprises squalene. In some embodiments, the adjuvant comprising squalene is Ribi (Sigma Adjuvant System Catalog No. S6322-1vl) or Addavax. TM MF59, AS03, or AF03 (see US9703095). In some embodiments, the adjuvant containing squalene is a nanoemulsion.

[0189] In some embodiments, the adjuvant comprises a polyacrylic acid polymer (PAA). In some embodiments, the adjuvant comprising PAA is SPA09 (see WO 2017218819).

[0190] In some embodiments, the adjuvant comprises a non-metabolizable oil. In some embodiments, the adjuvant is an incomplete Freund's adjuvant (IFA).

[0191] In some embodiments, the adjuvant comprises a non-metabolizable oil and killed Mycobacterium tuberculosis. In some embodiments, the adjuvant is a complete Freund's adjuvant (CFA).

[0192] In some embodiments, the adjuvant is a lipopolysaccharide. In some embodiments, the adjuvant is monophosphoryl A (MPL or MPLA). 3. Pharmaceutical Composition

[0193] In various embodiments, pharmaceutical compositions comprising the fusion proteins and / or related entities described herein are provided. In some embodiments, the pharmaceutical composition is an immunogenic composition (e.g., a vaccine) capable of eliciting an immune response (e.g., a protective immune response against a pathogen).

[0194] For example, in some embodiments, the pharmaceutical composition may comprise one or more of the following: (1) a fusion protein comprising (a) an Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gH polypeptide and / or (ii) an EBV gL polypeptide; and (b) ferritin of SEQ ID NO: 1 comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof; (2) a fusion protein comprising an Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gH polypeptide and / or (ii) an EBV gL polypeptide; and ferritin comprising the amino acid sequence of SEQ ID NO: 1 having at least one of the following amino acid substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof; (2) Glutamine at residue N145 of NO:1; and / or (iii) a bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO:2; (3) a fusion protein comprising an Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gp42 polypeptide and (ii) an EBV gH polypeptide and / or an EBV gL polypeptide; and ferritin comprising the amino acid sequence of SEQ ID NO:1, wherein the ferritin has at least one of the following amino acid substitutions: (i) glutamine at residue 102 corresponding to SEQ ID NO:1; (ii) glutamine at residue 145 corresponding to SEQ ID NO:1; and / or (iii) a bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO:2; (4) a fusion protein comprising an EBV gL polypeptide and an EBV gL polypeptide. Epstein-Barr virus (EBV) polypeptide gH polypeptide, wherein the EBV gL polypeptide comprises the sequence of residues 1-115 of SEQ ID NO: 11, and the EBV gH polypeptide comprises the sequence of residues 162-823 of SEQ ID NO: 11; ferritin polypeptide of SEQ ID NO: 1, or ferritin polypeptide having SEQ ID NO: 1 and at least one mutation selected from any one or more of the following: glutamine at the residue corresponding to position N18 of SEQ ID NO: 1; serine at the residue corresponding to position C30 of SEQ ID NO: 1;Glutamine at residue N102 corresponding to SEQ ID NO: 1; glutamine at residue N145 corresponding to SEQ ID NO: 1; and optionally a bullfrog ferritin extension sequence linked to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO: 2, and further optionally a mammalian leader sequence located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or 13; wherein the EBV gL polypeptide, the EBV gH polypeptide and the ferritin are arranged in the N-terminus to C-terminus sequence within the EBV polypeptide, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide; (5) a fusion protein comprising an Epstein-Barr virus (EBV) polypeptide containing an EBV gL polypeptide, an EBV gH polypeptide and an EBV gp42 polypeptide, wherein the EBV gL polypeptide comprises the amino acid sequence of residues 1-115 of SEQ ID NO: 11, the EBV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 11, and the EBV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or 13; wherein the EBV gL polypeptide comprises the amino acid sequence of residues 1-115 of SEQ ID NO: 11, ..., and the EBV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or 13, and the EBV gL polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or 13, and the EB The EBV gp42 polypeptide comprises the amino acid sequence of residues 162-823 of SEQ ID NO: 11, and the EBV gp42 polypeptide comprises the amino acid sequence of residues 856-1038 of SEQ ID NO: 11; comprises ferritin of SEQ ID NO: 24; and optionally a mammalian leader sequence located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or 13; wherein the EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide and the ferritin are arranged in the N-terminus to C-terminus sequence within the EBV polypeptide, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide; or (6) ferritin particles comprising any of the aforementioned polypeptides. In some embodiments, the pharmaceutical composition may comprise a fusion protein containing an antigenic EBV gL / gH polypeptide, a fusion protein containing an antigenic EBV gL / gH / gp42 polypeptide, and / or a fusion protein containing an antigenic EBV pg220 polypeptide, for example, wherein the fusion protein comprises a linker of at least 15 amino acids between the gL and gH polypeptide sequences and / or between the gH and gp42 sequences, and optionally wherein the fusion protein further comprises ferritin. In some embodiments, the ferritin comprises or consists of any one of the following amino acids: SEQ ID NO: 23, 24, 42, and 43.

[0195] In some embodiments, any of the pharmaceutical compositions described herein may be administered alone or in combination with one or more agents (e.g., the adjuvants described above) to enhance the immune response. In some embodiments, the pharmaceutical composition further comprises the adjuvants described above.

[0196] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or catalyst administered with the pharmaceutical composition. In exemplary embodiments, the carrier may comprise sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components. Pharmaceutically acceptable carriers may also include, but are not limited to, saline, buffered saline, dextran, glycerol, ethanol, and combinations thereof. As used herein, an excipient is any non-therapeutic agent that may be included in the pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, glycol, water, ethanol, etc. In various embodiments, the pharmaceutical composition is sterile.

[0197] In some embodiments, the pharmaceutical composition contains a small amount of a wetting agent or emulsifier, or a pH buffer. In some embodiments, the pharmaceutical composition may include any of a variety of additives, such as stabilizers, buffers, or preservatives. Additionally, it may include adjuvants, stabilizers, thickeners, lubricants, and colorants.

[0198] The described pharmaceutical composition can be administered at any dose suitable for achieving the desired outcome. In some embodiments, the desired outcome is the induction of a durable adaptive immune response against a pathogen due to the presence of nonferritin peptides in the fusion protein of the composition. In some embodiments, the desired outcome is a reduction in the intensity, severity, frequency, and / or delay of the onset of one or more infectious symptoms. In some embodiments, the desired outcome is the suppression or prevention of infection. The required dose will vary from subject to subject based on: subject species, age, weight and general condition, severity of the infection being prevented or treated, the specific composition used, and its administration method.

[0199] In some embodiments, the pharmaceutical composition according to the invention is administered in a single dose or multiple doses. In some embodiments, the pharmaceutical composition is administered in multiple doses on different days (e.g., a prime-boost vaccination strategy). In some embodiments, the pharmaceutical composition is administered as part of a booster regimen.

[0200] In various embodiments, the pharmaceutical composition is co-administered with one or more additional therapeutic agents. Co-administration does not require simultaneous administration of the therapeutic agents to achieve a combined therapeutic effect if the timing of the therapeutic agent administration causes a temporal overlap between the pharmacological activities of the additional therapeutic agent and one or more active ingredients in the pharmaceutical composition. Typically, each agent will be administered at a dosage and schedule determined for that agent. 4. Nucleic acid / mRNA

[0201] Nucleic acids encoding any of the fusion proteins described herein and in the BE section are also provided. In some embodiments, the nucleic acid used herein encodes a fusion protein comprising the amino acid sequence of any one of SEQ ID NO: 9-11, 14, 25, or 35-41. In some embodiments, the nucleic acid used herein encodes a fusion protein consisting of the amino acid sequence of any one of SEQ ID NO: 9-11, 14, 25, or 35-41. In some embodiments, the nucleic acid used herein encodes ferritin comprising or consisting of the amino acids of any one of SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the nucleic acid is mRNA. For the purposes of this disclosure, any nucleic acid capable of undergoing translation to produce a polypeptide is considered mRNA. In some embodiments, the nucleic acid comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 19-22. In some embodiments, the nucleic acid comprises or consists of the sequence of any one of SEQ ID NO: 19-22. 5. Expression vehicle

[0202] This document also provides expression vectors and cells containing (e.g., encoding) the fusion proteins described herein. Vectors used herein can be any suitable recipient nucleic acid molecule modified to contain or incorporate the provided nucleic acid sequence (e.g., a nucleic acid sequence encoding the fusion protein disclosed herein). Such suitable vectors include, but are not limited to, nucleic acid vectors (e.g., plasmids), viral vectors (e.g., AAV vectors), bacterial vectors, and / or yeast vectors (as the case may be). Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) can integrate into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome.

[0203] Vectors may include at least one restriction endonuclease recognition sequence, for example, for inserting a transgene into the vector. Vectors may include at least one gene sequence conferring antibiotic resistance or selectable characteristics to aid in the selection of host cells carrying the vector-transgene construct. Vectors may be single-stranded or double-stranded nucleic acid molecules and may be linear or circular nucleic acid molecules. Donor nucleic acids used for gene editing methods employing zinc finger nucleases, TALEN, or CRISPR / Cas can be one type of vector. One type of vector is a plasmid, which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be linked to a transgene and is capable of replicating in a host cell and transcribing and / or translating the transgene. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be linked to a transgene. The viral backbone sequence may be modified to disable infection but retain the viral backbone and the insertion of the co-linked transgene into the host cell genome. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, multivaccinia viruses, vaccinia viruses, herpes simplex viruses, and Epstein-Barr virus vectors. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome upon introduction and thus replicate along with the host genome. Vectors can be transfer vectors, i.e., compositions of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into the cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0204] The vector can be (alternatively or additionally) an expression vector, i.e., a vector containing a recombinant polynucleotide containing an expression control sequence operatively linked to the nucleotide sequence to be expressed. Such expression control sequences can direct the expression of the inserted gene to which they are operatively linked. The expression vector may contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Further examples of regulatory sequences and other elements for expression are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, California, and Baron et al., 1995, Nucleic Acids Res., 23:3605-3606. Expression vectors include all those known in the art, including viscera, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides. Standard techniques can be used for the engineering of vectors, for example, as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)), which is incorporated herein by reference for any purpose.

[0205] The vectors used herein may contain selection markers, such as any suitable selection markers known in the art. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR). Transfected host cells can be subjected to selection pressure, where only the transformants are uniquely adapted to survive due to the selectable gene present in the vector. Selection pressure is applied by culturing transfected cells under conditions of progressively increasing concentrations of the selector in the culture medium, resulting in the amplification of both the selectable gene and the DNA encoding another target nucleic acid sequence (such as the fusion protein disclosed herein). Therefore, the host cells used herein may be dihydrofolate reductase (DHFR) deficient, and the expression vector may contain a polynucleotide sequence encoding DHFR. In such embodiments, recombinant cells may be cultured in the presence of a DHFR selector (e.g., methotrexate) to select cells that have been successfully transfected and express the target nucleic acid sequence (such as the fusion protein disclosed herein).

[0206] In some embodiments, the expression vector used herein comprises a nucleic acid disclosed herein, such as a nucleic acid encoding a fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 9-11, 14, 25, or 35-41, and wherein the ferritin of the fusion protein comprises or is composed of the amino acids of any one of SEQ ID NOs: 23, 24, 42, and 43. In some embodiments, the expression vector comprises a nucleic acid having a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 19-21. In some embodiments, the expression vector comprises a nucleic acid having a sequence of any one of SEQ ID NOs: 19-21. In some embodiments, the expression vector comprises a nucleic acid consisting of a sequence of any one of SEQ ID NO: 19-21.

[0207] In some embodiments, the expression vector used herein comprises a nucleic acid encoding a second fusion protein as disclosed herein, such as a nucleic acid encoding a second fusion protein comprising an EBV gp220 polypeptide and optionally a metronidazole, such as a second fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, 25, or 35. In some embodiments, the ferritin of the second fusion protein comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the expression vector comprises a nucleic acid comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 22. In some embodiments, the expression vector comprises a nucleic acid comprising the sequence of SEQ ID NO: 22. In some embodiments, the expression vector comprises a nucleic acid comprising the sequence of SEQ ID NO: 22. Such second fusion proteins can be encoded on the same or different expression vectors as the fusion proteins disclosed herein. 6. Cells and methods for preparing cells

[0208] This document also provides cells comprising nucleic acids as disclosed herein (e.g., recombinant cells), and methods for preparing such cells. Host cells used herein include any suitable cell that can be used to generate recombinant proteins (e.g., fusion proteins disclosed herein) encoded by an expression vector (e.g., the expression vector disclosed herein) or to propagate expression vectors introduced into the host cell. Recombinant cells comprise heterologous expression vectors (e.g., the expression vectors disclosed herein), which may or may not be integrated into the host cell chromosome. In some embodiments, the host cell has been transfected with an expression vector containing a polynucleotide sequence encoding a fusion protein disclosed herein, and such recombinant cells are capable of producing fusion proteins as disclosed herein.

[0209] The host cells that can be used in this disclosure may be eukaryotic cells, fungal cells, insect cells, prokaryotic cells (e.g., bacterial or archaea cells), or cells derived from multicellular organisms (e.g., cell lines) cultured as single-celled entities. In some embodiments, the cells may be eukaryotic cells, such as 293T cells (e.g., 293 expi cells or HEK293T cells). Exemplary mammalian cells include, but are not limited to: Chinese hamster ovary (CHO) cells, CHO-K1 cells, CHO-derived cells, human embryonic kidney (HEK) cells (e.g., HEK293 cells expressing functional adenovirus E1 and HEK-derived cells such as HEK293T or HEK293F), Maddard's canine kidney (MDCK) cells, Vero cells, EB66 cells, chicken embryo cells, RK cells, RAF cells, PK15 cells, MRC-5 cells, A549 cells, WEHI cells, 3T3 cells, 10T1 / 2 cells, BHK cells, COS 1 cells, COS 7 cells, BSC 1 cells, BSC 40 cells, and BMT cells. 10 cells, WI38 cells, HeLa cells, Saos cells, C2C12 cells, L cells, HT1080 cells, HepG2 cells, NS-1 cells, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals (including humans, monkeys, mice, rats, rabbits, and hamsters). The choice of the species of host cells (e.g., mammalian species) is not a limitation of the invention; nor is the type of mammalian cell (i.e., fibroblasts, hepatocytes, tumor cells, etc.) a limitation of the invention. Those skilled in the art will understand that the conditions used in the methods disclosed herein will depend on the host cells used. Typical conditions (e.g., the culture medium or temperature to be used) are well known in the art. In one embodiment, culture is performed by incubating mammalian packaging cell lines (e.g., HEK293 or CHO cells) under humidified conditions. In a particular embodiment, humidified conditions include incubating transfected cells at about 37°C and about 5% CO2. In some embodiments, the cell culture comprises a culture medium suitable for culturing CHO cells, such as a chemically defined CHO cell culture medium. In some embodiments, the cell culture is serum-free. Exemplary commercially available cell culture media include, but are not limited to, Durbeco Modified Eagle Medium (e.g., including serum, such as 10% fetal bovine serum) (Gibco), BalanCD Medium (e.g., BalanCD CHO Medium; Fujifilm and Irvine Scientific), CD CHO AGT Medium (Gibco), or ExpiCHO™ Stable Production Medium (Gibco).

[0210] Cells can be transfected using any method known in the art, such as with one or more (e.g., one, two, three, or four) expression vectors (e.g., one, two, three, four, or more expression vectors as disclosed herein) or other nucleic acids. Transfection methods have been described in the art and include, for example, calcium phosphate coprecipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transfection, or nucleic acid delivery using high-speed microbolites. Other suitable transfection media include strontium phosphate, polycationic polymers (e.g., Superfect™ (Qiagen)), liposomes, and cationic polymers (e.g., polyethyleneimine (PEI)). In the disclosed embodiments, the term transfection covers any means of introducing nucleic acids into the host cell, including but not limited to transduction or infection with DNA or RNA viruses or viral vectors. The resulting cells can be transiently transfected with exogenous nucleic acid molecules, i.e., the exogenous DNA will not integrate into the genome of the transfected cells but can exist in a free state. Alternatively, the resulting cells can be stably transfected, i.e., nucleic acid molecules are covalently linked to the host cell genome or maintained and replicated as free units that can be passed on to progeny cells (e.g., capable of extrachromosomal replication at a sufficient rate).

[0211] In some embodiments, the cell (e.g., a recombinant cell) comprises any nucleic acid disclosed herein and / or any expression vector disclosed herein. In some embodiments, the cell (e.g., a recombinant cell) expresses a fusion protein as disclosed herein and optionally a second fusion protein. In some embodiments, the recombinant cell expresses a fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 9-11, 14, 25, or 35-41, wherein the ferritin of the fusion protein comprises or is composed of any one of the amino acids of SEQ ID NO: 23, 24, 42, and 43. In some embodiments, the recombinant cells further express a second fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, 25, or 35, wherein the ferritin of the fusion protein comprises or is composed of any one of the amino acids in SEQ ID NO: 23, 24, 42, and 43.

[0212] This document also provides methods for preparing recombinant cells expressing the fusion protein disclosed herein and optionally a second fusion protein disclosed herein. In some embodiments, the method of preparing such cells includes transfecting a host cell population with one or more expression vectors (e.g., one, two, three, four, or more) containing a polynucleotide sequence encoding the disclosed fusion protein and optionally a second fusion protein; culturing the transfected host cells; and obtaining recombinant cells expressing the fusion protein. In a particular embodiment, the host cell is a CHO cell. In another particular embodiment, the host cell (e.g., a CHO cell) is DHFR-deficient. In some such embodiments, the expression vector further contains a polynucleotide sequence encoding DHFR. In some such embodiments, the host cell (e.g., a CHO cell) may be cultured in the presence of a DHFR selector, optionally wherein the DHFR selector comprises methotrexate.

[0213] This document also provides methods for preparing the disclosed fusion proteins (e.g., fusion proteins comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 9-11, 14, 25, or 35-41). Further, fusion proteins obtainable or produced by the disclosed methods are provided. In some embodiments, the method for preparing the disclosed fusion protein comprises culturing host cells as disclosed herein (e.g., host cells comprising nucleic acids encoding the disclosed fusion protein, or host cells comprising an expression vector containing nucleic acids encoding the disclosed fusion protein; and optionally further comprising nucleic acids encoding a second fusion protein as disclosed herein) (under conditions in which the fusion protein (and optionally the second fusion protein) is expressed in the host cells), and recovering the fusion protein (and optionally the second fusion protein) from the cultured host cell culture. In some embodiments, the host cell is a CHO cell. In some embodiments, the host cell is DHFR-deficient; and the expression vector further comprises a polynucleotide sequence encoding DHFR. Therefore, some embodiments of the disclosed method include culturing host cells in the presence of a DHFR selector. In a particular embodiment, the DHFR selector comprises methotrexate. In a particular embodiment, the expression vector comprises a polynucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41. In another particular embodiment, the expression vector comprises a second polynucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 14, 25 or 35. In a particular embodiment, the expression vector comprises a polynucleotide sequence of any one of SEQ ID NO: 19-22. 7. Reagent kit

[0214] This document also provides kits comprising one or more fusion proteins, nucleic acids, antigenic ferritin particles, compositions, or pharmaceutical compositions described herein. In some embodiments, the kit further comprises one or more of a solvent, solution, buffer, instruction manual, or desiccant.

[0215]

[0216] This specification and exemplary embodiments should not be considered restrictive. For the purposes of this specification and the appended claims, unless otherwise indicated, all figures representing quantities, percentages, or proportions, as well as other numerical values ​​used in the specification and claims, should in all cases be understood to be modified by the term "about" (unless such figures and values ​​are so modified). "About" indicates a degree of difference that does not substantially affect the nature of the described subject matter, for example, within 10%, 5%, 2%, or 1%. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At least, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted, taking into account at least the number of reported significant digits and by applying common rounding techniques.

[0217] It is worth noting that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” as well as any singular use of any word, include multiple references unless specifically and expressly limited to one reference. As used herein, the term “comprising” and its grammatical variations are intended to be non-restrictive, such that the listing of items does not exclude other similar items that may replace or be added to the listed items. Unless the context otherwise requires, the term “or” is used in an inclusive sense, equivalent to “and / or.” Example

[0218] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of this disclosure. Example 1: Generation of host cells that produce C16, C17, and C18 constructs (nanoparticles)

[0219] A fusion protein (antigenic peptide) that induces antibodies against EBV was developed. Self-assembled ferritin nanoparticles displaying EBV gH, gL, and gp42 peptides as single chains were also developed.

[0220] CHO cells producing EBV ferritin gp42_gH_gL precursor candidates C16, C17, and C18 (SEQ ID NO: 9-11, as shown in Table 2, respectively) were generated by expressing the corresponding recombinant fusion protein in the internally derived CHO DXB11 8D6 host cell line. CHO cell pools were generated using a CD (chemically defined) cell line development platform without ADC (animal-derived components). The generated CHO cell pools were cryopreserved as a research cell bank (RCB). Protein titers in the pools were also determined. Materials and methods

[0221] The following plasmid constructs were used in this example: plasmid DNA encoding codon-optimized EBV ferritin gp42_gH_gL_C16 (Thermo Fisher Scientific construct, lot number 2647668); plasmid DNA encoding codon-optimized EBV ferritin gp42_gH_gL_C17 (Thermo Fisher Scientific construct, lot number 2647665); and plasmid DNA encoding codon-optimized EBV ferritin gp42_gH_gL_C18 (Thermo Fisher Scientific construct, lot number 2647662).

[0222] CHO DXB11 8D6 parental (host) cells were thawed, cultured, and transfected with one of the plasmids described above (via electroporation), and the resulting granules (C16, C17, and C18) were harvested. Transfected cells were selected using methotrexate (MTX) prior to harvest. Cells were passaged in CD CHO supplemented with 4 mM L-Gln and MTX (two rounds of selection).

[0223] The cell culture materials used included CD CHO medium, L-glutamine, CD DG44 medium, Prönkel F-68, DMSO, and methotrexate. Cell culture harvests were clarified by centrifugation. EBV ferritin gp42 titers in the clarified harvests were analyzed using an Octet Red 96 (US Breakthrough Lab, Cambridge). Results and Discussion Development of EBV ferritin gp42 cell line: Transfection and generation of CHO cell pools

[0224] The amino acid sequences of EBV ferritin gp42 gH_gL_C16, gp42 gH_gL_C17, and gp42 gH_gL_C18 were sent to Thermo Fisher Scientific for DNA sequence optimization and synthesis. Codon optimization only altered the DNA sequence of the ORF, keeping the amino acid sequence unchanged. As part of ORF optimization, a co-occurring Kozak sequence was included at the 5' end of the coding sequence. Restriction endonuclease (RE) sites were added to the outermost 5' and 3' ends of the coding sequence to facilitate molecular cloning.

[0225] Optimized transgenic expression cassettes containing ORFs and custom flanking regions are subcloned into the vector backbone pGZ729 (see, for example, U.S. Patent No. 10,317,329 B2 for the pGZ729 backbone). Transfection-quality DNA is prepared for these plasmids.

[0226] Each plasmid DNA was linearized by restriction endonuclease digestion and prepared for transfection under aseptic conditions. The linearized plasmid DNA was transfected into the CHO DXB11 8D6 parental cell line via electroporation. Five replicate pools of transfected cells were generated. Immediately after transfection, cells were seeded in parental cell growth medium (CD DG44 / 4 mM L-glutamine / 0.18% (v / v) Pluronic® F-68) for recovery.

[0227] Two days after transfection, the transfected cell pools began a DHFR-based selection process (two rounds of MTX selection). Cell counts were very low throughout the selection process, and therefore, pools had to be merged during passage. For example, pool C16 1 / 2 / 3 was formed by combining pools 1, 2, and 3. All pools were cryopreserved upon completion of each round of selection. To determine EBV ferritin gp42 production levels, the pools were seeded into batch cultures in spin tubes. Batch culture parameters are summarized in Table 3. Titers of the culture harvest on day 5 were determined. Day 5 titers for each pool are shown in Table 4. Table 3. Rotary tube cell culture conditions used for titer determination Table 4. EBV ferritin gp42 titer in 5-day batch culture.

[0228] CHO pools expressing EBV ferritin gp42 were generated using an ADC-free CHO cell line development platform and cryopreserved as RCBs. For each candidate molecule, the 5 nM MTX pool had a higher titer than the 50 nM MTX pool (as determined by Octet analysis). Example 2: Generation of host cells producing gp220-ferritin nanoparticles

[0229] CHO cells producing EBV ferritin gp220 bfp_Ferr (LC81213) were generated by expressing a recombinant fusion protein in an internally derived CHO DXB11 8D6 host cell line. The amino acid sequence of the gp220 bfp_Ferr protein was used to construct the corresponding expression vector for transfection. Cell lines were generated using a CD (chemically defined) cell line development platform without ADC (animal-derived component). CHO cell pools were generated, and CHO clones were subsequently isolated and screened. Materials and methods

[0230] In this example, plasmid DNA encoding codon-optimized EBV ferritin gp220_bfpFerr (Thermo Fisher Scientific construct, lot number 2349061) was used. CHO DXB11 8D6 parental (host) cells were thawed, cultured, and transfected with the plasmid described above (via electroporation), and the resulting particles (SEQ ID NO: 14, as shown in Table 2) were harvested. Prior to harvesting, transfected cells were selected using methotrexate (MTX). Cells were passaged in CD CHO medium supplemented with 4 mM L-Gln and MTX (two rounds of selection). Specifically, the cell culture materials used were: CD CHO medium, L-glutamine, CD DG44 medium, Prönkel F-68, DMSO, methotrexate, EfficientFeed B, DMEM:F12, and HEPES.

[0231] For flow cytometry sorting and analysis, FITC-labeled anti-CD52 was used. In FACS, cells expressing the highest CD52 reporter gene were collected from one or more pools, and one cell per well was deposited into a 96-well plate. For clonal screening, selection, and amplification, the wells of the plated cells were imaged daily on day 0 and after cell deposition. Selected clones were amplified using spin tube and shake flask cultures (using methotrexate as described above). The cell culture harvest was clarified by centrifugation. The EBV ferritin gp220 titer of the clarified harvest was analyzed using Octet Red96 (Breakthrough Laboratories, Cambridge, USA). Results and Discussion Development of EBV ferritin gp220 cell line: Transfection and generation of CHO cell pools

[0232] The amino acid sequence of EBV ferritin gp220 bfp_Ferr (LC81213) was sent to Thermo Fisher Scientific for DNA sequence optimization and synthesis. Codon optimization only altered the DNA sequence of the ORF, leaving the amino acid sequence (SEQ ID NO:35) unchanged. As part of ORF optimization, a co-occurring Kozak sequence was included at the 5' end of the coding sequence. Restriction endonuclease (RE) sites were added to the 5' and 3' ends of the coding sequence to facilitate molecular cloning.

[0233] An optimized transgenic expression cassette containing an ORF and a custom flanking region was subcloned into the vector backbone pGZ729. Transfection-quality DNA was prepared for this plasmid.

[0234] Each plasmid DNA was linearized by restriction endonuclease digestion and prepared for transfection under aseptic conditions. The linearized plasmid DNA was transfected into the CHO DXB11 8D6 parental cell line via electroporation. Five replicate pools of transfected cells were generated. Immediately after transfection, cells were seeded in parental cell growth medium (CD DG44 / 4 mM L-glutamine / 0.18% (v / v) Pluronic® F-68) for recovery.

[0235] Two days post-transfection, the transfected cell pools underwent a DHFR-based selection process (two rounds of MTX selection). All pools were cryopreserved upon completion of each round of selection. To determine EBV ferritin gp220 production levels, the pools were seeded into batch cultures in spin tubes. Batch culture parameters are summarized in Table 3 above. Titers of the culture harvested on day 5 were measured. Day 5 titers for each pool are shown in Table 5. Table 5. EBV ferritin gp220 pool titers during 5-day batch culture. Generation of CHO cell clones

[0236] Pools 5, 3, and 2 (50 nM MTX stage) were selected for clone isolation. One vial from each pool was thawed and amplified for clone generation. Cells were amplified for 1 week in MTX-free medium (CDCHO supplemented with 4 mM L-glutamine) prior to sorting day. For clone isolation, cells from each pool were incubated with FITC-labeled anti-CD52 antibody (CLD, Sanofi) without ADC and sorted using a BD Influx with an automated cell deposition unit (ACDU). Cells expressing the highest percentage of CD52 were collected from each sort. Cells isolated from pools 5, 3, and 2 by sorting (via ACDU) were deposited in 96-well plates at 1 cell per well. Pool 5 generated clone sets A and H, pool 3 generated clone sets B and I, and pool 2 generated clone sets C and J.

[0237] The 96-well plates were imaged, fed with fresh cell culture medium, and screened for CD52 reporter gene expression by flow cytometry. The steps in this process are further described below. For clonal recording, the plates were imaged four times on a CellMetric® CLD (Solentim) at high resolution (2 μm / pixel) within the first seven days before deposition and after seeding. These scans were performed on day 0 (before deposition), day 0 (after deposition), day 3, and day 7. The wells were fed with growth medium, imaged for confluence, and screened by flow cytometry. No clones were observed in the 96-well plates for the C and J sets sorted from pool 2, and therefore no clones were generated from pool 2.

[0238] Clonal screening by flow cytometry produced a geometric mean fluorescence intensity (GMFI) value for each clone based on its relative CD52 reporter gene expression. Clones were ranked based on their GMFI values, and the top-ranked clones from each set were selected for amplification. After initial lead candidate clones were identified, well images acquired via Cell Metric® CLD were reviewed to assess the monoclonalness of the lead clones (discussed further below).

[0239] The clones were scaled up, inoculated into batch cultures, and cryopreserved. All clones that completed the scale-up process were evaluated in the batch cultures to determine the EBV ferritin gp220 titer. Each clone was inoculated at 1 x 10⁻⁶ cells / year. 6 The cells were seeded at a density of vc / mL in 10 mL of CD CHO / 30% Efficient Feed B supplemented with 4 mM L-glutamine in ST (see Table 3 for cell culture conditions). Cell counting and sample harvesting were performed on day 52, and culture was terminated. Results are shown in... Figure 1A -B in.

[0240] For batch culture settings, a research cell bank (RCB) was prepared for each clone (4 vials per clone). The cryopreservation medium was CD CHO supplemented with 4 mM L-glutamine and 7% (v / v) DMSO. For each clone, 1 x 10⁻⁶ cells were cryopreserved per vial. 7 Each cell.

[0241] Based on the batch culture results, 24 clones were selected for fed-batch screening in the Ambr® 15 system. Based on the fed-batch culture results ( Figure 2 Six lead candidate clones were selected, and a vial of each corresponding RCB was thawed for pre-MCB preparation. Table 6 lists the six lead candidate clones and final titers (measured using the Octet method (Octet Red 96, Breakthrough Laboratories, Cambridge)) from the fed-batch clone screening. Table 6. gp220-ferritin leader candidate clones.

[0242] Cell Metric® CLD scan images from day 0 to day 7 were used for clonal recording to help select leading candidate clones for further evaluation. Only clones that clearly showed a single cell on day 0 of the plate and showed only colony growth derived from that single cell on subsequent scan days were selected as candidates for further evaluation.

[0243] A pool of CHO cells expressing EBV ferritin gp220 (bfpFerr) was generated using an ADC-free CHO cell line development platform. The CHO pool was selected for reporter gene-based flow cytometry sorting to isolate clones and generate CHO clones producing EBV ferritin gp220. Twenty-four clones were selected for fed-batch screening based on clone evaluation in batch culture. Six clones were selected for preparation based on cell culture performance in Ambr® fed-batch screening. Example 3: Analysis of antibody binding between C18 gL / gH / gp42 nanoparticles confirms antigenic epitopes.

[0244] The gL / gH / gp42 NP construct (“C18”, encoding the amino acid sequence of SEQ ID NO: 40) was expressed and purified in 293 expi cells. The binding kinetics of the nanoparticles were evaluated using a Sartorius Octet® R8 protein assay system with either an AMQ anti-mouse FC sensor (Satorius 18-5022) or an AHC2 anti-human FC sensor (Satorius 18-5142). Antibodies (Ammo1 human mAB anti-gL / gH / gp42 (Creative Biolabs PABC-311), E1D1 mouse mAB anti-gL (Creative Biolabs PABC-524), or 1D8 human mAB anti-gL / gH (purified)) were loaded onto the appropriate sensors at 15 µg / ml. After equilibrating the sensors in kinetic buffer for 12 min, a regeneration cycle was run, followed by a baseline step. Antibody loading was performed for 300 seconds, followed by another baseline loading for 100 seconds. Antigen association was then measured for 300 seconds, followed by dissociation in baseline buffer for 300 seconds. The readings were normalized using a reference well in the buffer. Figure 3A -C shows the antigenic epitope confirmed by octet analysis of the binding of C18 gL / gH / gp42 nanoparticles (SEQ ID NO: 11) to the antibody. Figure 3A -C shows the binding curves of C18 gL / gH / gp42 nanoparticles using Ammo1, E1D1, and 1D8 antibodies, respectively. Each antibody binds to different epitopes on the C18 antigen. Ammo1 binds to epitopes across gH, gL, and gp42; E1D1 binds to epitopes in gL; and 1D8 binds to epitopes in both gH and gL. The binding curves show strong interactions between these epitopes at various antigen concentrations, confirming the integrity of the antigen structure. Example 4: Immunization against C18 gL / gH / gp42 nanoparticles

[0245] Mice were immunized with a composition containing C18 gL / gH / gp42 ferritin nanoparticles (SEQ ID NO: 11) or gp350D123 ferritin nanoparticles (SEQ ID NO: 14). Figures 4A-4B The results showed that on days 0 and 21, 1 µg of C18 gL / gH / gp42 ferritin nanoparticles were used. Figure 4A (SEQ ID NO: 11) or gp350D123 ferritin nanoparticles ( Figure 4BELISA of serum from mice immunized with SEQ ID NO: 14. Controls were pre-collected blood samples from mice before immunization. Five mice were in each group. ELISA plates were coated with a His-labeled monomer of the antigen at a concentration of 2 µg / ml. Mouse serum was serially diluted 5-fold, starting at a 1 / 10 dilution, and the IC50 was determined from the curve. Mouse serum was detected using HRP-conjugated anti-mouse FC antibody (Jackson 115-035-071) at a 1:5000 dilution and KPL SureBlue TMB substrate. Absorbance was measured at 650 nm on a plate reader. Results showed strong immune responses to both nanoparticles 3 weeks after the second dose. Example 5: Electron microscopy imaging of C18 gL / gH / gp42 nanoparticles

[0246] Imaging of C18 gL / gH / gp42 ferritin nanoparticles (SEQ ID NO: 11) and gp350D123 ferritin nanoparticles (SEQ ID NO: 14) on a nitrocellulose-supported continuous carbon layer on a 400-mesh copper grid using negative staining electron microscopy. The grid was prepared by applying 3 μL of sample suspension to a clean grid, blotting it dry with filter paper, and immediately staining with uranyl formate. Electron microscopy analysis was performed using a Thermo Fisher Scientific Glacios cryo-transmission electron microscope (Cryo-TEM) (operated at 200 kV and equipped with a Falcon 3 direct electron detector). The grid was clamped in a cassette, transferred to a cartridge, and then transferred to a Glacios autoloader while maintaining the grid at a low temperature (i.e., below -170°C). Automated data collection was performed using Leginon software (Suloway et al., Struct Biol, 2005, 151(1):41-60 and Cheng et al., Protein Sci, 2021, 30(1):136-150), where high-magnification images were acquired by selecting targets at lower magnifications. Images of each grid were acquired at multiple scales to assess the overall distribution of the samples. After identifying potentially suitable target regions for imaging at lower magnifications, high-magnification images were acquired at nominal magnifications of 73,000x (0.201 nm / pixel) and 28,000x (0.519 nm / pixel). Images were acquired at nominal underfocus from -5.5 μm to -3.5 μm and at an electron dose of approximately 10⁻²⁵ e⁻ / Ų. Samples were prepared and imaged using NIS Nano Imaging Service. Figure 5A -B shows the results of negative staining analysis of the nanoparticles using electron microscopy. Figure 5AThe gL / gH / gp42 C18 ferritin nanoparticles were displayed, and Figure 5B The gp350D123 ferritin nanoparticles are shown. Uniform particle formation with very few aggregates can be observed.

Claims

1. A fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gH polypeptide and / or (ii) an EBV gL polypeptide; and (b) Ferritin of SEQ ID NO: 1, comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof.

2. The fusion protein of claim 1, wherein the EBV polypeptide further comprises the EBV gp42 polypeptide.

3. A fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising: (i) an EBV gp42 polypeptide and (ii) an EBV gH polypeptide and / or an EBV gL polypeptide; and (b) Ferritin of SEQ ID NO: 1, comprising at least one of the following substitutions: (i) glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; (ii) glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; or (iii) a combination thereof.

4. The fusion protein according to any one of claims 1-3, wherein the ferritin comprises glutamine at a residue corresponding to N102 of SEQ ID NO: 1 and glutamine at a residue corresponding to N145 of SEQ ID NO: 1, optionally wherein the ferritin further comprises glutamine at a residue corresponding to N18 of SEQ ID NO: 1 and serine at a residue corresponding to C30 of SEQ ID NO:

1.

5. The fusion protein according to any one of claims 1-4, wherein the bullfrog ferritin extension sequence is attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO:

2.

6. The fusion protein according to any one of claims 1-5, wherein the EBV polypeptide comprises: (i) EBV gp42 polypeptide, (ii) EBV gH polypeptide, and (iii) EBV gL polypeptide.

7. The fusion protein of claim 6, wherein the EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide and the ferritin are arranged in order from the N-terminus to the C-terminus.

8. The fusion protein of any one of claims 1-7, further comprising a linker between each EBV peptide.

9. The fusion protein of claim 8, wherein the linker between the EBV gL peptide and the EBV gH peptide is 44 to 48 amino acids long, and the linker between the EBV gH peptide and the EBV gp42 peptide is 32 amino acids long.

10. The fusion protein of claim 9, wherein the linker between the EBV gL polypeptide and the EBV gH polypeptide is 46 amino acids long, and the linker between the EBV gH polypeptide and the EBV gp42 polypeptide is 32 amino acids long.

11. The fusion protein of claim 8, wherein the linker separates the EBV gL polypeptide and the EBV gH polypeptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 3; and / or wherein the linker separates the EBV gH polypeptide and the EBV gp42 polypeptide and consists of an amino acid sequence having at least 80% identity with SEQ ID NO:

4.

12. The fusion protein according to any one of claims 1-11, further comprising an additional linker separating the ferritin and the EBVgp42 polypeptide.

13. The fusion protein of claim 12, wherein the additional linker has a length of 88 amino acids.

14. The fusion protein of claim 13, wherein the additional linker consists of an amino acid sequence having at least 80% identity with SEQ ID NO:

5.

15. The fusion protein according to any one of claims 1-14, wherein the ferritin comprises the amino acid sequence of SEQ ID NO:

23.

16. The fusion protein according to any one of claims 1-15, wherein the EBV gp42 polypeptide comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with residues 856-1038 of any one of SEQ ID NO: 9-11.

17. The fusion protein of claim 16, wherein the EBV gH polypeptide comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with residues 162-823 of any one of SEQ ID NO: 9-11; optionally wherein the EBV gH polypeptide comprises leucine at residue 189 corresponding to any one of SEQ ID NO: 9-11; glutamic acid at residue 368 corresponding to any one of SEQ ID NO: 9-11; and glutamic acid at residue 429 corresponding to any one of SEQ ID NO: 9-11.

18. The fusion protein of claim 16 or 17, wherein the EBV gL polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with residues 1-115 of any one of SEQ ID NO: 9-11; optionally wherein the EBV gL polypeptide comprises alanine at residue 36 corresponding to any one of SEQ ID NO: 9-11; glutamine at residue 47 corresponding to any one of SEQ ID NO: 9-11; and glutamine at residue 105 corresponding to any one of SEQ ID NO: 9-11.

19. The fusion protein according to any one of claims 1-18, comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the following: (a) amino acid residues 1-1133 of any one of SEQ ID NO: 9-11, (b) amino acid residues 1-1148 of any one of SEQ ID NO: 36, 38, or 40, or (c) amino acid residues 1-1156 of any one of SEQ ID NO: 37, 39, or 41.

20. The fusion protein of any one of claims 1-19, comprising a mammalian leader sequence located at the N-terminus of the fusion protein, optionally wherein the fusion protein comprises an EBV gL polypeptide, an EBV gH polypeptide, an EBV gp42 polypeptide, and ferritin, wherein the EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide, and the ferritin are arranged in N-terminus to C-terminus within the EBV polypeptide, and the mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide.

21. The fusion protein of claim 20, wherein the mammalian leader sequence is selected from SEQ ID NO: 12 and SEQ ID NO:

13.

22. A fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising an EBV gL polypeptide and an EBV gH polypeptide, wherein the EBV gL polypeptide comprises the sequence of residues 1-115 of SEQ ID NO: 11, and the EBV gH polypeptide comprises the sequence of residues 162-823 of SEQ ID NO:

11. (b) The ferritin polypeptide of SEQ ID NO: 1, or a ferritin polypeptide having SEQ ID NO: 1 and at least one mutation, wherein the at least one mutation is selected from one or more of the following: glutamine at a residue corresponding to position N18 of SEQ ID NO: 1; serine at a residue corresponding to position C30 of SEQ ID NO: 1; glutamine at a residue corresponding to position N102 of SEQ ID NO: 1; and glutamine at a residue corresponding to position N145 of SEQ ID NO: 1; and (c) A bullfrog ferritin extension sequence attached to the N-terminus of the ferritin, wherein the bullfrog ferritin extension sequence comprises the amino acid sequence of SEQ ID NO: 2, and optionally a mammalian leader sequence located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; The EBV gL polypeptide, the EBV gH polypeptide, and the ferritin are arranged sequentially from the N-terminus to the C-terminus within the EBV polypeptide, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide.

23. A fusion protein comprising: (a) An Epstein-Barr virus (EBV) polypeptide comprising an EBV gL polypeptide, an EBV gH polypeptide and an EBV gp42 polypeptide, wherein the EBV gL polypeptide comprises the amino acid sequence of residues 1-115 of SEQ ID NO: 11, the EBV gH polypeptide comprises the amino acid sequence of residues 162-823 of SEQ ID NO: 11, and the EBV gp42 polypeptide comprises the amino acid sequence of residues 856-1038 of SEQ ID NO: 11; (b) Ferritin containing SEQ ID NO: 24; and (c) Optionally located at the N-terminus of the fusion protein, wherein the mammalian leader sequence comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; The EBV gL polypeptide, the EBV gH polypeptide, the EBV gp42 polypeptide, and the ferritin are arranged in the EBV polypeptide in the order from the N-terminus to the C-terminus, and the optional mammalian leader sequence is located at the N-terminus of the EBV gL polypeptide.

24. The fusion protein of claim 22 or 23, wherein the linker separates the EBV gL polypeptide and the EBV gH polypeptide, and comprises the amino acid sequence of SEQ ID NO:

3.

25. The fusion protein of any one of claims 23-24, wherein the linker separates the EBV gH polypeptide and the EBVgp42 polypeptide, and comprises the amino acid sequence of SEQ ID NO:

4.

26. The fusion protein according to any one of claims 23-25, wherein the linker separates the EBV gp42 polypeptide from the ferritin and comprises the amino acid sequence of SEQ ID NO:

5.

27. The fusion protein according to any one of claims 22-26, wherein the ferritin consists of the amino acid sequence of SEQ ID NO:

24.

28. A fusion protein comprising the amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41.

29. A fusion protein comprising the amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41.

30. A fusion protein comprising (i) an EBV gp220 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO: 14, and (ii) ferritin comprising an amino acid sequence of any one of SEQ ID NO: 23, 24, 42, and 43.

31. The fusion protein of claim 30, wherein the amino acid sequence of the fusion protein consists of SEQ ID NO: 25 or 35.

32. A ferritin nanoparticle comprising a fusion protein as described in any one of claims 1-29.

33. A composition or kit comprising a first ferritin nanoparticle as described in claim 32, and optionally a second ferritin nanoparticle comprising a second fusion protein containing an EBV gp220 polypeptide and ferritin.

34. The composition or kit of claim 33, wherein the EBV gp220 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 1-425 of SEQ ID NO:

14.

35. The composition or kit of claim 33 or 34, wherein the ferritin in the second fusion protein contains a serine or cysteine ​​residue at position 110 of SEQ ID NO: 1; optionally, wherein the ferritin contains an amino acid sequence having at least 80% identity with residues 435-600 of SEQ ID NO:

14.

36. The composition or kit of claim 33, wherein the second fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, optionally wherein position C544 of SEQ ID NO: 14 is substituted with serine.

37. A composition or kit comprising: (a) a first ferritin nanoparticle comprising a fusion protein consisting of an amino acid sequence of any one of SEQ ID NO: 9-11; (b) a second ferritin nanoparticle comprising a second fusion protein consisting of an amino acid sequence of SEQ ID NO: 14; or (c) a second ferritin nanoparticle comprising a second fusion protein consisting of an amino acid sequence of SEQ ID NO: 14, wherein position C544 of SEQ ID NO: 14 is substituted with a serine residue.

38. A composition or kit comprising first ferritin nanoparticles and second ferritin nanoparticles, the first ferritin nanoparticles comprising a fusion protein consisting of the amino acid sequence of SEQ ID NO: 11, and the second ferritin nanoparticles comprising a second fusion protein consisting of the amino acid sequence of SEQ ID NO:

14.

39. A pharmaceutical composition comprising a fusion protein as described in any one of claims 1-31, or ferritin nanoparticles as described in claim 32, or a composition as described in any one of claims 33-38; and a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable carrier comprises an adjuvant.

41. A bivalent vaccine comprising the composition of any one of claims 33-38, wherein the fusion protein and the second fusion protein are present in a 1:1 weight ratio, or wherein the fusion protein and the second fusion protein are present in a 1:1 molar ratio.

42. The fusion protein of any one of claims 1-31, the composition of any one of claims 33-38, the ferritin particles of claim 32, the pharmaceutical composition of claim 39 or claim 40, or the bivalent vaccine of claim 41, for use in methods of inducing an immune response to EBV in a subject, protecting a subject from EBV infection, or preventing EBV-related diseases in a subject.

43. A method for inducing an immune response to EBV in a subject, protecting the subject from EBV infection, or preventing EBV-related diseases in a subject, the method comprising administering to the subject a fusion protein as described in any one of claims 1-31, a composition as described in any one of claims 33-38, ferritin particles as described in claim 32, a pharmaceutical composition as described in claim 39 or claim 40, or a bivalent vaccine as described in claim 41.

44. A nucleic acid encoding a fusion protein as described in any one of claims 1-31.

45. An expression vector comprising the nucleic acid as described in claim 44; and optionally further comprising the nucleic acid encoding a second fusion protein of the composition as described in any one of claims 32-38.

46. ​​A host cell comprising the nucleic acid as described in claim 44 or the expression vector as described in claim 45.

47. A method for preparing a fusion protein as described in any one of claims 1-31, the method comprising culturing a host cell under conditions in which the fusion protein is expressed in a host cell as described in claim 46, and recovering the fusion protein from the cultured host cell culture.

48. The method of claim 47, wherein the host cells are Chinese hamster ovary cells.

49. The method of claim 47 or claim 48, wherein the host cells are dihydrofolate reductase (DHFR) deficient; and the expression vector further comprises a polynucleotide sequence encoding DHFR.

50. The method of claim 49, further comprising culturing the host cells in the presence of a DHFR selector; optionally wherein the DHFR selector comprises methotrexate.

51. The method of any one of claims 47-50, wherein the expression vector comprises a polynucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 9-11 or 36-41.

52. The method of claim 51, wherein the expression vector comprises a polynucleotide sequence of any one of SEQ ID NO: 19-22.

53. A fusion protein which can be obtained or generated by the method of any one of claims 47-52.

54. A ferritin consisting of the amino acid sequence of SEQ ID NO: 24.

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