Antigenic epstein barr virus polypeptides

Abstract

To provide Epstein-Barr virus (EBV) polypeptides and their use in eliciting antibodies against EBV.SOLUTION: Antigenic Epstein-Barr virus (EBV) polypeptides comprising an EBV gL polypeptide and an EBV gH polypeptide, a linker having a length of at least 15 amino acids separating the EBV gL polypeptide and the EBV gH polypeptide, are provided. Antigenic EBV polypeptides comprising an EBV polypeptide and a ferritin protein are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 652,201, filed April 3, 2018, which is incorporated herein by reference in its entirety.

[0002] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on March 27, 2019, is named 2019-03-27_01121-0032-00PCT_SL_ST25.txt, and is 377,803 bytes in size. [Background technology]

[0003] Despite numerous successes in the field of vaccinology, novel breakthroughs are needed to protect humans from many life-threatening infectious diseases. Many currently licensed vaccines rely on decades-old technologies to produce live-attenuated or inactivated-killed vaccines, which have inherent safety concerns, often stimulate only short-lived and weak immune responses, and require the administration of multiple doses. Advances in genetic and biochemical engineering have made it possible to develop therapeutics against challenging disease targets, but their application in vaccinology has yet to be fully realized. Recombinant protein technology now allows for the design of optimal antigens. Additionally, nanoparticles are increasingly demonstrating their potential for optimal antigen presentation and targeted drug delivery. Nanoparticles conjugated with multiple antigens have been shown to have increased binding avidity through the multivalent presentation of their molecular cargo and the ability to cross biological barriers more efficiently due to their microscopic size. Helicobacter pylori (H. pylori) ferritin nanoparticles fused to the influenza virus hemagglutinin (HA) protein enabled improved antigen stability and increased immunogenicity in a mouse influenza model (see Non-Patent Document 1). This fusion protein self-assembles into octahedral symmetric nanoparticles displaying eight trimeric HA spike structures, and when used with adjuvants, confers potent immune responses in various preclinical models.

[0004] Epstein-Barr virus (EBV) infects approximately 95% of the world's adult population and is known to be associated with two B-cell lymphomas, Burkitt lymphoma and Hodgkin lymphoma. The virus can also infect epithelial cells and has been associated with nasopharyngeal carcinoma. Additionally, EBV causes the majority of cases of infectious mononucleosis in developed countries, primarily affecting children and young adults. Infectious mononucleosis can result in a long recovery period, lasting up to one month. There is currently no approved vaccine on the market, and therefore there is a strong need for a preventative vaccine.

[0005] This paper presents a set of novel polypeptides, nanoparticles, compositions, methods, and uses involving EBV polypeptides. Novel EBV single-chain gL and gH (sometimes referred to as gL / gH or gH / gL) polypeptides were produced, as were antigenic polypeptides comprising these novel EBV polypeptides and ferritin. It was observed that antigenic polypeptides and nanoparticles comprising single-chain gL and gH polypeptides can contain a relatively long linker between the gL and gH sequences, which provides increased immunogenicity. Antigenic ferritin polypeptides and nanoparticles comprising EBV gp220 polypeptides were also produced. Furthermore, self-adjuvanting antigenic polypeptides comprising the described EBV polypeptides and ferritin were developed, in which an immunostimulatory moiety, such as an adjuvant, was chemically linked directly to the antigenic polypeptide. Direct conjugation of the immunostimulatory moiety to the antigenic polypeptide allows for targeted co-delivery of the immunostimulatory moiety and EBV polypeptide as a single polymeric entity, thereby greatly reducing the potential for systemic toxicity that is a concern with conventional vaccines that contain immunostimulatory moieties such as antigens and adjuvants as separate molecules. Co-delivery of the immunostimulatory moiety with the EBV polypeptide as a polymeric entity, and its multivalent presentation, can also reduce the overall dose required to induce protection and reduce manufacturing burden and costs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kanekiyo et al., Nature 499:102-106 (2013) Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present disclosure to provide compositions, kits, methods, and uses that can provide one or more of the above advantages, or that at least provide the public with a useful choice. [Means for solving the problem]

[0008] Embodiment 1 is an antigenic Epstein-Barr virus (EBV) polypeptide comprising an EBV gL polypeptide and an EBV gH polypeptide, wherein a linker having a length of at least 15 amino acids separates the EBV gL polypeptide and the EBV gH polypeptide.

[0009] Embodiment 2 is an antigenic Epstein-Barr virus (EBV) polypeptide comprising an EBV gL polypeptide, an EBV gH polypeptide, and an EBV gp42 polypeptide, wherein a linker having a length of at least 15 amino acids separates the EBV gL polypeptide and the EBV gH polypeptide.

[0010] Embodiment 3 is the antigenic EBV polypeptide of embodiment 1 or 2, further comprising ferritin.

[0011] Embodiment 4 is an antigenic EBV polypeptide comprising an EBV polypeptide and a ferritin protein, wherein the ferritin protein comprises a mutation that replaces a surface-exposed amino acid with a cysteine.

[0012] Embodiment 5 is the antigenic EBV polypeptide of embodiment 4, wherein the EBV polypeptide comprises an EBV gL polypeptide, an EBV gH polypeptide, or an EBV gp220 polypeptide.

[0013] Embodiment 6 is the antigenic EBV polypeptide of embodiment 5, wherein the EBV polypeptide comprises a gL polypeptide and the polypeptide further comprises an EBV gH polypeptide.

[0014] Embodiment 7 is the antigenic EBV polypeptide of any one of embodiments 1 or 3-6, wherein the polypeptide further comprises an EBV gp42 polypeptide.

[0015] Embodiment 8 is a composition comprising a first antigenic EBV polypeptide and a second antigenic EBV polypeptide, wherein the first antigenic EBV polypeptide comprises a ferritin heavy chain and the first EBV polypeptide, and the second antigenic EBV polypeptide comprises a ferritin light chain and a second EBV polypeptide, and wherein the first and second EBV polypeptides are different.

[0016] Embodiment 9 is the composition of embodiment 8, wherein the first EBV polypeptide or the second EBV polypeptide comprises a gp220 polypeptide.

[0017] Embodiment 10 is a composition according to embodiment 8 or 9, wherein (i) the first antigenic EBV polypeptide comprises one or both of a gL polypeptide and a gH polypeptide, and the second antigenic EBV polypeptide comprises a gp220 polypeptide, or (ii) the first antigenic EBV polypeptide comprises a gp220 polypeptide and the second antigenic EBV polypeptide comprises one or both of a gL polypeptide and a gH polypeptide.

[0018] Embodiment 11 is a composition according to any one of embodiments 8 to 10, wherein the first antigenic EBV polypeptide comprises a gL polypeptide and a gH polypeptide, or the second antigenic EBV polypeptide comprises a gL polypeptide and an EBV gH polypeptide.

[0019] Embodiment 12 is the composition of embodiment 10 or 11, wherein the antigenic EBV polypeptides comprising a gL polypeptide and / or a gH polypeptide further comprise a gp42 polypeptide.

[0020] Embodiment 13 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 12, comprising gH and gL polypeptides, wherein the gH polypeptide is C-terminal to the gL polypeptide, optionally comprising a gp42 polypeptide, and the gp42 polypeptide is C-terminal to the gH polypeptide.

[0021] Embodiment 14 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 13, comprising a gp42 polypeptide, wherein the gp42 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 239 or 240.

[0022] Embodiment 15 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 14, comprising an EBV gH polypeptide and an EBV gp42 polypeptide, wherein a linker having a length of at least 15 amino acids separates the EBV gH polypeptide and the EBV gp42 polypeptide, optionally the linker having 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, and further optionally the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 234.

[0023] Embodiment 16 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 15, comprising a linker, the linker having a length of at least 15 amino acids, and optionally separating the first EBV polypeptide and the second EBV polypeptide.

[0024] Embodiment 17 is an antigenic EBV polypeptide or composition according to embodiment 16, wherein the linker has 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.

[0025] Embodiment 18 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 17, wherein the EBV polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 36.

[0026] Embodiment 19 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 18, wherein the EBV polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 37.

[0027] Embodiment 20 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 19, wherein the polypeptide comprises a linker comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 30, and optionally the linker separates the first EBV polypeptide and the second EBV polypeptide.

[0028] Embodiment 21 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 20, wherein the EBV polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 38.

[0029] Embodiment 22 is an antigenic EBV polypeptide or composition according to any one of embodiments 3 to 21, further comprising an additional linker separating the EBV polypeptide and the ferritin.

[0030] Embodiment 23 is an antigenic EBV polypeptide or composition according to any one of embodiments 3 to 22, comprising an EBV gp42 polypeptide located N-terminal to ferritin and C-terminal to the gH polypeptide, wherein a linker separates the EBV gp42 polypeptide and ferritin, and optionally the linker is at least 15 amino acids in length, or 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 in length, and further optionally the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 233, 234, 235, 236, 237, or 238.

[0031] Embodiment 24 is an antigenic EBV polypeptide or composition according to embodiment 22 or 23, wherein the linker comprises a cysteine.

[0032] Embodiment 25 is an antigenic EBV polypeptide or composition according to any one of embodiments 22 to 24, wherein the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 33.

[0033] Embodiment 26 is an antigenic EBV polypeptide or composition according to embodiment 24 or 25, wherein the cysteine ​​is conjugated to an immunostimulatory moiety, and optionally the immunostimulatory moiety is an agonist of TLR2, TLR7 / 8, TLR9, or STING.

[0034] Embodiment 27 is an antigenic EBV polypeptide or composition according to any one of embodiments 3 to 26, wherein the ferritin comprises one or more of the E12C, S26C, S72C, A75C, K79C, S100C, and S111C mutations of H. pylori ferritin, or one or more corresponding mutations in a non-H. pylori ferritin, as determined by pairwise or structural alignment.

[0035] Embodiment 28 is an antigenic EBV polypeptide or composition described in any one of embodiments 3 to 27, wherein the ferritin comprises a mutation replacing a surface-exposed asparagine with a non-asparagine amino acid, optionally with the asparagine at position 19 of H. pylori ferritin or at an analogous position in a non-H. pylori ferritin as determined by pairwise or structural alignment.

[0036] Embodiment 29 is an antigenic EBV polypeptide or composition according to any one of embodiments 3 to 28, wherein the ferritin comprises a mutation replacing an internal cysteine ​​with a non-cysteine ​​amino acid, and optionally the internal cysteine ​​is at position 31 of H. pylori ferritin or at a position corresponding to position 31 of H. pylori ferritin as determined by pairwise or structural alignment.

[0037] Embodiment 30 is an antigenic EBV polypeptide or composition according to any one of embodiments 3 to 29, wherein the ferritin comprises an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 201-207 or 211-215.

[0038] Embodiment 31 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 30, wherein the antigenic EBV polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to amino acids 23 to 1078 of SEQ ID NO: 226.

[0039] Embodiment 32 is an antigenic EBV polypeptide or composition according to any one of embodiments 1 to 31, wherein the antigenic EBV polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to any of SEQ ID NOs: 226-231 or 241-242, and optionally lacks a leader sequence.

[0040] Embodiment 33 is a ferritin particle comprising the antigenic EBV polypeptide or the first and second polypeptides according to any one of embodiments 3 to 32.

[0041] Embodiment 34 is a composition comprising an antigenic EBV polypeptide or ferritin particle according to any one of embodiments 1 to 33 and a pharmaceutically acceptable carrier.

[0042] Embodiment 35 is the composition of embodiment 34, wherein the ferritin particle comprises an EBV gL polypeptide and an EBV gH polypeptide, and the composition further comprises a second ferritin particle comprising a gp220 polypeptide.

[0043] Embodiment 36 is an antigenic EBV polypeptide, ferritin particle, or composition according to any one of embodiments 1 to 35 for use in a method of inducing an immune response against influenza or in protecting a subject from infection by EBV.

[0044] Embodiment 37 is a method for inducing an immune response against EBV or protecting a subject against EBV infection, comprising administering to a subject one or more antigenic EBV polypeptides, ferritin particles, or compositions described in any one of embodiments 1 to 36.

[0045] Embodiment 38 is the antigenic EBV polypeptide, ferritin particle, composition, or method of embodiment 36 or 37, wherein the subject is a human.

[0046] Embodiment 39 is a nucleic acid encoding an antigenic EBV polypeptide according to any one of embodiments 1 to 32, optionally wherein the nucleic acid is mRNA.

[0047] Additional objects and advantages will be set forth in the description which follows and / or will be obvious from the description or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0048] The foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims.

[0049] The following drawings, which are incorporated in and constitute a part of this specification, illustrate specific embodiments and, together with the description, serve to explain the principles described herein. [Brief explanation of the drawings]

[0050] [Figure 1-1] Figures 1A-1B show the single-chain gL and gH monomers (Figure 1A) (SEQ ID NO: 6) and trimers (Figure 1B) (SEQ ID NO: 11) with and without His-tag removal by Coomassie and Western blot analysis. Figure 1B also shows UV absorbance traces of fractions from size-exclusion column (Superose® 6) purification. [Figure 1-2] Continued from Figure 1-1. [Figure 2-1] Figures 2A-2E show the purification and characterization of single-chain gL / gH-ferritin nanoparticles (SEQ ID NO: 14). UV absorbance traces of Superose® 6 purified fractions (Figure 2A) and Coomassie (Figure 2B) and Western blot (Figure 2C) analyses of selected fractions from the purification (L indicates a molecular weight ladder; the positions of the 150 and 250 kDa bands are indicated to the right of Figure 2B) are shown. Dynamic light scattering (Figure 2D) and electron microscopy (Figure 2E) analyses of the single-chain nanoparticles are also presented. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3]Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 3] FIG. 1 shows different representative single-chain gL / gH-ferritin constructs. [Figure 4] Figure 1 shows antibody titers after immunization of mice with single-chain gL / gH trimers or nanoparticles (NPs) mixed with AF03 adjuvant, a squalene emulsion-based adjuvant. *p-value=<0.05 when comparing NP constructs with their corresponding trimer constructs. From left to right, the constructs were SEQ ID NOs: 16, 10, 11, 13, 12, and 14. [Figure 5] Figures 5A-5B show the anti-gL / gH antibody response in mice to a bivalent composition containing both gp220 nanoparticles (SEQ ID NO: 1) and single-chain gL / gH nanoparticles ("gL_gH_C5 NP," SEQ ID NO: 19), compared with single-chain gL / gH nanoparticles and a negative control, naked ferritin (i.e., ferritin not conjugated to any non-ferritin polypeptides or immunostimulatory moieties). The results show that the use of the bivalent composition does not interfere with the anti-gL / gH antibody response, compared with the single-chain gL / gH results with the negative control, naked ferritin. Both compositions contained AF03 adjuvant. ELISA results at individual dilutions (Figure 5A) and binding titers (Figure 5B) are shown. [Figure 6] Figures 6A-6B show anti-gp220 antibody responses to a bivalent composition containing both gp220 nanoparticles and single-chain gL / gH nanoparticles, as described for Figures 5A-5B. The results demonstrate that the use of a bivalent composition does not result in interference with the anti-gp220 antibody response, compared to the results of gp220 nanoparticles with naked ferritin as a negative control. Both compositions contained AF03 adjuvant. ELISA results at individual dilutions (Figure 6A) and binding titers (Figure 6B) are shown. [Figure 7]Figure 7A shows the design of a nanoparticle comprising an EBV polypeptide and ferritin, containing mutations that replace surface-exposed amino acids with cysteines for conjugation to an immunostimulatory moiety, such as a Toll-like receptor (TLR) agonist. See SEQ ID NO: 14 for an exemplary sequence corresponding to this design. Here, the single-chain gL / gH antigen is attached to ferritin via a flexible 46-amino acid linker. Figure 7B shows a representative Toll-like receptor agonist (SM7 / 8a with a PEG4-maleimide linker) suitable for conjugation to the construct according to Figure 7A. Figure 7C shows an electron micrograph (EM) image of a gL / gH nanoparticle with SM7 / 8a conjugated via a PEG4-maleimide linker and a cysteine ​​on the ferritin surface. [Figure 8A] FIG. 8A is a diagram showing the structure of a portion of ferritin containing a mutation that replaces a surface-exposed amino acid with a cysteine, with the location of the cysteine ​​indicated. [Figure 8B] Figure 8B shows the conjugation of CpG adjuvant (SEQ ID NO: 247) to ferritin by adjacently arranging the ferritin, linker, and CpG adjuvant, which are oriented to show the portions of each moiety that become bound in close proximity to each other. [Figure 9A] 9A and 9B show mass spectrometry (MS) spectra of the unconjugated (FIG. 9A) and SM7 / 8a-conjugated (FIG. 9B) forms of gL / gH-ferritin. The difference in mass of the main peak is 711 Da, which roughly corresponds to the difference expected from the conjugate of SM7 / 8a with the linker. [Figure 9B] 9A and 9B show mass spectrometry (MS) spectra of the unconjugated (FIG. 9A) and SM7 / 8a-conjugated (FIG. 9B) forms of gL / gH-ferritin. The difference in mass of the main peak is 711 Da, which roughly corresponds to the difference expected from the conjugate of SM7 / 8a with the linker. [Figure 10A]10A and 10B show mass spectrometry (MS) spectra of the unconjugated (FIG. 10A) and SM7 / 8a-conjugated (FIG. 10B) forms of gp220-ferritin. The difference in mass of the main peak is 714.7 Da, which roughly corresponds to the difference expected from conjugation of SM7 / 8a with a linker. [Figure 10B] 10A and 10B show mass spectrometry (MS) spectra of the unconjugated (FIG. 10A) and SM7 / 8a-conjugated (FIG. 10B) forms of gp220-ferritin. The difference in mass of the main peak is 714.7 Da, which roughly corresponds to the difference expected from conjugation of SM7 / 8a with a linker. [Figure 11] Figures 11A-11D show electron microscopy (EM) images of unconjugated (Figure 11A,C) and conjugated (Figure 11B,D) single-chain gL / gH (Figure 11A,B) and gp220 (Figure 11C,D) ferritin nanoparticles, demonstrating that conjugation of SM7 / 8a to these nanoparticles did not disrupt the nanoparticle structure. [Figure 12] 12A-12B show antibody responses in mice after treatment with single-chain gL / gH without conjugated SM7 / 8a or other adjuvants, AF03 adjuvant as a separate molecule, or ferritin nanoparticles containing conjugated SM7 / 8a. ELISA results are shown as individual dilutions (FIG. 12A) and binding titers (FIG. 12B). [Figure 13] 13A-13B show antibody responses in mice after treatment with nanoparticles containing either AF03 adjuvant alone, as a separate molecule, or conjugated SM7 / 8a. ELISA results are shown as individual dilutions (FIG. 13A) and binding titers (FIG. 13B). [Figure 14]14A-14B show anti-gL / gH antibody responses in mice after treatment with SM7 / 8a-conjugated gp220 nanoparticles and SM7 / 8a-conjugated single-chain gL / gH ferritin nanoparticles compared to treatment with SM7 / 8a-conjugated single-chain gL / gH ferritin nanoparticles and naked ferritin, as measured by ELISA. Results are shown for experiments without (FIG. 14A) or with (FIG. 14B) mixed AF03. [Figure 15] 15A-15B show anti-gp220 antibody responses in mice after treatment with SM7 / 8a-conjugated gp220 nanoparticles and single-chain gL / gH nanoparticles conjugated to SM7 / 8a compared to treatment with SM7 / 8a-conjugated gp220 nanoparticles and naked ferritin, as measured by ELISA. Results are shown for experiments without (FIG. 15A) or with (FIG. 15B) mixed AF03. [Figure 16] FIG. 1 shows anti-gL / gH antibody responses in mice after treatment with single-chain gL / gH nanoparticles (gL / gH_C5, SEQ ID NO: 19) and naked ferritin, with or without mixed AF03 adjuvant and / or SM7 / 8a conjugated to single-chain gL / gH nanoparticles, as measured by ELISA endpoint titers. [Figure 17] FIG. 1 shows anti-gp220 antibody responses in mice after treatment with gp220 nanoparticles and naked ferritin, with or without mixed AF03 adjuvant and / or SM7 / 8a conjugated to gp220 nanoparticles. [Figure 18] 1 shows anti-gL / gH antibody responses in mice after treatment with a bivalent composition comprising gp220 nanoparticles and single-chain gL / gH nanoparticles. As indicated in the legend, some nanoparticles were conjugated to SM7 / 8a and some nanoparticles were mixed with AF03. The top-to-bottom symbol order of the key corresponds to the left-to-right symbol order of the graph. [Figure 19] Figure 1 shows anti-gp220 antibody responses in mice after treatment with bivalent compositions containing single-chain gL / gH nanoparticles and gp220 nanoparticles and / or naked ferritin. As indicated in the legend, some nanoparticles were conjugated to SM7 / 8a and some nanoparticles were mixed with AF03. The order of symbols from top to bottom in the key corresponds to the order of symbols from left to right on the graph. [Figure 20-1] 20A-20D show antibody responses in mice after treatment with gL_gH_C7 nanoparticles (SEQ ID NO: 20) with or without conjugation to mixed AF03 and / or SM7 / 8a (FIGS. 20A, 20C, or 20D) or CpG oligodeoxynucleotides (FIG. 20B). Shown are endpoint titers from prime bleeds measured by ELISA (FIG. 20A), ELISA results at individual dilutions from booster bleeds (FIG. 20B), and endpoint titers measured by ELISA from booster bleeds (FIG. 20C) and terminal bleeds (FIG. 20D). [Figure 20-2] Continuation of Figure 20-1. [Figure 21] FIG. 1 shows antibody responses in mice after treatment with gL_gH_C5 nanoparticles (SEQ ID NO: 19), with or without conjugation to mixed AF03 adjuvant and / or SM7 / 8a, as endpoint titers measured by ELISA from prime, boost, and terminal bleeds. [Figure 22] FIG. 1 shows antibody responses in mice after treatment with gp220 nanoparticles with or without conjugation to mixed AF03 adjuvant and / or SM7 / 8a as endpoint titers measured by ELISA from prime, boost, and terminal bleeds. [Figure 23A]Figure 23A shows the light and heavy chains of T. ni ferritin, with or without fusion to either gp220 or gL / gH, visualized by Coomassie staining (Figure 23A). The 20, 25, 75, 100, and 150 kDa markers in the rightmost lane of Figure 23A are labeled. [Figure 23B] Figure 23B provides examples of constructs containing the light and heavy chains of T. ni ferritin, with or without fusion to either gp220 or gL / gH. [Figure 24-1] Figures 24A-24D demonstrate ion exchange (Q column) and size exclusion chromatography (SEC) purification of gp220-T. ni ferritin. Absorbance traces from the Q column at pH 7 (Figure 24A) and SEC Superose® 6,16 / 600 (Figure 24B), Coomassie stain of fractions from the Q column at pH 7 (Figure 24C) (lanes from the left are input ("In"), flow-through ("FT"), molecular weight ladder (sizes in kD on the left), and selected fractions), and Coomassie stain of fractions from SEC Superose® 6,16 / 600 (Figure 24D) (lanes from the left are molecular weight ladder (sizes in kD on the left) and selected fractions) are shown. Figure 24E shows an example construct. [Figure 24-2] Continuation of Figure 24-1. [Figure 24-3] Continued from Figure 24-2. [Figure 25-1]Figures 25A-25D demonstrate ion exchange (Q column) and size exclusion chromatography (SEC) purification of gL / gH(light) / gp220(heavy)-T. ni ferritin. Absorbance traces from the Q column at pH 7 (Figure 25A) and SEC Superose® 6,16 / 600 (Figure 25B), Coomassie stain of fractions from the Q column at pH 7 (Figure 25C) (lanes from the left are input ("In"), flow-through ("FT"), molecular weight ladder (sizes in kD on the left), and selected fractions), and Coomassie stain of fractions from SEC Superose® 6,16 / 600 (Figure 25D) (lanes from the left are molecular weight ladder (sizes in kD on the left) and selected fractions) are shown. Figure 25E shows an example of a construct. [Figure 25-2] Continuation of Figure 25-1. [Figure 25-3] Continued from Figure 25-2. [Figure 26-1] Figures 26A-26H show gp220-T. ni ferritin or gL / gH (light chain) / gp220 (heavy chain)-T. ni ferritin constructs visualized by Coomassie staining (Figures 26A and 26E), illustrated diagrammatically (Figures 26B and 26F), characterized by dynamic light scattering (DLS) (Figures 26D and 26H), and visualized by electron micrographs (Figures 26C and 26G). Figures 26A-26D show data using gp220 fused to both the light and heavy chains (Figure 26B). Figures 26E-26H show data using gp220 fused to the heavy chain and gL / gH fused to the light chain (Figure 26F). [Figure 26-2] Continuation of Figure 26-1. [Figure 26-3] Continued from Figure 26-2. [Figure 26-4] Continued from Figure 26-3. [Figure 27-1] Figures 27A-27C show naked T. ni ferritin particles (i.e., not fused to non-ferritin polypeptides) visualized by Coomassie staining (Figure 27A), visualized in electron micrographs (Figure 27B), and characterized by DLS (Figure 27C). [Figure 27-2] Continuation of Figure 27-1. [Figure 28A] Figure 28A shows an SDS-denaturing Coomassie-stained gel (left) using purified gH / gL / gp42 NP construct (SEQ ID NO: 227) expressed in 293 expi cells, and (right) a size-exclusion chromatography (SEC) peak of gH / gL / gp42 NP. The horizontal axis of the SEC chromatogram is in mL. [Figure 28B] Figure 28B shows gH / gL / gp42 NP purified from a CHO pool to have a dynamic light scattering radius of approximately 26.2 nm. [Figure 29] Figures 29A-B show evaluation of immune responses elicited by monovalent gH / gL / gp42 nanoparticle compositions combined with naked ferritin nanoparticles or bivalent compositions (gH / gL / gp42 nanoparticles combined with gp220). Figure 29A shows B cell neutralization. Figure 29B shows epithelial cell neutralization. [Figure 30-1] Figures 30A-E show endpoint binding titers against the indicated antigens. Figures 30F-G show EBV virus neutralization assays of sera from ferrets vaccinated as indicated. Prime=Inj.1, boost=Inj.2. [Figure 30-2] Continuation of Figure 30-1. [Figure 30-3] Continuation of Figure 30-2. [Figure 30-4] Continuation of Figure 30-3. [Figure 31A] Figure 31A shows the purification of gH / gL / gp42_NP_C12 (SEQ ID NO: 228) using Superose 6 size exclusion chromatography. Arrows indicate fractions collected from the peak by denaturing Coomassie gel analysis and Western blot analysis using anti-ferritin antibody. [Figure 31B] FIG. 31B is a dynamic light scattering analysis of the sample in FIG. 31A showing a particle size radius of 20.6 nm. [Figure 32A]Figure 32A shows the purification of gH / gL / gp42_NP_C13 (SEQ ID NO: 229) using Superose 6 size exclusion chromatography. Arrows indicate fractions collected from the peak by denaturing Coomassie gel analysis and Western blot analysis using anti-ferritin antibody. [Figure 32B] FIG. 32B is a dynamic light scattering analysis of the sample in FIG. 32A showing a particle size radius of 17.1 nm. [Figure 33A] Figure 33A shows the purification of gH / gL / gp42_NP_C14 (SEQ ID NO: 230) using Superose 6 size exclusion chromatography. Arrows indicate fractions collected from the peak by denaturing Coomassie gel analysis and Western blot analysis using anti-ferritin antibody. [Figure 33B] FIG. 33B is a dynamic light scattering analysis of the sample in FIG. 33A showing a particle size radius of 16.9 nm. [Figure 34] The SDS-reducing Coomassie gel on the left shows the purified single-chain gH / gL / gp42-His product (SEQ ID NO: 226). The protein was purified using nickel affinity chromatography. On the right is the 2.9 Å crystal structure of the single-chain gH / gL / gp42-His product (SEQ ID NO: 226). Gp42 (dark gray and indicated by an arrow) interacts with the gH / gL heterodimer. [Figure 35]Figures 35A-E show depictions of single-chain constructs of gH / gL / gp42 fused to ferritin (as in each of SEQ ID NOS: 227-231) shown in Figure 35A. Fusion between each protein is via a flexible amino acid linker or the rigid amino acid linker specified above. Single-chain gH / gL / gp42 molecules confirm a 1:1:1 ratio of heterotrimer formation on nanoparticles. The crystal structure of this heterotrimer has been solved to show that single-chain gH / gL / gp42 can adopt a heterotrimer formation similar to that of wild-type gH, gL, and gp42 proteins found in nature (Figure 35B; see also Figure 34). Figure 35C is a model of how this single-chain gH / gL / gp42 heterotrimer is displayed on a nanoparticle via fusion with ferritin. There are 24 copies of single-chain gH / gL / gp42 displayed on a single nanoparticle. Figure 35D shows purification after expression of SEQ ID NO:227 in 293Expi cells. A denaturing SDS Coomassie gel shows that gH / gL / gp42 fused to ferritin exceeds 150 kD due to glycosylation. Figure 35E shows negative staining electron microscopy analysis of the purified product, demonstrating that single-chain gH / gL / gp42 fused to ferritin can successfully form nanoparticles that display gH / gL / gp42 antigens on their surface. DETAILED DESCRIPTION OF THE INVENTION

[0051] EBV polypeptides are provided that can be antigenic when administered alone, with an adjuvant as a separate molecule, and / or as part of nanoparticles (e.g., ferritin particles or lumazine synthase particles), which can be self-adjuvanting. Such polypeptides and compositions containing such polypeptides can be used to induce antibody responses against Epstein-Barr virus (EBV). EBV polypeptides can include gL, gH, gL / gH, gp220, or gp42 polypeptides, or combinations thereof, and multimerization domains such as ferritin. Ferritin can contain mutations that replace surface-exposed amino acids with cysteines, which can facilitate conjugation of immunostimulatory moieties to ferritin via the cysteines. Such conjugation can eliminate or reduce the need for separately administered adjuvants, potentially reducing the amount of adjuvant / immunostimulatory moiety required to induce an immune response against the EBV polypeptide. In some embodiments, an antigenic EBV polypeptide is provided that includes (i) an EBV polypeptide and (ii-a) a ferritin comprising a surface-exposed cysteine, or (ii-b) a ferritin and an N-terminal or C-terminal linker comprising a cysteine. Any of the EBV polypeptides described herein can be combined with any of the ferritins described below. Nucleic acids encoding the polypeptides described herein are also provided.

[0052] A.Definition 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, or gp220 amino acid sequence encoded by EBV, respectively. For example, a polypeptide having at least 80% identity to an EBV-encoded polypeptide necessarily contains a portion of an 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 an EBV polypeptide as part or all of the antigenic polypeptide can confer protection against infection with EBV. Unless the context states otherwise, any polypeptide disclosed herein, including an EBV polypeptide, may include all or part of multiple sequences encoded by EBV (e.g., all or part of EBV gL and gH, or all or part of EBV gL, gH, and gp42).

[0053] As used herein, "monomer" or "monomeric construct" refers to a construct expressed as a single-chain protein. A monomer can include EBV gL and gH expressed as a single chain, or EBV gL, gH, and gp42 expressed as a single chain.

[0054] As used herein, "trimer" or "trimeric construct" refers to a construct comprising EBV gL and / or gH together with a trimerization domain, such as the Foldon trimerization domain derived from T4 phage fibritin. Other trimerization domains, such as the human collagen XVIII trimerization domain (see, e.g., Alvarez-Cienfuegos et al., Scientific Reports 2016;6:28643) and the L1ORF1p trimerization domain (see, e.g., Khazina et al., Proc Natl Acad Sci USA 2009 Jan 12;106(3):731-36), are also known in the art and can be used in the trimerization construct.

[0055] "Ferritin" or "ferritin protein," as used herein, refers to a protein that has detectable sequence identity to H. pylori ferritin (SEQ ID NO: 208 or 209), or another ferritin discussed herein, such as P. furiosus ferritin, Trichoplusia ni ferritin, or human ferritin, and that serves to store iron, for example, within cells or tissues, or to transport iron to the bloodstream. Exemplary such ferritins, including ferritins that exist as two polypeptide chains known as heavy and light chains (e.g., Trichoplusia ni and human ferritin), are discussed in detail below. In some embodiments, the ferritin comprises a sequence having at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to a ferritin sequence disclosed herein, for example, in Table 2 (Sequence Listing). The ferritin can be a fragment of a full-length naturally occurring sequence.

[0056] "Wild-type ferritin," as used herein, refers to ferritin whose sequence consists of a naturally occurring sequence. Ferritin also includes full-length ferritin or fragments of ferritin that have one or more differences in their amino acid sequence from wild-type ferritin.

[0057] As used herein, "ferritin monomer" refers to a single ferritin molecule (e.g., a single ferritin heavy or light chain, if applicable) that is not assembled with other ferritin molecules. "Ferritin multimer" includes multiple associated ferritin monomers. "Ferritin protein" includes monomeric ferritin and multimeric ferritin.

[0058] As used herein, "ferritin particle" refers to ferritin that has self-assembled into a spherical form. Ferritin particles are sometimes referred to as "ferritin nanoparticles" or simply "nanoparticles." In some embodiments, a ferritin particle comprises 24 ferritin monomers (or 24 total heavy and light chains, if applicable).

[0059] As used herein, "hybrid ferritin" refers to a ferritin comprising H. pylori ferritin with an amino-terminal extension of bullfrog ferritin. An exemplary sequence used as the amino-terminal extension of bullfrog ferritin is set forth as SEQ ID NO: 217. In hybrid ferritins, the amino-terminal extension of bullfrog ferritin can be fused to H. pylori ferritin so that the binding sites for the immunostimulatory moieties are uniformly distributed on the surface of the ferritin particle. A "bullfrog linker," as used herein, is a linker comprising the sequence of SEQ ID NO: 217. Hybrid ferritins are also sometimes referred to as "bfpFerr" or "bfp ferritin." Any construct containing bullfrog sequences can also be provided without the bullfrog sequences, for example, without a linker or alternative linker. Exemplary bullfrog linker sequences are provided in Table 2. While Table 2 shows bullfrog linkers, the same constructs can be made without the linker or alternative linker.

[0060] "N-glycan," as used herein, refers to a carbohydrate chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. Thus, an N-glycan is formed by the process of N-glycosylation. The glycan may be a polysaccharide.

[0061] "Glycosylation," as used herein, refers to the addition of carbohydrate units to a protein.

[0062] As used herein, an "immune response" refers to the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells, to a stimulus, such as an antigen or a vaccine. An immune response can include any cell of the body involved in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. An immune response includes, but is 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 prevents the development of a disease associated with infection). Methods for measuring immune responses are well known in the art and include, for example, measuring lymphocyte (e.g., B or T cell) proliferation and / or activity, cytokine or chemokine secretion, inflammation, antibody production, etc. An "antibody response" is an immune response in which antibodies are produced.

[0063] As used herein, "antigen" refers to an agent that, when exposed to or administered to an organism, elicits an immune response and / or to which a T cell receptor (e.g., when presented by an MHC molecule) binds or to which an antibody (e.g., when produced by a B cell) binds. In some embodiments, the antigen elicits a humoral response in the organism (e.g., including the production of antigen-specific antibodies). Alternatively, or in addition, in some embodiments, the antigen elicits a cellular response in the organism (e.g., including T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or some members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism's species. In some embodiments, the 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 species. In some embodiments, the antigen binds to an antibody and / or a T-cell receptor but may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to an antibody and / or a T-cell receptor in vitro, regardless of whether such an interaction occurs in vivo. In some embodiments, the antigen reacts with products of specific humoral or cellular immunity, including products induced by heterologous immunogens. Antigens include antigenic ferritin proteins, including ferritin (e.g., containing one or more mutations) and non-ferritin polypeptides described herein.

[0064] "Immunostimulatory moiety," as used herein, refers to a moiety that can be covalently attached to ferritin or an antigenic ferritin polypeptide and activate a component of the immune system (alone or when attached to a ferritin or antigenic ferritin polypeptide). Exemplary immunostimulatory moieties include agonists of toll-like receptors (TLRs), e.g., TLR4, 7, 8, or 9. In some embodiments, the immunostimulatory moiety is an adjuvant.

[0065] "Adjuvant," as used herein, refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of antigen-adsorbed minerals (e.g., alum, aluminum hydroxide, or phosphates), water-in-oil or oil-in-water emulsions in which an antigen solution is emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant), and sometimes killed mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, for example, 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 can also include biological molecules such as Toll-Like Receptor (TLR) agonists and costimulatory molecules.Adjuvants can be administered as separate molecules in compositions or covalently bound (conjugated) to ferritin or antigenic ferritin polypeptides.

[0066] As used herein, "antigenic EBV polypeptide" refers to a polypeptide comprising all or a portion of an EBV amino acid sequence of sufficient length that the molecule is antigenic with respect to EBV. The antigenicity can be a feature of the EBV sequence as part of a construct that further comprises heterologous sequences, such as ferritin or lumazine synthase protein, and / or an immunostimulatory moiety. That is, when the EBV sequence is part of a construct that further comprises heterologous sequences, it is sufficient that the construct be able to serve as an antigen to generate anti-EBV antibodies, regardless of whether the EBV sequence without the heterologous sequences is able to do so.

[0067] "Antigenic ferritin polypeptide" and "antigenic ferritin protein" are used interchangeably herein and refer to a polypeptide comprising ferritin and non-ferritin polypeptides (e.g., EBV polypeptides) of sufficient length that the molecule is antigenic with respect to the non-ferritin polypeptide. The antigenic ferritin polypeptide may further comprise an immunostimulatory moiety. Antigenicity may be a feature of the non-ferritin sequence as part of a larger construct. That is, it is sufficient that the construct be able to serve as an antigen for the non-ferritin polypeptide, regardless of whether the non-ferritin polypeptide (and immunostimulatory moiety, if applicable) without ferritin is able to do so. In some embodiments, the non-ferritin polypeptide is an EBV polypeptide, in which case the antigenic ferritin polypeptide is also an "antigenic EBV polypeptide." However, for clarity, the antigenic EBV polypeptide need not include ferritin. "Antigenic polypeptide," as used herein, refers to a polypeptide that is either or both of an antigenic ferritin polypeptide and an antigenic EBV polypeptide.

[0068] "Self-adjuvanting," as used herein, refers to a composition or polypeptide comprising ferritin and an immunostimulatory moiety directly conjugated to the ferritin, such that the ferritin and immunostimulatory moiety are in the same molecular entity. Antigenic ferritin polypeptides, including non-ferritin polypeptides, may be conjugated to an immunostimulatory moiety to produce a self-adjuvanting polypeptide.

[0069] A "surface-exposed" amino acid, as used herein, refers to an amino acid residue in a protein (e.g., ferritin) that has a side chain that is accessible to solvent molecules when the protein is in its native three-dimensional conformation after multimerization, if applicable. Thus, for example, in the case of ferritin that forms a 24-mer, a surface-exposed amino acid residue is one whose side chain is accessible to solvent when the ferritin assembles as a 24-mer, e.g., as a ferritin multimer or ferritin particle.

[0070] As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the non-human subject is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, the subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments of the present invention, the subject is an adult, adolescent, or juvenile. In some embodiments, the terms "individual" or "patient" are used interchangeably with "subject" and are intended to be interchangeable.

[0071] As used herein, the term "vaccination" or "vaccinate" refers to the administration of a composition intended to generate an immune response, for example, against a pathogen. Vaccination can be administered before, during, and / or after exposure to the pathogen and / or the onset of one or more symptoms, and in some embodiments, can be administered before, during, and / or immediately after exposure to the pathogen. In some embodiments, vaccination involves multiple administrations of the vaccinating composition at appropriate intervals.

[0072] The present disclosure describes nucleic acid and amino acid sequences that have a certain degree of identity to a given nucleic acid or amino acid sequence, respectively (a reference sequence).

[0073] "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides identified between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0074] The term "% identical", "% identity", or similar terms is intended to refer to the percentage of nucleotides or amino acids that are identical, particularly in optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may be distributed over the entire length of the sequences being compared, but are not necessarily distributed randomly. Comparison of two sequences is usually carried out by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison may be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0075] The percentage identity is obtained by determining the number of identical positions to which the compared sequences correspond, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0076] 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 over the entire length of the reference sequence.

[0077] A nucleic acid sequence or amino acid sequence having a certain degree of identity to a predetermined nucleic acid sequence or amino acid sequence, respectively, can have at least one functional property of the predetermined sequence, for example, in some cases, it is functionally equivalent to the predetermined sequence. One important property includes, in particular, the ability to act as a cytokine when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a certain degree of identity to a predetermined nucleic acid sequence or amino acid sequence is functionally equivalent to the predetermined sequence.

[0078] 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 associated materials, such as solvents, solutions, buffers, instructions, or desiccants.

[0079] B. Antigenic EBV polypeptides, including gL and gH polypeptides EBV contains three glycoproteins, glycoprotein B (gB), gH, and gL, which form the core membrane fusion apparatus and enable viral penetration into cells. gL and gH have been previously described, for example, in Matsuura et al., Proc Natl Acad Sci USA. 2010 Dec 28;107(52):22641-6. Monomers and trimers of gL and gH for use as vaccines have been described, for example, in Cui et al., Vaccine. 2016 Jul 25;34(34):4050-5. The gH and gL proteins associate to form a heterodimeric complex that is thought to be necessary for efficient membrane fusion and epithelial cell receptor binding required for viral entry.

[0080] Disclosed herein are antigenic polypeptides comprising EBV gL and EBV gH. In some embodiments, the polypeptides exist as a single chain. In some embodiments, the polypeptides form trimers by trimerization of a trimerization domain, such as the T4 phage fibritin trimerization domain. In some embodiments, the polypeptides form nanoparticles (e.g., ferritin or lumazine synthase particles) by multimerization of ferritin or lumazine synthase. In some embodiments, the antigenic EBV polypeptides disclosed herein comprise an EBV gL polypeptide and an EBV gH polypeptide, and a linker having a length of at least 15 amino acids separating the EBV gL polypeptide and the EBV gH polypeptide. It has been found that longer linkers provide improved expression and / or immunogenicity.

[0081] In some embodiments, the EBV gH and / or gL polypeptides are full-length gH and / or gL (see GenBank Accession Nos. CEQ35765.1 and YP_001129472.1, respectively, for exemplary full-length sequences). In some embodiments, the EBV gH and / or gL polypeptides are fragments of gH and / or gL. In some embodiments, the gL polypeptide is a gL(D7) construct, which has a 7-amino acid deletion at the end of the gL C-terminus. In some embodiments, the gH polypeptide comprises a mutation at C137, such as a C137A mutation. In some embodiments, the C137 mutation removes a naturally unpaired cysteine ​​and prevents non-specific conjugation. In some embodiments, the gH polypeptide comprises a mutation that removes a cysteine ​​corresponding to cysteine ​​137 of SEQ ID NO: 37, such as a C137A mutation. In some embodiments, the C137 mutation removes a naturally unpaired cysteine ​​and prevents non-specific conjugation.

[0082] In some embodiments, the EBV gL polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 36. In some embodiments, the EBV gH polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 37.

[0083] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is added to an EBV polypeptide, such as a gH or gL polypeptide, at its N-terminus, e.g., at the N-terminus of the polypeptide. In some embodiments, the mammalian leader sequence provides for the secretion of the protein when expressed in mammalian cells.

[0084] The sequence of native EBV gH and / or gL is shown in GenBank Accession No. NC_009334.1 (Human Herpesvirus 4, Complete Genome, dated March 26, 2010). For some of the constructs disclosed herein, amino acids 23-137 of the gL amino acid sequence in NC_009334.1 were used as the gL polypeptide, and the native signal peptide (amino acids 1-22 of the NCBI sequence) was replaced with an IgGκ leader sequence. For some of the constructs, amino acids 19-678 of the gH amino acid sequence in NC_009334.1 were used as the gH polypeptide. In some embodiments, gL and gH were linked via a linker, as shown in the sequence tables herein.

[0085] In some embodiments, the gL and gH polypeptides are expressed as a single-chain monomer. In some embodiments, the monomer composition comprises or consists of a sequence shown in the Sequence Listing and annotated with the description "monomer." A single chain comprising gL and gH polypeptides is referred to as "gL / gH," which can be used interchangeably as "gH_gL," "gL_gH," or "gL / gH."

[0086] In some embodiments, gL and gH are provided as trimers. In some embodiments, a trimerization domain is placed after the gH sequence (C-terminus), which in some embodiments is followed by a His6 (SEQ ID NO: 243) sequence. The Foldon trimerization domain is exemplary, as any trimerization domain known in the art can be used, such as the collagen or L1ORF1p trimerization domains referenced herein. Using peripheral blood human naive B cells, gL and gH trimers have been shown to elicit higher serum neutralization titers compared to gL and gH monomers (see, e.g., Cui et al., Vaccine. 2016 Jul 25;34(34):4050-5).

[0087] In some embodiments, the gL / gH trimer has an amino acid sequence that comprises or consists of the sequence shown in the Sequence Listing and annotated with the entry as "trimer."

[0088] The gL / gH polypeptide can also be combined with any of the ferritins or lumazine synthases discussed herein. For example, in some embodiments, the antigenic EBV polypeptide comprises a monomeric or trimeric gL / gH polypeptide (+ / - gp42 and / or gp220) and i) a heavy or light chain ferritin (e.g., T. nii heavy or light chain ferritin), or ii) a ferritin optionally containing a surface-exposed cysteine.

[0089] Additionally, in some embodiments, any antigenic EBV polypeptide, including EBV gL / gH polypeptides and ferritin, may be present in a composition that includes another polypeptide disclosed herein.

[0090] C. Antigenic EBV polypeptides, including gp220 polypeptides In some embodiments, the antigenic EBV polypeptide comprises a gp220 polypeptide. gp220 hybrid bullfrog / H. pylori ferritin nanoparticles have been previously described by Kanekiyo, Cell. 2015 Aug 27;162(5):1090-100. Among other differences from certain ferritins described herein, these nanoparticles did not contain mutations providing surface-exposed cysteines or linkers containing cysteines.

[0091] In some embodiments, the gp220 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:38.

[0092] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is added to the N-terminus of the gp220 polypeptide, hi some embodiments, the mammalian leader sequence provides for the secretion of the protein when expressed in mammalian cells.

[0093] The gp220 polypeptide can be combined with any of the ferritin or lumazine synthase polypeptides discussed herein. For example, in some embodiments, the antigenic EBV polypeptide comprises a gp220 polypeptide (+ / - gL / gH and / or gp42) and i) a heavy or light chain ferritin (e.g., T. nii heavy or light chain ferritin), or ii) a ferritin optionally containing a surface-exposed cysteine ​​as described herein.

[0094] Additionally, in some embodiments, any antigenic EBV polypeptide, including gp220 polypeptide and ferritin, may be present in a composition that includes another polypeptide disclosed herein.

[0095] D. Antigenic EBV Polypeptides, Including gp42 Polypeptides In some embodiments, the antigenic EBV polypeptide comprises a gp42 polypeptide. An exemplary gp42 sequence is provided as SEQ ID NO: 34. A further exemplary gp42 sequence suitable for inclusion in a fusion with, e.g., gL and gH polypeptides is provided as SEQ ID NO: 239. Another exemplary gp42 sequence suitable for inclusion in a fusion with, e.g., gL and gH polypeptides is provided as SEQ ID NO: 240.

[0096] In some embodiments, the gp42 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In some embodiments, the gp42 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 239. In some embodiments, the gp42 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 240.

[0097] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is added to the N-terminus of the gp42 polypeptide. In some embodiments, the mammalian leader sequence results in secretion of the protein when expressed in mammalian cells. An exemplary leader sequence is amino acids 1-22 of SEQ ID NO:226.

[0098] In some embodiments, an antigenic EBV polypeptide comprising a gH and / or gL polypeptide further comprises a gp42 polypeptide. Any of the above-described EBV polypeptides comprising a gH and / or gL polypeptide may further comprise a gp42 polypeptide. In some embodiments, the gp42 polypeptide is located C-terminal to the gH and / or gL polypeptide, as exemplified in SEQ ID NOs: 21 and 226-231. In some embodiments, the gp42 polypeptide is located N-terminal to ferritin, as exemplified in SEQ ID NOs: 21 and 227-231. Thus, for example, an antigenic EBV polypeptide may comprise, in an N- to C-terminal orientation, a gL polypeptide, a gH polypeptide, a gp42 polypeptide, and optionally ferritin. A linker, as described herein, can separate the gp42 polypeptide from the EBV polypeptide and / or ferritin located at its N- and / or C-terminus. In some embodiments, a linker separates each EBV polypeptide (e.g., a gL polypeptide, a gH polypeptide, and a gp42 polypeptide) in the antigenic ferritin polypeptide, and a linker, if present, may also be present between the ferritin and an adjacent EBV polypeptide (e.g., a gp42 polypeptide).

[0099] In some embodiments, a linker having a length of at least 15 amino acids separates the EBV gH polypeptide and the EBV gp42 polypeptide. Such a linker may have a length of 15-60 amino acids, 20-60 amino acids, 30-60 amino acids, 40-60 amino acids, 30-50 amino acids, or 40-50 amino acids. In some embodiments, the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:234.

[0100] In some embodiments, when gp42 and ferritin are present in a polypeptide, a linker separates the EBV gp42 polypeptide from the ferritin. Such a linker may be at least 15 amino acids in length, or may be 15-60 amino acids, 20-60 amino acids, 30-60 amino acids, 40-60 amino acids, 30-50 amino acids, or 40-50 amino acids in length. In some embodiments, such a linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 233, 234, 235, 236, 237, or 238.

[0101] A gp42 polypeptide can be combined with any of the ferritins or lumazine synthases discussed herein. For example, in some embodiments, the polypeptide comprises a gp42 polypeptide (+ / - gL / gH and / or gp220) and i) a heavy or light chain ferritin (e.g., T. nii heavy or light chain ferritin), or ii) a ferritin optionally containing a surface-exposed cysteine ​​as described herein.

[0102] In some embodiments, the antigenic EBV polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to amino acids 23-1078 of SEQ ID NO: 226. In some embodiments, the antigenic EBV polypeptide comprises a sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to amino acids 1-1078 of SEQ ID NO: 226. In some embodiments, the antigenic EBV polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 226, 227, 228, 229, 230, or 231, optionally lacking a leader sequence (e.g., lacking any or all of amino acids 1-22 of these sequences).

[0103] Additionally, in some embodiments, any antigenic EBV polypeptide, including gp42 polypeptide and ferritin, may be present in a composition that includes another polypeptide disclosed herein.

[0104] E. Linker In some embodiments, the antigenic EBV polypeptide comprises a linker between the gL and gH polypeptides. In some embodiments, the antigenic EBV polypeptide comprises a linker between the EBV polypeptide and ferritin or lumazine synthase. While the following features are described for any such linker, the present invention provides that a relatively long linker between the gL and gH sequences can increase immunogenicity. Any linker may be used; for example, in some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the linker is about 2-4, 2-6, 2-8, 2-10, 2-12, or 2-14 amino acids in length. In some embodiments, the linker is a peptide linker, which can facilitate expression of the antigenic ferritin polypeptide as a fusion protein (e.g., from a single open reading frame). In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is GS, GGGS (SEQ ID NO: 244), 2xGGGS (i.e., GGGSGGGS) (SEQ ID NO: 245), or 5xGGGS (SEQ ID NO: 246). In some embodiments, the linker between the EBV polypeptide and ferritin is GS, GGGS (SEQ ID NO: 244), 2xGGGS (i.e., GGGSGGGS) (SEQ ID NO: 245), or 5xGGGS (SEQ ID NO: 246).

[0105] In some embodiments, the linker is at least 15 amino acids in length. In some embodiments, the linker is at least 25 amino acids in length. In some embodiments, the linker is at least 30 amino acids in length. In some embodiments, the linker is at least 35 amino acids in length. In some embodiments, the linker is at least 40 amino acids in length. In some embodiments, the linker is 60 amino acids or less in length. In some embodiments, the linker is 50 amino acids or less in length. In some embodiments, the linker is about 16, 28, 40, 46, or 47 amino acids in length. In some embodiments, the linker is flexible. In some embodiments, the linker comprises a cysteine, e.g., for use as a site for conjugation of an immunostimulatory moiety (e.g., an adjuvant); an exemplary linker comprising a cysteine ​​is provided as SEQ ID NO: 225. In some embodiments, the linker comprises a sequence having at least 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 225, and further comprises a cysteine ​​corresponding to the cysteine ​​in SEQ ID NO: 225. In some embodiments, the linker comprises at least 25 amino acids (e.g., 25-60 amino acids), with the cysteine ​​located within the range of the 8th amino acid from the N-terminus to the 8th amino acid from the C-terminus or within the central 10 amino acids or at the point of attachment of the linker.

[0106] In some embodiments, the linker comprises glycine (G) and / or serine (S) amino acids. In some embodiments, the linker comprises or consists of glycine (G), serine (S), asparagine (N), and / or alanine (A) amino acids, and optionally cysteines as discussed above. In some embodiments, the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 222. In some embodiments, the linker comprises GGGGSGGGGSGGGGSG (SEQ ID NO: 28), GGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 29), GGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 30), or GS. In some embodiments, the linker comprises FR1 (SEQ ID NO: 31) or FR2 (SEQ ID NO: 32). In some embodiments, the linker comprises SEQ ID NOs: 233-238.

[0107] In some embodiments, linkers containing cysteines as conjugation sites for immunostimulatory moieties, such as adjuvants, are used in constructs comprising ferritin molecules that lack a surface-exposed unpaired cysteine ​​or that comprise a surface-exposed unpaired cysteine.

[0108] In some embodiments, the linker is a cysteine-thrombin-histidine linker. In some embodiments, this linker is used to directly conjugate an EBV polypeptide to ferritin via click chemistry. An exemplary sequence comprising a cysteine-thrombin-histidine linker is SEQ ID NO: 39. Click chemistry suitable for conjugation reactions involving cysteine-thrombin-histidine linkers is discussed herein.

[0109] In some embodiments, the construct does not include a linker. In some embodiments, the construct includes one linker. In some embodiments, the construct includes two or more linkers.

[0110] In some embodiments, the construct includes a linker between gH and gL but not between the polypeptide and ferritin, or vice versa, In some embodiments, the construct includes a linker only between the polypeptide and ferritin.

[0111] F. Antigenic EBV Polypeptides, Including EBV Polypeptides and Ferritin or Lumazine Synthase In some embodiments, antigenic EBV polypeptides are provided, including an EBV polypeptide and ferritin. The EBV polypeptide can be any of the EBV polypeptides described herein, such as gL, gH, gL / gH, gp220, or gp42 polypeptides, or a combination thereof. The ferritin component of the polypeptide can be ferritin from any species, with or without mutations, such as those described herein substituting surface-exposed amino acids with cysteines. In some embodiments, the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-27.

[0112] In some embodiments, the ferritin in the polypeptide is wild-type ferritin. In some embodiments, the ferritin is derived from bacteria, insects, fungi, birds, or mammals. In some embodiments, the ferritin is derived from humans. In some embodiments, the ferritin is derived from bacteria.

[0113] In some embodiments, the ferritin is a light chain and / or a heavy chain ferritin. In some embodiments, the ferritin is an insect ferritin, such as Trichoplusia nii heavy chain ferritin (SEQ ID NO: 211) or Trichoplusia nii light chain ferritin (SEQ ID NO: 212). In some embodiments, the ferritin is a human ferritin, such as human heavy chain ferritin (SEQ ID NO: 214 or FTH1, GENE ID No: 2495) or human light chain ferritin (SEQ ID NO: 215 or FTL, GENE ID No: 2512). In some embodiments, the ferritin nanoparticles contain all 24 subunits of heavy and light chain ferritin, such as human or Trichoplusia nii ferritin nanoparticles. T. nii ferritin nanoparticles may contain 12 subunits of heavy chain ferritin and 12 subunits of light chain ferritin.

[0114] In some embodiments, the antigenic EBV polypeptide comprises a light chain ferritin and an EBV polypeptide. In some embodiments, the antigenic EBV polypeptide comprises a heavy chain ferritin and an EBV polypeptide. In some embodiments, an antigenic EBV polypeptide comprising a light chain ferritin and an EBV polypeptide can assemble with a heavy chain ferritin that is not linked to an EBV polypeptide. In some embodiments, an antigenic EBV polypeptide comprising a heavy chain ferritin and an EBV polypeptide can assemble with a light chain ferritin that is not linked to an EBV polypeptide. Ferritin that is not linked to an EBV polypeptide (or more generally, a non-ferritin polypeptide) may be referred to as "naked ferritin."

[0115] In some embodiments, an antigenic polypeptide comprising a heavy chain ferritin and a polypeptide assembles with an antigenic polypeptide comprising a light chain ferritin and an EBV polypeptide, allowing the display of two identical or different non-ferritin polypeptides on a single ferritin nanoparticle. In some embodiments, the two different non-ferritin polypeptides are EBV polypeptides. In some embodiments, the two different non-ferritin polypeptides are encoded by EBV and different infectious agents. In some embodiments, the different non-ferritin polypeptides from different infectious agents are derived from viruses or bacteria.

[0116] In some embodiments, an antigenic polypeptide comprising a heavy chain ferritin and a non-ferritin polypeptide can assemble with a polypeptide comprising a light chain ferritin and a non-ferritin polypeptide to generate a bivalent composition.

[0117] In some embodiments, the antigenic polypeptide comprises a light chain ferritin and a gp220 and / or gp42 polypeptide. In some embodiments, the antigenic polypeptide comprises a heavy chain ferritin and a gp220 and / or gp42 polypeptide.

[0118] In some embodiments, the antigenic polypeptide comprises a light chain ferritin and single chain gL and gH polypeptides. In some embodiments, the antigenic polypeptide comprises a heavy chain ferritin and single chain gL and gH polypeptides.

[0119] In some embodiments, antigenic polypeptides comprising ferritin light chain and gp220 and / or gp42 polypeptides are assembled with antigenic polypeptides comprising ferritin heavy chain and single chain gL and gH polypeptides.

[0120] In some embodiments, antigenic polypeptides comprising heavy chain ferritin and gp220 and / or gp42 polypeptides are assembled with antigenic polypeptides comprising light chain ferritin and single-chain gL and gH polypeptides. In some embodiments, as in assembled T. nii ferritin nanoparticles, 12 gp220 and / or gp42 polypeptides and 12 single-chain gL and gH polypeptides are included in the assembled ferritin nanoparticles.

[0121] Any type of ferritin nanoparticle containing both gp220 and / or gp42 and single-chain gL and gH polypeptides may be referred to as a "bivalent" or "bivalent EBV" particle or construct. A composition containing gL and gH trimers together with ferritin containing gp220 and / or gp42 would also be a bivalent EBV composition.

[0122] In some embodiments, the ferritin is H. pylori ferritin, optionally with one or more mutations as described herein (see SEQ ID NO: 208 or 209 for exemplary H. pylori ferritin sequences). In some embodiments, the lower sequence homology between H. pylori ferritin (or other bacterial ferritins) and human ferritin may reduce the likelihood of autoimmunity when used as a vaccine platform (see Kanekiyo et al., Cell 162, 1090-1100 (2015)).

[0123] In some embodiments, nanoparticles are provided comprising an antigenic EBV polypeptide disclosed herein, including an EBV polypeptide and ferritin.

[0124] 1. Ferritin mutation In some embodiments, the present specification discloses ferritin containing one or more mutations. In some embodiments, the one or more mutations include, for example, a change in the amino acid sequence of wild-type ferritin and / or an insertion at the N- or C-terminus. In some embodiments, one, two, three, four, five, or more different amino acids are mutated in ferritin compared to wild-type ferritin (in some embodiments, in addition to any N-terminal insertion). One or more mutations can change the functional properties of ferritin, for example, as discussed in detail below. Generally, a mutation simply refers to a sequence difference (such as a substituted, added, or deleted amino acid residue or residues) compared to the corresponding wild-type ferritin.

[0125] 2. Cysteine ​​for conjugation In some embodiments, ferritin is mutated to provide a chemical handle for conjugation of an immunostimulatory moiety and / or an EBV polypeptide. This can be achieved by a mutation that replaces a surface-exposed non-cysteine ​​amino acid with a cysteine. For the avoidance of doubt, phrases such as "replace a surface-exposed amino acid with a cysteine" necessarily imply that the surface-exposed amino acid in the wild-type or pre-mutated sequence is not a cysteine. Another approach to providing a chemical handle for conjugation of an immunostimulatory moiety or an EBV polypeptide is to include a linker-like amino acid segment at the N- or C-terminus of ferritin, where the amino acid segment contains a cysteine. In some embodiments, this cysteine ​​(replacing a surface-exposed amino acid or in the N- or C-terminal linker) is unpaired, meaning that it does not have a suitable partner cysteine ​​to form a disulfide bond. In some embodiments, this cysteine ​​does not alter the secondary structure of ferritin. In some embodiments, this cysteine ​​does not alter the tertiary structure of ferritin.

[0126] In some embodiments, this cysteine ​​can be used to conjugate an agent, such as an immunostimulatory moiety, to ferritin. In some embodiments, this cysteine ​​provides a reactive free thiol group. In some embodiments, the agent conjugated to this cysteine ​​on ferritin is exposed on the surface of the assembled ferritin particle. In some embodiments, this cysteine ​​can interact with molecules and cells of interest during the assembly of the ferritin particle after administration.

[0127] In some embodiments, the presence of this cysteine ​​allows for conjugation of one or more immunostimulatory moieties, such as adjuvants, hi some embodiments, conjugation of immunostimulatory moieties does not occur in the absence of this cysteine.

[0128] In some embodiments, the non-cysteine ​​amino acid to be replaced with cysteine ​​is selected from E12, S72, A75, K79, S100, and S111 of H. pylori ferritin. Thus, in some embodiments, the surface-exposed amino acid to be replaced with cysteine ​​is the amino acid residue corresponding to E12, S26, S72, A75, K79, S100, or S111 of H. pylori ferritin. Similar amino acids can be found in non-H. pylori ferritin by pairwise or structural alignment. In some embodiments, the non-cysteine ​​amino acid to be replaced with cysteine ​​can be selected from the amino acids corresponding to S3, S19, S33, I82, A86, A102, and A120 of human light chain ferritin. In some embodiments, the surface-exposed amino acid to be replaced with a cysteine ​​is selected with the understanding that when the original amino acid is replaced with a cysteine, it will be reactive in the assembled ferritin multimer or particle, and / or the cysteine ​​will not interfere with the stability of the ferritin multimer or particle, and / or the cysteine ​​will not result in a reduction in the expression level of ferritin.

[0129] In some embodiments, the ferritin comprises an E12C mutation. In some embodiments, the E12C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the E12C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the E12C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 E12C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0130] In some embodiments, the ferritin comprises an S26C mutation. In some embodiments, the S26C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the S26C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the S26C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S26C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0131] In some embodiments, the ferritin comprises an S72C mutation. In some embodiments, the S72C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the S72C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the S72C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S72C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0132] In some embodiments, the ferritin comprises an A75C mutation. In some embodiments, the A75C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the A75C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the A75C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 A75C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0133] In some embodiments, the ferritin comprises a K79C mutation. In some embodiments, the K79C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the K79C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the K79C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 K79C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0134] In some embodiments, the ferritin comprises an S100C mutation. In some embodiments, the S100C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the S100C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the S100C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S100C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0135] In some embodiments, the ferritin comprises an S111C mutation. In some embodiments, the S111C residue can be used to conjugate an agent (e.g., an immunostimulatory moiety and / or an EBV polypeptide) to ferritin. In some embodiments, the S111C residue provides a reactive free thiol group. In some embodiments, an agent conjugated to the S111C residue on a ferritin monomer is expressed on the surface of an assembled ferritin multimer or particle. In some embodiments, 24 S111C residues (one from each monomer) are present on the surface of the ferritin multimer or particle.

[0136] 3. Removal of Internal Cysteines In some embodiments, the ferritin contains a mutation that replaces an internal cysteine ​​with a non-cysteine ​​amino acid. By removing the original internal cysteine ​​residue, only one unpaired cysteine ​​is present per ferritin monomer, avoiding undesirable reactions such as disulfide formation and resulting in more stable and efficient results (e.g., adjuvant presentation). In some embodiments, C31 of H. pylori ferritin is replaced with a non-cysteine ​​amino acid. In some embodiments, C31 of H. pylori ferritin is replaced with serine (C31S), although any non-cysteine ​​residue, such as alanine, glycine, threonine, or asparagine, may also be used. Similar amino acids can be found in non-H. pylori ferritins by pairwise or structural alignment. Thus, in some embodiments, the internal cysteine ​​replaced with a non-cysteine ​​is an amino acid residue that aligns with C31 of H. pylori ferritin. Exemplary ferritin sequences exhibiting the C31S mutation are set forth in SEQ ID NOs: 201-207. In some embodiments, when more than one internal cysteine ​​is present in ferritin, two or more (e.g., each) internal cysteine ​​is replaced with a non-cysteine ​​amino acid, such as serine or an amino acid selected from serine, alanine, glycine, threonine, or asparagine.

[0137] 4. Glycosylation Human-compatible glycosylation can contribute to the safety and efficacy of recombinant drug products. Regulatory approval may be contingent on demonstrating appropriate glycosylation as a critical quality attribute (see Zhang et al., Drug Discovery Today 21(5):740-765 (2016)). N-glycans can result from glycosylation of asparagine side chains and may differ in structure between humans and other organisms, such as bacteria and yeast. Thus, it may be desirable to reduce or eliminate non-human glycosylation and / or N-glycan formation in ferritin according to the present disclosure. In some embodiments, controlling glycosylation of ferritin improves the efficacy and / or safety of the composition, particularly when used for human vaccination.

[0138] In some embodiments, the ferritin is mutated to inhibit N-glycan formation. In some embodiments, the mutated ferritin has reduced glycosylation compared to its corresponding wild-type ferritin.

[0139] In some embodiments, the ferritin comprises a mutation that replaces a surface-exposed asparagine with a non-asparagine amino acid. In some embodiments, the surface-exposed asparagine is N19 of H. pylori ferritin, or a position corresponding to position 31 of H. pylori ferritin, as determined by pairwise or structural alignment. In some embodiments, mutating such an asparagine, e.g., N19 of H. pylori ferritin, reduces glycosylation of ferritin. In some embodiments, the mutation replaces the asparagine with glutamine. In some embodiments, the ferritin is H. pylori ferritin comprising an N19Q mutation. SEQ ID NOs: 201-207 are exemplary ferritin sequences comprising an N19Q mutation.

[0140] Mammals exposed to glycosylated proteins produced in bacteria or yeast may develop an immune response to the glycosylated protein because the glycosylation pattern of a given protein in bacteria or yeast may differ from the glycosylation pattern of the same protein in a mammal. Thus, some glycosylated therapeutic proteins may not be suitable for production in bacteria or yeast.

[0141] In some embodiments, reducing glycosylation of ferritin by amino acid mutations facilitates protein production in bacteria or yeast. In some embodiments, reducing glycosylation of ferritin reduces the likelihood of side effects in mammals when mutant ferritin expressed in bacteria or yeast is administered. In some embodiments, the reactogenicity of mutant ferritin produced in bacteria or yeast in human subjects is reduced due to reduced glycosylation. In some embodiments, the incidence of hypersensitivity reactions in human subjects is lower after treatment with mutant ferritin having reduced glycosylation compared to wild-type ferritin.

[0142] In some embodiments, a composition comprising a mutant ferritin with reduced glycosylation is degraded slower in a subject than a composition comprising wild-type ferritin or a corresponding ferritin with wild-type glycosylation. In some embodiments, a composition comprising a mutant ferritin with reduced glycosylation has reduced clearance in a subject than a composition comprising wild-type ferritin or a corresponding ferritin with wild-type glycosylation. In some embodiments, a composition comprising a mutant ferritin with reduced glycosylation has a longer serum half-life than a composition comprising wild-type ferritin or a corresponding ferritin with wild-type glycosylation.

[0143] 5. Combination of mutations In some embodiments, the ferritin comprises more than one type of mutation described herein. In some embodiments, the ferritin comprises one or more mutations independently selected from a mutation that reduces glycosylation, a mutation that removes an internal cysteine, and a mutation that creates a surface-exposed cysteine. In some embodiments, the ferritin comprises a mutation that reduces glycosylation, a mutation that removes an internal cysteine, and a mutation that creates a surface-exposed cysteine.

[0144] In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and a mutation that creates a surface-exposed cysteine. In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and an E12C mutation. In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and an S72C mutation. In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and an A75C mutation. In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and a K79C mutation. In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and an S100C mutation. In some embodiments, the ferritin comprises an N19Q mutation, a C31S mutation, and an S111C mutation. In some embodiments, the ferritin comprises mutations corresponding to any of the aforementioned sets of mutations, where the corresponding mutations change N to Q, C to S, and a non-cysteine ​​surface-exposed amino acid to cysteine ​​at positions determined by pairwise alignment of the ferritin amino acid sequence with the H. pylori ferritin amino acid sequence (SEQ ID NO: 208 or 209).

[0145] Exemplary ferritins containing more than one type of mutation are provided in SEQ ID NOs: 201-207.

[0146] 6. Structural Alignment As discussed herein, the positions of mutations corresponding to those described for a given polypeptide (e.g., H. pylori ferritin) can be identified by pairwise or structural alignment. Structural alignment is appropriate for large protein families, such as ferritin, where proteins share similar structures despite considerable sequence variation and many members of the family have been structurally characterized, and can also be used to identify corresponding positions in different forms of other polypeptides described herein, such as EBV polypeptides (e.g., gL, gH, gp220, or gp42). The Protein Data Bank (PDB) contains 3D structures for many ferritins, including the ferritins listed below with their accession numbers.

[0147] 2jd6,2jd7-PfFR - Pyrococcus furiosus. 2jd8-PfFR + Zn. 3a68-soFR from gene SferH4 - soybean. 3a9q-soFR from gene SferH4 (mutant). 3egm,3bvf,3bvi,3bvk,3bvl-HpFR - Helicobacter pylori. 5c6f-HpFR (mutant) + Fe. 1z4a,1vlg-FR - Thermotoga maritime. 1s3q,1sq3,3kx9-FR - Archaeoglubus fulgidus, 1krq-FR - Campylobacter jejuni. 1eum-EcFR - Escherichia coli. 4reu-EcFR + Fe. 4xgs-EcFR (mutant) + Fe2O2. 4ztt-EcFR (mutant) + Fe2O + Fe2 + Fe + O2. 1qgh-LiFR - Listeria innocua. 3qz3-VcFR - Vibrio cholerae. 3vnx-FR - Ulva pertusa. 4ism, 4isp, 4itt, 4itw, 4iwj, 4iwk, 4ixk, 3e6s-PnmFR - Pseudo-nitschia multiseries. 4zkh, 4zkw, 4zkx, 4zl5, 4zl6, 4zlw, 4zmc - PnmFR (mutant) + Fe. 1z6o-FR - Nettle root looper. 4cmy-FR + Fe - Green sulfur bacteria (Chlorobaculum tepidum). Ferritin light chain (FTL). 1lb3,1h96-mFTL - Mouse. 1rcc,1rcd,1rci - bFTL + tartrate + Mg. 1rce,1rcg - bFTL + tartrate + Mn. 3noz,3np0,3np2,3o7r - hoFTL (mutant) - Horse. 3o7s,3u90 - hoFTL. 4v1w-hoFTL - CryoEM. 3rav,3rd0 - hoFTL + barbiturate. Ferritin light chain + heavy chain: 5gn8 - hFTH + Ca.

[0148] Structural alignment involves identifying corresponding residues across two (or more) polypeptide sequences by (i) using the known structure of the second sequence to model the structure of the first sequence, or (ii) comparing the structures of the first and second sequences that are both known, and identifying the residue in the first sequence that is most similar to the residue of interest in the second sequence.In some algorithms, corresponding residues are identified based on minimizing the distance between alpha carbons in the superimposed structures (for example, which pair of alpha carbons provides the smallest mean square deviation for alignment).When identifying the position in non-H. pylori ferritin that corresponds to the position described for H. pylori ferritin, H. pylori ferritin can be the "second" sequence. When the non-H. pyrolori ferritin of interest does not have a known structure available, but is more closely related to another non-H. pyrolori ferritin with a known structure than H. pyrolori ferritin, it may be most effective to use the known structure of the closely related non-H. pyrolori ferritin to model the non-H. pyrolori ferritin of interest, and then compare this model with the H. pyrolori ferritin structure to identify the desired corresponding residue in the ferritin of interest.There is a wide range of literature on structural modeling and alignment, and representative disclosures include U.S. Patent No. 6,859,736; U.S. Patent No. 8,738,343; and the disclosures cited in Aslam et al., Electronic Journal of Biotechnology 20 (2016) 9-13.For the discussion of structural modeling based on known related structures or multiple structures, see, for example, Bordoli et al., Nature Protocols 4 (2009) 1-13, and the references cited therein.

[0149] 7. Lumazine synthase In some embodiments, the antigenic polypeptide comprises a lumazine synthase protein. Lumazine synthase can form higher-order structures, such as a 60-subunit lumazine synthase particle. Exemplary lumazine synthases are Aquifex aeolicus lumazine synthase (SEQ ID NO: 40) and E. coli lumazine synthase (SEQ ID NO: 41). In some embodiments, the lumazine synthase has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 40 or 41. The lumazine synthase can be located C-terminal to the EBV polypeptide and separated from the EBV polypeptide by a linker, as discussed herein.

[0150] G. Mutations in the sequences of gL, gH, gp42, linker, and / or ferritin to eliminate potential oxidation, deamidation, or isoaspartate formation sites In some embodiments, the antigenic EBV polypeptide comprises one or more mutations to remove potential oxidation, deamidation, or isoaspartate formation sites, such as the exemplary mutations set forth in Table 1 below.

[0151] For example, in some embodiments, the gL sequence includes one or more mutations to remove potential succinimide / isoaspartate or deamidation sites. For example, the gL sequence may include a G to A mutation at a position corresponding to position 36 of SEQ ID NO: 227, an N to Q mutation at a position corresponding to position 47 of SEQ ID NO: 227, or an N to Q mutation at a position corresponding to position 105 of SEQ ID NO: 227. A position in an amino acid sequence "corresponds" to a given position in SEQ ID NO: 227 if aligned to that position according to a standard sequence alignment algorithm, such as the Smith-Waterman algorithm using default parameters.

[0152] In some embodiments, the linker contains one or more mutations to remove potential deamidation sites, for example, the linker sequence may contain an N to G mutation at a position corresponding to 132 or 141 of SEQ ID NO:227.

[0153] In some embodiments, the gH sequence contains one or more mutations to remove potential succinimide / isoaspartate or oxidation sites. For example, the gH sequence can contain an M to L mutation at a position corresponding to 189, 401, or 729 of SEQ ID NO:227, a D to E mutation at a position corresponding to 368 of SEQ ID NO:227, an M to I mutation at a position corresponding to 499 or 639 of SEQ ID NO:227, or an N to Q mutation at a position corresponding to 653 of SEQ ID NO:227.

[0154] In some embodiments, the gp42 sequence includes one or more mutations to remove potential deamidation sites. For example, the gp42 sequence can include an N to Q mutation at a position corresponding to position 959 or 990 of SEQ ID NO:227, or an N to S mutation at a position corresponding to position 988 of SEQ ID NO:227.

[0155] In some embodiments, the ferritin sequence includes one or more mutations to remove potential deamidation, oxidation, or isoaspartate formation sites. For example, the ferritin sequence can include a Q to S mutation at position 1150 of SEQ ID NO:227, an M to I mutation at position 1168 of SEQ ID NO:227, an M to L mutation at position 1177 of SEQ ID NO:227, a G to A mutation at position 1188 of SEQ ID NO:227, or an N to Q mutation at position 1253 or 1296 of SEQ ID NO:227.

[0156] Exemplary mutations are shown below in Table 1. Position numbering corresponds to SEQ ID NO:227.

[0157] [Table 1]

[0158] H. Immunostimulatory moieties; adjuvants; conjugated EBV polypeptides In some embodiments, the immunostimulatory moiety, such as an EBV polypeptide and / or an adjuvant, is bound to a surface-exposed amino acid. In some embodiments, the surface-exposed amino acid is a cysteine, for example, resulting from a mutation as discussed above. In some embodiments, the surface-exposed amino acid is a lysine, aspartate, or glutamate. Conjugation procedures using glutaraldehyde (for conjugating lysine to an amino-bearing linker or moiety) or carbodiimides (e.g., 1-cyclohexyl-3-(2-morpholin-4-yl-ethyl)carbodiimide, or 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC; EDAC) for conjugating aspartate or glutamate to an amino-bearing linker or moiety, or lysine to a carboxyl-bearing linker or moiety) are described, for example, in Chapter 4 of Holtzhauer, M., Basic Methods for the Biochemical Lab, Springer 2006, ISBN 978-3-540-32785-1, available from www.springer.com.

[0159] In some embodiments, an immunostimulatory moiety, such as an adjuvant, is attached to a surface-exposed amino acid on ferritin. In some embodiments, more than one immunostimulatory moiety, such as an adjuvant, is attached to a surface-exposed amino acid on ferritin. In some embodiments, 24 immunostimulatory moieties are attached to a ferritin multimer or particle (e.g., one moiety of each monomer in an H. pylori ferritin particle). In some embodiments where multiple immunostimulatory moieties are attached to ferritin nanoparticles, the immunostimulatory moieties are all identical. In some embodiments where multiple immunostimulatory moieties are attached to ferritin nanoparticles, the immunostimulatory moieties are not all identical.

[0160] 1. Type of immunostimulatory moiety; adjuvant Immunostimulatory moieties capable of binding to surface-exposed amino acids (e.g., cysteine) can be used in ferritin in accordance with the present disclosure. In some embodiments, the immunostimulatory moiety is a B-cell agonist.

[0161] In some embodiments, the immunostimulatory moiety is not hydrophobic. In some embodiments, the immunostimulatory moiety is hydrophilic. In some embodiments, the immunostimulatory moiety is polar. In some embodiments, the immunostimulatory moiety is capable of hydrogen bonding or ionic bonding, including, for example, a hydrogen bond donor, a hydrogen bond acceptor, a cationic moiety, or an anionic moiety. A moiety is considered to be cationic or anionic if it is ionized in aqueous solution at a physiologically relevant pH, such as pH 6, 7, 7.4, or 8.

[0162] In some embodiments, the immunostimulatory moiety is an adjuvant. In some embodiments, the adjuvant comprises a pathogen-associated molecular pattern (PAMP). In some embodiments, the adjuvant is a toll-like receptor (TLR) agonist or a stimulator of interferon genes (STING) agonist. In some embodiments, the adjuvant activates TLR signaling in B and / or T cells. In some embodiments, the adjuvant modulates adaptive immune responses.

[0163] a) TLR2 agonist In some embodiments, the immunostimulatory moiety is a TLR2 agonist. In some embodiments, the immunostimulatory moiety stimulates TLR2 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand of TLR2. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist of TLR2 signaling.

[0164] In some embodiments, the TLR2 agonist is PAM2CSK4, FSL-1, or PAM3CSK4.

[0165] b) TLR7 / 8 agonists In some embodiments, the immunostimulatory moiety is a TLR7 and / or TLR8 agonist (i.e., an agonist of at least one of TLR7 and TLR8). In some embodiments, the immunostimulatory moiety stimulates TLR7 and / or TLR8 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand of TLR7 and / or TLR8. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist of TLR7 and / or TLR8 signaling.

[0166] In some embodiments, the TLR7 and / or TLR8 agonist is a single-stranded (ssRNA). In some embodiments, the TLR7 and / or TLR8 agonist is an imidazoquinoline. In some embodiments, the TLR7 and / or TLR8 agonist is a nucleoside analog.

[0167] In some embodiments, the TLR7 and / or TLR8 agonist is an imidazoquinoline amine Toll-like receptor (TLR) agonist, such as 3M-012 (3M Pharmaceuticals). The structure of free 3M-012 is: [ka] It is understood that an immunostimulatory moiety, such as 3M-012 or any moiety discussed herein, can be conjugated to ferritin by substituting a suitable terminal atom (e.g., hydrogen) of the moiety, for example, at the sulfur of a surface-exposed cysteine ​​to bond with ferritin as described herein, or by a linker that binds to such sulfur. Thus, when conjugated to ferritin, the structure of the immunostimulatory moiety differs slightly from that of the free molecule.

[0168] In some embodiments, the TLR7 and / or TLR8 agonist is SM7 / 8a. The structure of free SM7 / 8a is: [ka] is.

[0169] See, e.g., Nat Biotechnol. 2015 Nov;33(11):1201-10. doi:10.1038 / nbt.3371.

[0170] c) TLR9 agonist In some embodiments, the immunostimulatory moiety is a TLR9 agonist. In some embodiments, the immunostimulatory moiety stimulates TLR9 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand of TLR9. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist of TLR9 signaling.

[0171] In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (ODN). In some embodiments, the TLR9 agonist is an unmethylated CpG ODN. In some embodiments, the CpG ODN comprises a partial or complete phosphorothioate (PS) backbone instead of the natural phosphodiester (PO) backbone found in normal DNA.

[0172] In some embodiments, the CpG ODN is a class B ODN, which contains one or more hexamer CpG motifs containing 5' purine (Pu)-pyrimidine (Py)-CG-Py-Pu 3'; has a fully phosphorothioated (i.e., PS-modified) backbone; and is 18-28 nucleotides in length. In some embodiments, the CpG ODN comprises the sequence of SEQ ID NO: 210, optionally including phosphorothioate linkages in the backbone.

[0173] In some embodiments, the TLR9 agonist comprises an immunostimulatory sequence (ISS). In some embodiments, the TLR9 agonist is ISS-1018 (Dynavax) (SEQ ID NO: 210).

[0174] d) STING agonists In some embodiments, the immunostimulatory moiety is a STING (stimulator of interferon genes, also known as endoplasmic reticulum IFN stimulator) agonist. In some embodiments, the immunostimulatory moiety stimulates STING signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule ligand of STING. In some embodiments, the immunostimulatory moiety is a synthetic small molecule agonist of STING signaling.

[0175] In some embodiments, STING agonist is cyclic dinucleotide (CDN).For example, see Danilchanka et al., Cell 154:962-970 (2013).Exemplary CDNs include cdA, cdG, cAMP-cGMP, and 2'-5',3'-5'cGAMP (for structure, see Danilchanka et al.).STING agonist also includes synthetic agonist such as DMXAA. [ka]

[0176] 2. Conjugated EBV Polypeptides In some embodiments, the EBV polypeptide is conjugated to an amino acid exposed on the surface of ferritin. In some embodiments, the EBV polypeptide makes the ferritin protein antigenic. In some embodiments, the EBV polypeptide is antigenic alone, while in some embodiments, the EBV polypeptide is antigenic due to its association with ferritin. In some embodiments, the EBV polypeptide is any one of the EBV polypeptides described herein.

[0177] 3. Conjugation In some embodiments, an immunostimulatory moiety such as an adjuvant or an EBV polypeptide is conjugated to ferritin using a surface-exposed cysteine ​​(e.g., due to a mutation described herein) or a cysteine ​​in a peptide linker attached to ferritin (e.g., the N-terminus of ferritin). In some embodiments, a linker is conjugated to such a cysteine, which can then be conjugated to an immunostimulatory moiety such as an adjuvant or an EBV polypeptide. In some embodiments, such a cysteine ​​creates a chemical handle for a conjugation reaction that joins the adjuvant, linker, or EBV polypeptide. In some embodiments, a bioconjugate is produced, and an immunostimulatory moiety such as an adjuvant or an EBV polypeptide is linked to ferritin after reduction of such cysteine. In some embodiments, the cysteine ​​is an unpaired surface-exposed cysteine, i.e., a cysteine ​​lacking a partner cysteine ​​in the appropriate position to form a disulfide bond. In some embodiments, the cysteine ​​is an unpaired cysteine ​​containing a free thiol side chain.

[0178] a) Types of conjugation chemistry Any type of chemistry can be used to conjugate an immunostimulatory moiety such as an adjuvant or an EBV polypeptide to ferritin, for example, via reaction of a surface-exposed amino acid such as cysteine ​​or another amino acid such as Lys, Glu, or Asp.

[0179] In some embodiments, conjugation is carried out using click chemistry. As used herein, "click chemistry" refers to a reaction between a pair of functional groups that react (i.e., "click") with each other rapidly and selectively. In some embodiments, click chemistry can be carried out under mild aqueous conditions. In some embodiments, the click chemistry reaction utilizes cysteines on the surface of ferritin, such as cysteines resulting from mutation of surface-exposed amino acids, and click chemistry is carried out using functional groups that can react with cysteines.

[0180] A variety of reactions that meet the criteria of click chemistry are known in the art, and one of skill in the art can use any of several published methodologies (see, e.g., Hein et al., Pharm Res 25(10):2216-2230 (2008)). A wide range of commercially available reagents can be used for click chemistry, such as reagents from Sigma Aldrich, Jena Bioscience, or Lumiprobe. In some embodiments, conjugation is carried out using click chemistry, as described in the examples below.

[0181] In some embodiments, click chemistry occurs after reduction of ferritin.

[0182] In some embodiments, the click chemistry can be a one-step click reaction. In some embodiments, the click chemistry can be a two-step click reaction.

[0183] In some embodiments, the reaction comprises metal-free click chemistry, hi some embodiments, the reaction comprises thiol-maleimide and / or disulfide exchange.

[0184] Metal-free click chemistry Metal-free click chemistry can be used for conjugation reactions to avoid possible oxidation of proteins. Metal-free click chemistry has been used to form antibody conjugates (see van Geel et al., Bioconjugate Chem. 2015, 26, pp. 2233-2242).

[0185] In some embodiments, metal-free click chemistry is used in the reaction of binding an adjuvant to ferritin. In some embodiments, copper-free conjugation is used in the reaction of binding an adjuvant to ferritin. In some embodiments, metal-free click chemistry uses bicyclo[6.1.0]nonyne (BCN). In some embodiments, metal-free click chemistry uses dibenzoazacyclooctyne (DBCO). In some embodiments, BCN or DBCO reacts with an azide group.

[0186] DBCO has high specificity for azide groups through a strain-promoted click reaction in the absence of a catalyst, leading to high yields of stable triazoles. In some embodiments, DBCO reacts with azides in the absence of a copper catalyst.

[0187] In some embodiments, metal-free click chemistry is used in a one-step click reaction. In some embodiments, metal-free click chemistry is used in a two-step click reaction.

[0188] Thiol-maleimide and disulfide exchange As used herein, ferritin can contain cysteines containing thiols, also known as sulfhydryls, which are available (or can be made available through reduction) for reaction with sulfhydryl-reactive chemical groups. Thus, cysteines allow for chemoselective modification to add immunostimulatory moieties, such as adjuvants, to ferritin. Under basic conditions, cysteines are deprotonated to generate thiolate nucleophiles, which can react with weak electrophiles, such as maleimides and iodoacetamides. Reaction of cysteines with maleimides or iodoacetamides results in carbon-sulfur bonds.

[0189] In some embodiments, the sulfhydryl-reactive chemical group reacts with a surface-exposed cysteine ​​or a cysteine ​​in the linker of ferritin. In some embodiments, the sulfhydryl-reactive chemical group is a haloacetyl, maleimide, aziridine, acryloyl, arylating agent, vinyl sulfone, pyridyl disulfide, or TNB-thiol.

[0190] In some embodiments, the sulfhydryl-reactive chemical group is conjugated to the sulfhydryl of a cysteine ​​by alkylation (i.e., forming a thioether bond). In some embodiments, the sulfhydryl-reactive chemical group is conjugated to the sulfhydryl of a cysteine ​​by disulfide exchange (i.e., forming a disulfide bond).

[0191] In some embodiments, the reaction to conjugate an immunostimulatory moiety, such as an adjuvant, to ferritin is a thiol-maleimide reaction.

[0192] In some embodiments, the sulfhydryl-reactive chemical group is maleimide. In some embodiments, the reaction of maleimide with cysteine ​​results in the formation of a stable thioester bond, for example, that is not reversible. In some embodiments, maleimide does not react with tyrosine, histidine, or methionine in ferritin. In some embodiments, unreacted maleimide is quenched at the end of the reaction, for example, by adding an excess of free thiol.

[0193] In some embodiments, the reaction for conjugating an immunostimulatory moiety, such as an adjuvant, to ferritin is a thiol-disulfide exchange, also known as disulfide interchange. In some embodiments, the reaction involves the formation of a mixed disulfide that contains a portion of the original disulfide. In some embodiments, the original disulfide is a cysteine ​​that was introduced into ferritin by mutation of a surface-exposed amino acid or by addition of an N-terminal linker.

[0194] In some embodiments, the sulfhydryl-reactive chemical group is a pyridyldithiol. In some embodiments, the sulfhydryl-reactive chemical group is a TNB-thiol group.

[0195] b) Linker In some embodiments, an immunostimulatory moiety, such as an adjuvant, or an EBV polypeptide is attached to ferritin via a linker covalently attached to a surface-exposed amino acid, such as a cysteine. In some embodiments, the linker comprises a polyethylene glycol (e.g., PEG) linker. In some embodiments, the polyethylene glycol (e.g., PEG) linker increases the water solubility and ligation efficiency of ferritin linked to an immunostimulatory moiety, such as an adjuvant. PEG linkers are between 2 and 18 PEG lengths, e.g., PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, and PEG18.

[0196] In some embodiments, the linker comprises a maleimide. In some embodiments, the linker comprises an immunostimulatory moiety (ISM)-linker-maleimide component. In some embodiments, the ISM-linker-maleimide is conjugated to ferritin in one-step click chemistry by reaction of the maleimide with a cysteine ​​on ferritin. In some embodiments, the ISM of the adjuvant-linker-maleimide is SM7 / 8a. In some embodiments, the linker of the ISM-linker-maleimide is PEG4. In some embodiments, the ISM-linker-maleimide is SM7 / 8a-PEG4-maleimide.

[0197] In some embodiments, a two-step click chemistry protocol is used with a linker that contains a sulfhydryl-reactive chemical group at one end and an amine-reactive group at the other end.In this two-step click chemistry protocol, the sulfhydryl-reactive chemical group reacts with the cysteine ​​of ferritin, while the amine-reactive group reacts with the reagent bound to the ISM.In this way, the ISM is conjugated to ferritin via a set of two click chemistry reagents.

[0198] In some embodiments of the two-step click chemistry protocol, the sulfhydryl-reactive chemical group is a maleimide, which reacts with a cysteine ​​introduced into ferritin by mutation of a surface-exposed amino acid or addition of an N-terminal linker.

[0199] In some embodiments of the two-step click chemistry protocol, the amine reactive group is DBCO. In some embodiments of the two-step click chemistry protocol, DBCO reacts with an azide group attached to an ISM.

[0200] In some embodiments, maleimide-linker-DBCO is used. In some embodiments, the maleimide-linker-DBCO is conjugated to ferritin after reduction of ferritin. In some embodiments, the maleimide-linker-reagent is conjugated to ferritin in the first step by reacting maleimide with cysteine ​​of ferritin. In some embodiments, DBCO is used to link to an ISM linked to an azide. In some embodiments, the ISM linked to the azide is ISS-1018. In some embodiments, the adjuvant coupled to the azide is 3M-012 or CpG.

[0201] In some embodiments, a linker having a reactive group is attached to the ISM, hi some embodiments, the linker is a PEG4-azide linker or a PEG4-maleimide linker.

[0202] In some embodiments, the PEG4-azide linker is conjugated to 3M-012. An exemplary structure of 3M-012 conjugated to a PEG4-azide linker is: [ka] is.

[0203] In some embodiments, the PEG4-azide linker is conjugated to SM7 / 8a. An exemplary structure of SM7 / 8a conjugated to a PEG4-azide linker is: [ka] is.

[0204] In some embodiments, a PEG4-maleimide linker is conjugated to SM7 / 8a. An exemplary structure of SM7 / 8a conjugated to a PEG4-maleimide linker is: [ka] is.

[0205] In some embodiments, the azide group is conjugated to ISS-1018. An exemplary structure of ISS-1018 conjugated to an NHS ester-azide linker is: [ka] is.

[0206] I. Exemplary Compositions, Kits, Nucleic Acids, Uses, and Methods In some embodiments, the present invention provides a method for immunizing a subject against infection by EBV. The present invention further provides a method for inducing an immune response to EBV in a subject. In some embodiments, the method of the present invention comprises administering to the subject an effective amount of a pharmaceutical composition described herein. In some embodiments, the method of the present invention comprises administering to the subject an effective amount of an antigenic EBV polypeptide or nanoparticle described herein.

[0207] In some embodiments, compositions are provided that include any one or more of the antigenic EBV polypeptides described herein and a pharmaceutically acceptable vehicle, adjuvant, or excipient.

[0208] In some embodiments, an antigenic EBV polypeptide, nanoparticle, or composition described herein is administered to a subject, such as a human or any of the subjects discussed below, to immunize against infection caused by EBV. In some embodiments, an antigenic EBV polypeptide or nanoparticle described herein is administered to a subject, such as a human, to generate a protective immune response against future infection with EBV. In some embodiments, an antigenic EBV polypeptide is administered. In some embodiments, an antigenic EBV polypeptide is administered, including an EBV polypeptide and ferritin, where the ferritin can have one or more mutations described herein. In some embodiments, an antigenic EBV polypeptide or nanoparticle comprising any one of SEQ ID NOs: 1-27 is administered.

[0209] In some embodiments, the protective immune response reduces the incidence of hospitalization, hi some embodiments, the protective immune response reduces the incidence of EBV infection, mononucleosis, complications caused by mononucleosis (e.g., hepatitis, encephalitis, severe hemolytic anemia, or splenomegaly), nasopharyngeal carcinoma, gastric cancer, or B lymphoma (including Burkitt's or Hodgkin's lymphoma).

[0210] In some embodiments, the composition comprises one antigenic EBV polypeptide (e.g., a monovalent composition). In some embodiments, the composition comprises an antigenic EBV polypeptide comprising a gH polypeptide. In some embodiments, the composition comprises an antigenic EBV polypeptide comprising a gL polypeptide. In some embodiments, the composition comprises an antigenic EBV polypeptide comprising a gp220 polypeptide.

[0211] In some embodiments, the composition comprises two or more antigenic EBV polypeptides. In some embodiments, the composition comprises one or more antigenic EBV polypeptides comprising two or more polypeptides encoded by EBV (i.e., a multivalent composition). In some embodiments, the EBV vaccine comprises nanoparticles comprising a gp220 polypeptide and, separately, nanoparticles comprising gH and gL polypeptides.

[0212] In some embodiments, any one or more of the antigenic EBV polypeptides, nanoparticles, or compositions described herein are provided for use in immunizing against infection caused by EBV. In some embodiments, any one or more of the polypeptides, nanoparticles, or compositions described herein are provided for use in generating a protective immune response against future infection with EBV.

[0213] 1. Target In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0214] In some embodiments, the subject is an adult (age greater than or equal to 18 years). In some embodiments, the subject is a child or adolescent (age less than 18 years). In some embodiments, the subject is an elderly (age greater than 60 years). In some embodiments, the subject is a non-elderly adult (age greater than or equal to 18 years and less than or equal to 60 years).

[0215] In some embodiments, the composition is formulated appropriately for its intended route of administration, examples of suitable routes of administration include intramuscular, transdermal, subcutaneous, intranasal, oral, or transdermal.

[0216] In some embodiments, more than one dose of the composition is administered to the subject, hi some embodiments, a booster dose improves the immune response.

[0217] In some embodiments, one or more of the antigenic polypeptides or compositions described herein are for use in a mammal such as a primate (e.g., a non-human primate such as a monkey (e.g., a macaque such as a rhesus or cynomolgus monkey) or an ape), a rodent (e.g., a mouse or rat), or a domesticated mammal (e.g., a dog, rabbit, cat, horse, sheep, cow, goat, camel, or donkey).

[0218] 2. Adjuvants An adjuvant may be administered to a subject together with an antigenic EBV polypeptide and / or nanoparticle described herein, and administration of such a composition can result in the subject generating higher titers of antibodies against the EBV polypeptide compared to administration of the EBV polypeptide without the adjuvant. The adjuvant can promote an earlier, stronger, or longer-lasting immune response against the EBV polypeptide.

[0219] In some embodiments, the composition comprises one adjuvant, in some embodiments, the composition comprises more than one adjuvant, in some embodiments, the composition does not comprise an adjuvant.

[0220] In some embodiments, the adjuvant includes aluminum. In some embodiments, the adjuvant is aluminum phosphate. In some embodiments, the adjuvant is alum (Alyhydrogel'85 2%; Brenntag - Catalog No. 21645-51-2).

[0221] 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.

[0222] In some embodiments, the adjuvant comprises squalene. In some embodiments, the adjuvant comprising squalene is Ribi (Sigma adjuvant system catalog number S6322-1vl), Addavax™ MF59, AS03, or AF03 (see U.S. Pat. No. 9,703,095). In some embodiments, the adjuvant comprising squalene is a nanoemulsion.

[0223] In some embodiments, the adjuvant comprises a polyacrylic acid polymer (PAA). In some embodiments, the adjuvant comprising PAA is SPA09 (see WO2017218819).

[0224] In some embodiments, the adjuvant comprises a non-metabolizable oil. In some embodiments, the adjuvant comprises Freund's incomplete adjuvant (IFA).

[0225] In some embodiments, the adjuvant comprises a non-metabolizable oil and killed Mycobacterium tuberculosis. In some embodiments, the adjuvant is complete Freund's adjuvant (CFA).

[0226] In some embodiments, the adjuvant is lipopolysaccharide, hi some embodiments, the adjuvant is monophosphoryl A (MPL or MPLA).

[0227] 3. Pharmaceutical Compositions In various embodiments, pharmaceutical compositions are provided comprising the antigenic EBV polypeptides and / or related entities described herein. In some embodiments, the pharmaceutical composition is an immunogenic composition (e.g., a vaccine) capable of eliciting an immune response, such as a protective immune response, against a pathogen.

[0228] For example, in some embodiments, a pharmaceutical composition may include one or more of the following: (1) an antigenic EBV polypeptide comprising an EBV polypeptide and a ferritin comprising a mutation replacing a surface-exposed amino acid with a cysteine; (2) an antigenic EBV polypeptide comprising an EBV polypeptide and a ferritin comprising a mutation replacing a surface-exposed amino acid with a cysteine ​​and an antigenic moiety linked to the cysteine; (3) an antigenic EBV polypeptide comprising an EBV polypeptide and a ferritin comprising (i) a surface-exposed cysteine ​​and (ii) a peptide linker N-terminal to the ferritin protein, wherein the EBV polypeptide is N-terminal to the peptide linker; (4) an antigenic EBV polypeptide comprising an EBV polypeptide and a ferritin comprising (i) a surface-exposed cysteine ​​and (ii) a peptide linker N-terminal to the ferritin protein, wherein the EBV polypeptide is N-terminal to the peptide linker; and an antigenic EBV polypeptide comprising a ferritin containing (i) a mutation replacing a surface-exposed amino acid with a cysteine ​​and an immunostimulatory moiety linked to the cysteine, (ii) a mutation replacing an internal cysteine ​​at position 31 of H. pylori ferritin with a non-cysteine ​​amino acid, or a mutation of an internal cysteine ​​to a non-cysteine ​​amino acid at a position similar to position 31 of non-H. pylori ferritin as determined by pairwise or structural alignment, and (iii) a mutation replacing a surface-exposed asparagine with a non-asparagine amino acid, or (5) a ferritin particle comprising any of the above polypeptides. In some embodiments, the pharmaceutical composition may comprise an antigenic EBV gL / gH polypeptide, e.g., the polypeptide comprises a linker of at least 15 amino acids between the gL and gH polypeptide sequences.

[0229] In some embodiments, the present invention provides pharmaceutical compositions comprising antibodies or other agents related to the antigenic polypeptides described herein. In one embodiment, the pharmaceutical composition comprises an antibody that binds to and / or competes with the antigenic polypeptides described herein. Alternatively, the antibody may recognize a virus particle or bacteria that contains a non-ferritin polypeptide component of the antigenic polypeptides described herein.

[0230] In some embodiments, the pharmaceutical compositions described herein are administered alone or in combination with one or more agents for enhancing immune responses, such as adjuvants described herein. In some embodiments, the pharmaceutical composition further comprises an adjuvant as described above.

[0231] 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 vehicle with which the pharmaceutical composition is administered. In exemplary embodiments, the carrier may comprise a sterile liquid, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, 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, dextrose, glycerol, ethanol, and combinations thereof. As used herein, an excipient is any non-therapeutic agent that can be included in a 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, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, etc. In various embodiments, the pharmaceutical composition is sterile.

[0232] In some embodiments, pharmaceutical compositions contain trace amounts of wetting agents or emulsifiers, or pH buffering agents.In some embodiments, pharmaceutical compositions can contain any of a variety of additives, such as stabilizers, buffers, or preservatives.In addition, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents can be included.

[0233] In various embodiments, pharmaceutical compositions can be formulated to be compatible with any desired mode of administration.For example, pharmaceutical compositions can be in the form of liquid, suspension, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquid, gelatin capsules, powder, sustained-release preparations, suppositories, emulsions, aerosols, sprays, suspensions, freeze-dried powders, freeze-suspensions, dry powders, or any other form suitable for use.General discussions on pharmaceutical formulation and manufacturing can be found, for example, in Remington's Pharmaceutical Sciences, 1999, which is incorporated herein by reference. th ed., Mack Publishing Co., Easton, PA, 1995.

[0234] The pharmaceutical composition can be administered via any route of administration. Examples of routes of administration include oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, mucosal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, intratracheal instillation, bronchial instillation, inhalation, or topical administration. Administration can be local or systemic. In some embodiments, administration is oral. In other embodiments, administration is by parenteral injection. In some instances, administration results in the release of the antigenic ferritin polypeptides described herein into the bloodstream. The mode of administration can be left to the discretion of the physician.

[0235] In some embodiments, the pharmaceutical compositions are suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, and subcutaneous). Such compositions can be formulated, for example, as solutions, suspensions, dispersions, emulsions, etc. They may also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. For example, parenteral administration can be achieved by injection. In such embodiments, the injectables are prepared in conventional forms, i.e., liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. In some embodiments, injectable solutions and suspensions are prepared from sterile powders, lyophilized powders, or granules.

[0236] In further embodiments, the pharmaceutical composition is formulated for delivery by inhalation (e.g., direct delivery to the lungs and respiratory tract). For example, the composition may take the form of a nasal spray or any other known aerosol formulation. In some embodiments, the preparation for inhalation or aerosol delivery comprises a plurality of particles. In some embodiments, such preparations may have an average particle size of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13 microns. In some embodiments, the preparation for inhalation or aerosol delivery is formulated as a dry powder. In some embodiments, the preparation for inhalation or aerosol delivery is formulated as a wet powder, for example, by including a wetting agent. In some embodiments, the wetting agent is selected from the group consisting of water, saline, or other liquids at physiological pH.

[0237] In some embodiments, pharmaceutical compositions according to the invention are administered as nasal or oral drops, hi some embodiments, a dose may comprise multiple drops (e.g., 1-100, 1-50, 1-20, 1-10, 1-5 drops, etc.).

[0238] The pharmaceutical compositions of the present invention can be administered at any dose appropriate to achieve the desired outcome. In some embodiments, the desired outcome is the induction of a long-lasting adaptive immune response against a pathogen, such as the source of the non-ferritin polypeptide present in the antigenic ferritin polypeptide present in the composition. In some embodiments, the desired outcome is a reduction in the intensity, severity, frequency, and / or delay in the onset of one or more symptoms of an infectious disease. In some embodiments, the desired outcome is the inhibition or prevention of an infectious disease. The required dose will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the infection being prevented or treated, the specific composition used, and its mode of administration.

[0239] In some embodiments, the pharmaceutical compositions according to the invention are administered in a single or multiple doses. In some embodiments, the pharmaceutical compositions are administered in multiple doses administered on different days (e.g., a prime-boost vaccination strategy). In some embodiments, the pharmaceutical compositions are administered as part of a booster regimen.

[0240] 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, provided that the pharmacological activities of the additional therapeutic agents and the active ingredients in the pharmaceutical composition overlap in time, thereby providing a combined therapeutic effect. Generally, each agent is administered at a dose and on a time schedule determined for that agent.

[0241] 4. Nucleic acid / mRNA Also provided is a nucleic acid encoding the antigenic EBV polypeptide described herein.In some embodiments, the nucleic acid is mRNA.Any nucleic acid that can be translated to produce a polypeptide is considered to be mRNA for the purposes of this disclosure.

[0242] 5. Kit Also provided herein are kits comprising one or more antigenic EBV polypeptides, nucleic acids, antigenic ferritin particles, antigenic lumazine synthase particles, compositions, or pharmaceutical compositions described herein. In some embodiments, the kits further comprise one or more of a solvent, solution, buffer, instructions, or desiccant.

[0243] ***

[0244] The present description and exemplary embodiments should not be construed as limiting. For purposes of this specification and the appended claims, unless otherwise indicated, all numerical values ​​expressing quantities, percentages, or proportions, as well as other numerical values ​​used in this specification and claims, are understood to be modified in all instances, to the extent not already so modified, by the term "about." "About" indicates a degree of variation that does not substantially affect the properties of the described subject matter, for example, variations within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed by at least considering the number of reported significant digits and applying ordinary rounding techniques.

[0245] As used in this specification and the appended claims, it should be noted that the singular forms "a," "an," and "the," as well as the use of any singular form of other terms, include plural referents unless clearly and unambiguously limited to one referent. As used herein, the term "comprises" and its grammatical variations are intended to be open-ended, and the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items. The term "or" is used in an inclusive sense, i.e., equivalent to "and / or," unless the context indicates otherwise.

[0246] Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 [Table 20] [Table 21] [Table 22] [Example]

[0247] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0248] 1. Antigenic EBV polypeptides for eliciting antibodies against EBV We developed antigenic polypeptides that induce antibodies against EBV. We developed self-assembling ferritin nanoparticles that display EBV gL and gH polypeptides as single chains, and evaluated the immunogenicity of these nanoparticles in mice.

[0249] Monomeric and trimeric gL / gH constructs were expressed and purified. Figure 1A shows single-chain gL and gH monomers (SEQ ID NO: 6) with and without His-tag cleavage by Coomassie and Western blot (anti-His) analysis. Figure 1B shows the absorbance traces on a Superose® SEC column and the fractionation of gL and gH trimers (SEQ ID NO: 11) by Coomassie, along with a Western blot to confirm His-tag cleavage by thrombin protease. For the SEQ ID NO: 6 construct, the final sample concentration was 1 mg / mL, the total volume was 15 mL, and the endotoxin level was 1.48 EU / mL.

[0250] Single-chain gL / gH ferritin nanoparticles (SEQ ID NO: 14) were expressed and purified. Figures 2A-2E show the purification and characterization by Superose® 6 SEC fractionation (2A), Coomassie blotting of the SEC fraction (2B), Western blot of the SEC fraction with anti-ferritin primary antibody (2C), dynamic light scattering (DLS, 2D), and electron microscopy (2E).

[0251] Exemplary constructs of single-chain EBV gL and gH fused to ferritin are shown in Figure 3. Conjugation sites for immunostimulatory moieties, such as Toll-like receptor 7 / 8 agonist (TLR7 / 8a), can be present on the ferritin or on the linker (see, e.g., SEQ ID NOs: 14, 19, 22, 20, 23, and 33 for exemplary sequences).

[0252] Mice were injected with gL / gH trimers or nanoparticles containing various linkers, and immune sera were evaluated (Figure 4). Mice were administered two 2 μg injections, 3 weeks apart, with the adjuvant AF03, a squalene emulsion-based adjuvant. Anti-gL / gH antibody endpoint titers were measured by ELISA at week 6. For gH_16_gL, nanoparticles (SEQ ID NO: 10) were superior to the trimeric construct (SEQ ID NO: 16). gL_28_gH nanoparticles (SEQ ID NO: 13) did not perform significantly differently from the trimeric construct (SEQ ID NO: 11). gL_46_gH nanoparticles (SEQ ID NO: 14) were superior to the gL_46_gH trimer (SEQ ID NO: 12).

[0253] These data indicate that single-chain gL / gH nanoparticles can induce a potent immune response against EBV.

[0254] 2. Bivalent immunity against gL / gH and gp220 Bivalent immunizations were performed using a composition containing single-chain gL / gH nanoparticles and gp220 nanoparticles. The inclusion of gp220 nanoparticles (SEQ ID NO: 1) did not have a significant interfering effect on the immune response elicited by single-chain gL / gH nanoparticles (gL-gH_C5 NP [SEQ ID NO: 19]), as measured by ELISA binding assays using sera from mice vaccinated as described above (Figures 5A-5B, which show measurements at individual dilutions and binding titers, respectively). Similarly, when administered in combination with single-chain gL / gH nanoparticles, no interference was observed in the response to the immune response to gp220 nanoparticles, as measured by ELISA (Figures 6A-6B, which show measurements at individual dilutions and binding titers, respectively).

[0255] Thus, immunization with both single-chain gL / gH nanoparticles and gp220 nanoparticles did not reduce the immune response to either polypeptide.

[0256] 3. Conjugation of Adjuvants to Ferritin Nanoparticles Next, we evaluated the conjugation of adjuvants to ferritin nanoparticles. Figure 7A illustrates a construct in which ferritin contains a mutation that replaces a surface-exposed amino acid with a cysteine ​​available for conjugation. Figure 7B shows an exemplary immunostimulatory moiety (SM7 / 8a, a TLR-7 / 8 agonist) linked to a PEG4 linker and maleimide. This maleimide can be used to covalently conjugate the linker (itself bound to SM7 / 8a) to the surface-exposed cysteine ​​of ferritin. Figure 7C shows an electron micrograph of a polypeptide containing a single-chain gL / gH polypeptide fused to ferritin conjugated to SM7 / 8a.

[0257] The cysteines resulting from the mutation of surface-exposed amino acids are illustrated in the structure of the ferritin molecule in Figure 8 A. Conjugation of CpG adjuvant (SEQ ID NO: 230) to ferritin is illustrated in Figure 8B, with the ferritin, linker, and CpG adjuvant juxtaposed and oriented to show parts of each moiety bound in close proximity to each other.

[0258] gL / gH nanoparticles (SEQ ID NO: 19) were reduced with 2 mM TCEP and then oxidized by adding 1× PBS and removing the TCEP using a 100 kD microspin column. SM7 / 8a was then incubated with the gL / gH nanoparticles for conjugation. Excess SM7 / 8a was removed from the reaction using a 100 kD microspin column. Mass spectrometry (MS) data indicated that approximately 100% of the polypeptide containing single-chain gL / gH and ferritin (SEQ ID NO: 19) was conjugated to SM7 / 8a (FIG. 9B), based on the shift in the major MS peak relative to the spectrum of the unconjugated polypeptide (FIG. 9A). The mass difference between the conjugated and unconjugated polypeptides corresponds to the molecular weight of the SM7 / 8a-linker-maleimide adduct (711 Da).

[0259] gp220 nanoparticles (SEQ ID NO: 1) were reduced with 2 mM TCEP and then oxidized by adding 1× PBS and removing the TCEP using a 100 kD microspin column. SM7 / 8a was then incubated with gL / gH nanoparticles for conjugation. Excess SM7 / 8a was removed from the reaction using a 100 kD microspin column. MS data indicated that approximately 100% of the conjugated polypeptides, including gp220 and ferritin (SEQ ID NO: 1), were conjugated to SM7 / 8a (FIG. 10B), based on the shift in the major MS peak relative to the spectrum of the unconjugated polypeptide (FIG. 10A).

[0260] Electron microscopy (EM) data also confirmed that conjugation of SM7 / 8a to polypeptides containing single-chain gL / gH and ferritin (Figure 11B compared to the unconjugated sample in Figure 11A) or to polypeptides containing gp220 and ferritin (Figure 11D compared to the unconjugated sample in Figure 11C) did not disrupt nanoparticle assembly.

[0261] Antibody responses were assayed by ELISA after immunization with 1 μg of nanoparticles containing single-chain gL / gH (gL_gH_C5 NPs, Figures 12A and 12B) or nanoparticles containing gp220 (Figures 13A and 13B). The nanoparticles were combined with 1 μg of naked ferritin and were either conjugated to SM7 / 8a or not. Unconjugated nanoparticles were administered with or without the adjuvant AF03. Each mouse received 100 μL of the above nanoparticle composition. For mice receiving AF03 adjuvant, AF03 and nanoparticles were mixed at a 1:1 volume ratio. BALB / c mice (n = 5 per group) were immunized twice, with a 3-week interval between doses. Blood was collected for ELISA analysis at week 5. The strongest ELISA responses were observed with nanoparticles administered in AF03 adjuvant. Conjugation to SM7 / 8a resulted in a stronger ELISA response compared to unconjugated nanoparticles without adjuvant.

[0262] The effect of coadministration of 1 μg each of SM7 / 8a-conjugated gL_gH_C5 nanoparticles and SM7 / 8a-conjugated gp220 nanoparticles was also evaluated in Figures 14A-14B and 15A-15B compared to administration of either nanoparticle alone with naked ferritin nanoparticles. No interference was observed with the immune response to either single-chain gL / gH (Figures 14A-14B, without and with AF03, respectively) or gp220 (Figures 15A-15B, without and with AF03, respectively).

[0263] 4.Long-term immunogenicity A study was conducted to evaluate the immunogenicity of nanoparticles containing single-chain gL / gH (gL / gH_C5, SEQ ID NO: 19) 3 months after administration. BALB / c mice (n = 5 per group) were immunized twice, with a 3-week interval between doses. 1 μg of naked ferritin (i.e., ferritin not conjugated to any polypeptide or adjuvant) was administered with 1 μg of nanoparticles containing single-chain gL / gH. The nanoparticles were formulated with or without the adjuvant AF03. Blood was collected for ELISA analysis at week 13. For mice receiving the adjuvant AF03, the nanoparticle composition was mixed with AF03 at a volume ratio of 1:1. Each mouse received 100 μL of the nanoparticle composition described above. Some mice received nanoparticles containing single-chain gL / gH in which ferritin was conjugated to SM7 / 8a ("7 / 8a" in Figures 16-17).

[0264] Nanoparticles containing single-chain gL / gH conjugated to SM7 / 8a produced the greatest immune response when formulated in AF03, as shown in Figure 16. Strong immune responses were also observed with these nanoparticles, even in the absence of AF03.

[0265] Parallel experiments were performed using gp220 nanoparticles (SEQ ID NO: 1) (with or without conjugation to SM7 / 8a) instead of nanoparticles containing single-chain gL / gH. Similar results were observed with these nanoparticles, with the mixed AF03 formulation producing the strongest response, and a strong immune response was also observed with these nanoparticles, even in the absence of AF03 (Figure 17).

[0266] The immune response elicited by a bivalent composition containing nanoparticles containing single-chain gL / gH (gL / gH_C5, SEQ ID NO: 19) and nanoparticles containing gp220 (SEQ ID NO: 1) was evaluated. BALB / c mice (n=5 / group) were immunized at 3-week dosing intervals. 100 μL of nanoparticle composition containing 1 μg of each nanoparticle was administered. For mice receiving AF03 adjuvant, AF03 and the vaccine were mixed at a volume ratio of 1:1. Blood was collected for ELISA analysis at the final 13th week. The immune responses to single-chain gL / gH (FIG. 18) and gp220 (FIG. 19) were not interfered with by the administration of the combined nanoparticles compared to when either nanoparticle was administered in combination with naked ferritin.

[0267] Further experiments using gL / gH_C5 nanoparticles (SEQ ID NO: 19) confirmed that long-lasting immune responses were observed when the nanoparticles were conjugated to SM7 / 8a (7 / 8a) or formulated in AF03 (Figure 21). BALB / c mice (n=5 / group) were immunized at 3-week dosing intervals. 100 μL of nanoparticle composition containing 1 μg of nanoparticles was administered. For mice receiving AF03 adjuvant, AF03 and the vaccine were mixed at a 1:1 volume ratio. Blood was collected for ELISA analysis at 2 weeks (prime), 5 weeks (boost), and 13 weeks (final). Parallel experiments were performed using gp220 nanoparticles (SEQ ID NO: 1), and similar long-lasting responses were observed with the gp220 nanoparticles (Figure 22).

[0268] Various nanoparticles containing single-chain gL / gH (gL_gH-C7, SEQ ID NO: 20) were also evaluated. The gL_gH_C7 construct contains a flexible linker between the gH polypeptide and ferritin, which contains a cysteine ​​as a conjugation site for the immunostimulatory moiety. The linker may also be used with ferritin that does not have a surface-exposed cysteine ​​(as shown in SEQ ID NO: 20). SM7 / 8a was conjugated to gL_gH_C7 by reducing the protein with 2 mM TCEP, then oxidizing it by adding 1× PBS and removing the TCEP using a 100 kD microspin column. SM7 / 8a was then incubated with gL / gH nanoparticles. After conjugation, excess SM7 / 8a was removed from the reaction using a 100 kD microspin column.

[0269] Mice were administered 1 μg of these gL / gH nanoparticles, conjugated or unconjugated to SM7 / 8a, and 1 μg of naked ferritin. 100 μL of the nanoparticle composition containing 1 μg of nanoparticles was administered. BALB / c mice (n=5 / group) were immunized at 3-week dosing intervals. For mice receiving AF03 adjuvant, AF03 and the nanoparticle composition were mixed at a 1:1 volume ratio. Blood was collected for ELISA analysis at weeks 2 (prime), 5 (booster), and 13 (final). These nanoparticles, when formulated in AF03 or conjugated to SM7 / 8a, elicited an immune response, as measured by ELISA endpoint titers in the prime bleed (Figure 20A). Similar results were observed in samples from the booster bleed (Figure 20C) or final bleed (Figure 20D). These nanoparticles were also conjugated to CpG oligodeoxynucleotides and administered in a similar manner. Results for the CpG conjugates were similar to unconjugated nanoparticles at 5 weeks (FIG. 20B).

[0270] 5. Characterization of Trichoplusia ni Ferritin-containing Nanoparticles We have also developed nanoparticles containing Trichoplusia nii ferritin and gp220 and / or gL / gH polypeptides. The Trichoplusia nii ferritin nanoparticles contain self-assembled heavy and light chains in a 1:1 ratio. By combining one non-ferritin polypeptide with a light chain and another non-ferritin polypeptide with a heavy chain, we found that two different polypeptides could be displayed on the surface of individual nanoparticles. Thus, for example, self-assembled Trichoplusia nii ferritin nanoparticles could display both gp220 and gL / gH.

[0271] Trichoplusia nii ferritin nanoparticles were produced and purified using the heavy chain fused to gp220 (SEQ ID NO: 24) or the single-chain gL / gH (SEQ ID NO: 25) and the light chain fused to gp220 (SEQ ID NO: 26) or the single-chain gL / gH (SEQ ID NO: 27) (constructs are illustrated in Figure 23B and visualized by Coomassie gel staining in Figure 23A, showing the expected increase in molecular weight compared to the light and heavy chains alone). The combination of the light and heavy chains fused to gL / gH and gp220, respectively, or vice versa, produced individual multivalent nanoparticles capable of displaying two different EBV polypeptides.

[0272] Two T. nii ferritin nanoparticles were also produced: one containing only gp220 in both the heavy and light chains (shown in Figure 24E) and one containing gp220 in the heavy chain and gH_gL in the light chain (shown in Figure 25E). Purification followed two steps. The first purification step was an ion-exchange chromatography step (Q column; see Figure 24A and Coomassie results in Figure 24C, and Figure 24A and Coomassie results in Figure 25C). This step was followed by size-exclusion chromatography (see Figure 24B and Coomassie results in Figure 24D, and Figure 25B and Coomassie results in Figure 25D).

[0273] Nanoparticles containing Trichoplusia nii light and heavy chains fused to gp220 (SEQ ID NOS: 24 and 26, shown in Figure 26B) exhibited profiles consistent with the formation of nanoparticles containing heterologous gp220 polypeptides, based on Coomassie staining (Figure 26A), an increased DLS radius compared to naked T. nii ferritin (Figure 27C) (Figure 26D), and EM analysis (Figure 26C), which revealed a more peripheral density around the nanoparticle core compared to naked nanoparticles (Figure 27B). Similar results were observed for SEQ ID NOS: 24 and 27, indicating the presence of heterologous gL / gH and gp220 polypeptides in the nanoparticles (T. nii light chain containing gL / gH polypeptides and heavy chain containing gp220 polypeptides; see Figures 26E-H for visualization by Coomassie staining, construct illustration, electron micrograph, and DLS characterization, respectively). For comparison, Figures 27A-C show the Coomassie staining (Figure 27A), DLS radius (Figure 27B), and EM analysis (Figure 27C) for naked (i.e., not conjugated to any polypeptide) T. nii ferritin.

[0274] Thus, the use of T. nii ferritin allows the display of two polypeptides on separate nanoparticles.

[0275] 6. gH / gL / gp42 construct A schematic diagram of the single-chain constructs of gH / gL / gp42 fused to ferritin (SEQ ID NOS: 227-231 and 241-242, respectively) is shown in Figure 35A. Fusion between each protein is via a flexible or rigid amino acid linker. The single-chain gH / gL / gp42 molecules form heterotrimers on the nanoparticles in a 1:1:1 ratio.

[0276] The crystal structure of a gH / gL / gp42 His-tagged fusion (SEQ ID NO: 226) was solved, showing that single-chain gH / gL / gp42 can adopt a heterotrimeric conformation similar to the wild-type gH, gL, and gp42 proteins found in nature (Figures 34 and 35B). In Figures 34 and 35B, Gp42 (shown by the dark gray arrow in Figure 34) interacts with the gH / gL heterodimer. Figure 35C is a model of how this single-chain gH / gL / gp42 heterotrimer fused to ferritin is displayed on a nanoparticle. There are 24 copies of single-chain gH / gL / gp42 displayed on a single nanoparticle.

[0277] The gH / gL / gp42 NP construct (SEQ ID NO: 227) was expressed in 293expi cells and purified (Figure 28A). The gH / gL / gp42 NP purified from the CHO pool had a dynamic light scattering radius of approximately 26.2 nm (Figure 28B).

[0278] The immune responses elicited by monovalent (gH / gL / gp42 NPs + naked ferritin nanoparticles) or bivalent (gH / gL / gp42 NPs + gp220 NPs) compositions were evaluated. The gH / gL / gp42 NPs had the sequence of SEQ ID NO: 227, and the gp220 NPs had the sequence of SEQ ID NO: 1. BALB / c mice (n=5 / group) were immunized at 3-week intervals. 100 μL of the nanoparticle composition containing 1 μg of each nanoparticle was administered with AF03 adjuvant (AF03 mixed with the vaccine at a 1:1 volume ratio). The boost indicates serum collected 5 weeks after the second immunization. EBV virus neutralization assays were performed on B cells (Figure 29A) and epithelial cells (Figure 29B) using serum collected from mice at 5 weeks. No interference was observed with administration of nanoparticles in bivalent formulations compared with administration of monovalent forms (naked ferritin and gH / gL / gp42).

[0279] Bivalent immunization of ferrets was performed with compositions comprising single-chain gL / gH / nanoparticles (gL_gH_C137A_bfp Ferr nanoparticles N19Q / C31S / S111C [SEQ ID NO: 22]) and gp220 nanoparticles (SEQ ID NO: 1) in the presence of adjuvant AF03 (Figures 30A-30B) or gL / gH / gp42 NPs (SEQ ID NO: 227) and gp220 nanoparticles (SEQ ID NO: 1) in the presence of adjuvant AF03 (Figures 30C-30E). Inj.1 = Injection 1 (serum collected from 6 ferrets 2 weeks after Inj.1). Inj.2 = Injection 2 (serum collected from 6 ferrets 2 weeks after Inj.2). Endpoint binding titers measured by ELISA binding assay against the antigens shown in Figures 30A-30E, 30F-30G show EBV virus neutralization assays (in B cells and epithelial cells, respectively) of sera from ferrets vaccinated with bivalent gL / gH / gp42 NP (SEQ ID NO: 227) and gp220 nanoparticles (SEQ ID NO: 1) in the presence of adjuvant AF03. Prime = Inj. 1, Boost = Inj. 2.

[0280] gH / gL / gp42 NP_C12 (SEQ ID NO: 228) was expressed and purified using Superose 6 size exclusion chromatography (Figure 31A). Dynamic light scattering analysis of the sample in Figure 31A showed a particle radius of 20.6 nm (Figure 31B).

[0281] gH / gL / gp42 NP_C13 (SEQ ID NO: 229) was expressed and purified using Superose 6 size exclusion chromatography (Figure 32A). Dynamic light scattering analysis of the sample in Figure 32A showed a particle radius of 17.1 nm (Figure 32B).

[0282] gH / gL / gp42 NP_C14 (SEQ ID NO: 230) was expressed and purified using Superose 6 size exclusion chromatography (Figure 33A). Dynamic light scattering analysis of the sample in Figure 33A showed a particle radius of 16.9 nm (Figure 33B).

[0283] Figure 35D shows the purification of SEQ ID NO:227 after expression in 293Expi cells. A denaturing SDS Coomassie gel shows that gH / gL / gp42 fused to ferritin exceeds 150 kD due to glycosylation. Negative-stain electron microscopy analysis of the purified product shows that single-chain gH / gL / gp42 fused to ferritin successfully formed nanoparticles, displaying gH / gL / gp42 antigens on the surface (Figure 35E). Potential destabilizing sequences were identified by sequence analysis or mass spectrometry of single-chain gH / gL / gp42 nanoparticles of SEQ ID NO:227 after stress testing of temperature, oxidation, and / or deamidation at 0, 3, 7, or 14 days. To improve the stability of the vaccine, expression, and / or immunogenicity of this vaccine construct, conservative amino acid substitution mutations will be added to SEQ ID NO:227 in various combinations, particularly at the sites listed in Table 1. Conservative amino acid substitutions at each position in a particular gene will also be tested in SEQ ID NOs: 228-230, which differ from SEQ ID NO: 227 only in the linker sequence fusing the C-terminus of gp42 to the N-terminus of the ferritin sequence.

Claims

1. A polypeptide comprising EBV gL polypeptide, EBV gH polypeptide, EBV gp42 polypeptide, and ferritin, wherein a linker having at least 40 amino acids and a length of 50 amino acids or less separates the EBV gL polypeptide and the EBV gH polypeptide, wherein the polypeptide contains a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 227 to 231, or with SEQ ID NO. 241 lacking a leader sequence, and does not contain amino acids 31 to 1156 of SEQ ID NO.

242.

2. A polypeptide comprising EBV gL polypeptide, EBV gH polypeptide, EBV gp42 polypeptide, and ferritin, wherein a linker having a length of 15 to 60 amino acids separates the EBV gH polypeptide and the EBV gp42 polypeptide, and the polypeptide contains a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 227 to 231, or with SEQ ID NO. 241 lacking a leader sequence, and does not contain amino acids 31 to 1156 of SEQ ID NO.

242.

3. A linker having a length of 15 to 60 amino acids is formed between the EBV gH polypeptide and EBV The polypeptide according to claim 1, wherein the gp42 polypeptide is separated.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises a sequence having at least 95% sequence identity with respect to sequence number 227, which lacks a leader sequence.

5. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises a sequence having at least 98% sequence identity with respect to sequence number 227, which lacks a leader sequence.

6. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises a sequence having at least 99% sequence identity with respect to sequence number 227, which lacks a leader sequence.

7. The polypeptide comprises the amino acid sequence of SEQ ID NO: 227, which lacks a leader sequence, claim Polypeptides as described in item 2.

8. The polypeptide according to claim 2, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 227, which lacks a leader sequence.

9. The polypeptide according to any one of claims 1 to 3, wherein the EBV gL polypeptide comprises the amino acid sequence of amino acids 23 to 137 of SEQ ID NO: 227, the EBV gH polypeptide comprises the amino acid sequence of amino acids 184 to 845 of SEQ ID NO: 227, and the EBV gp42 polypeptide comprises the amino acid sequence of amino acids 878 to 1078 of SEQ ID NO:

227.

10. The polypeptide according to any one of claims 1 to 3, wherein the EBV gL polypeptide, the EBV gH polypeptide, and the EBV gp42 polypeptide are present in the polypeptide in the order from the N-terminus to the C-terminus.

11. The polypeptide according to claim 2 or 3, wherein a linker having at least 40 amino acids and a length of 50 amino acids or less separates the EBV gL polypeptide and the EBV gH polypeptide.

12. The polypeptide according to claim 10, wherein a linker having a length of 46 or 47 amino acids separates the EBV gL polypeptide and the EBV gH polypeptide.

13. The polypeptide according to claim 10, wherein a linker having a length of 15 to 60 amino acids separates the EBV gH polypeptide and the EBV gp42 polypeptide.

14. The polypeptide according to claim 11, wherein a linker having a length of 30 to 50 amino acids separates the EBV gH polypeptide and the EBV gp42 polypeptide.

15. (a) the linker separating the EBV gL polypeptide and the EBV gH polypeptide, (b) the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide, or (c) the linker in (a) and the linker in (b) each contain one or more glycine, asparagine, serine, and alanine, according to claim 3.

16. (a) the linker separating the EBV gL polypeptide and the EBV gH polypeptide, (b) the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide, or (c) the linker in (a) and the linker in (b) each contain one or more glycine, asparagine, serine, and alanine, according to claim 13.

17. The polypeptide according to claim 2 or 3, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide has the amino acid sequence of SEQ ID NO: 233 or 236; and the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide has the amino acid sequence of SEQ ID NO:

234.

18. The polypeptide according to any one of claims 1 to 3, further comprising a linker separating the EBV gp42 polypeptide and the ferritin, wherein the linker separating the EBV gp42 polypeptide and the ferritin has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

235.

19. The polypeptide according to claim 18, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide consists of an amino acid sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 222, 225, or 236; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs. 234; and the linker separating the EBV gp42 polypeptide and the ferritin consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs.

235.

20. The polypeptide according to claim 18, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 233; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 234; and the linker separating the EBV gp42 polypeptide and the ferritin consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

235.

21. The polypeptide according to claim 18, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide consists of an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 233; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide consists of an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 234; and the linker separating the EBV gp42 polypeptide and the ferritin consists of an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

235.

22. The polypeptide according to claim 18, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 233; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide comprises the amino acid sequence of SEQ ID NO: 234; and the linker separating the EBV gp42 polypeptide and the ferritin comprises the amino acid sequence of SEQ ID NO:

235.

23. The polypeptide according to claim 10, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide consists of an amino acid sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 222, 225, or 236; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs. 234; and the linker separating the EBV gp42 polypeptide and the ferritin consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs.

235.

24. The polypeptide according to claim 10, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 233; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 234; and the linker separating the EBV gp42 polypeptide and the ferritin consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

235.

25. The linker separating the EBV gL polypeptide and the EBV gH polypeptide has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

233. The polypeptide according to claim 10, comprising an acid sequence; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 234; and the linker separating the EBV gp42 polypeptide and the ferritin comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

235.

26. The polypeptide according to claim 10, wherein the linker separating the EBV gL polypeptide and the EBV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 233; the linker separating the EBV gH polypeptide and the EBV gp42 polypeptide comprises the amino acid sequence of SEQ ID NO: 234; and the linker separating the EBV gp42 polypeptide and the ferritin comprises the amino acid sequence of SEQ ID NO: 235.

Images