Antigenic Epstein-Barr Virus Polypeptides

Abstract

The present disclosure relates to antigenic EBV polypeptides and their use in eliciting antibodies to EBV. Antigenic polypeptides, including EBV polypeptides and ferritin proteins, are also disclosed.

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 by reference herein 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 many successes in the field of vaccinology, novel breakthroughs are needed to protect humans against 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 and often stimulate only short-lived and weak immune responses, requiring the administration of multiple doses. Advances in genetic and biochemical engineering have enabled the development of therapeutics against challenging disease targets, but their application in the field of vaccinology has yet to be fully realized. Recombinant protein technology now allows the design of optimal antigens. In addition, nanoparticles are increasingly demonstrating their potential for optimal antigen presentation and targeted drug delivery. Nanoparticles conjugated with multiple antigens have been shown to have the ability to cross biological barriers more efficiently due to the increased binding avidity afforded by the multivalent presentation of their molecular cargo and their microscopic size. Helicobacter pylori (H. pylori) ferritin nanoparticles fused to influenza virus hemagglutinin (HA) protein allowed improved antigen stability and increased immunogenicity in a mouse influenza model (see Non-Patent Document 1). The fusion protein self-assembles into octahedral symmetric nanoparticles displaying eight trimeric HA spike structures and confers potent immune responses in various preclinical models when used with adjuvants.

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

[0005] Herein, a set of novel polypeptides, nanoparticles, compositions, methods, and uses involving EBV polypeptides are presented. Novel EBV single chain gL and gH (sometimes referred to as gL / gH or gH / gL) polypeptides have been produced, as well as antigenic polypeptides comprising these novel EBV polypeptides and ferritin. It has been observed that antigenic polypeptides and nanoparticles comprising single chain gL and gH polypeptides can contain a relatively long linker between the sequences of gL and gH, which provides increased immunogenicity. Antigenic ferritin polypeptides and nanoparticles comprising EBV gp220 polypeptides have also been produced. In addition, self-adjuvanting antigenic polypeptides comprising the described EBV polypeptides and ferritin have been developed, in which immunostimulatory moieties, such as adjuvants, are directly chemically bound to the antigenic polypeptides. Direct conjugation of the immunostimulatory moiety to the antigenic polypeptide allows for targeted co-delivery of the immunostimulatory moiety and the EBV polypeptide as a single polymeric entity, which can greatly reduce 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 an antigenic EBV polypeptide according to embodiment 1 or 2, further comprising ferritin.

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

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

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

[0014] Embodiment 7 is an antigenic EBV polypeptide according to 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 described in 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 a gH and a gL polypeptide, wherein the gH polypeptide is C-terminal to a 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 described in 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 15-60 amino acids, 20-60 amino acids, 30-60 amino acids, 40-60 amino acids, 30-50 amino acids, or 40-50 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 described in 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 described in 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 the ferritin, and optionally the linker has a length of at least 15 amino acids, or a length of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, or 40 to 50 amino acids, 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 described in 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 described in 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 described in 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 to 207 or 211 to 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 an 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 pharma- ceutically 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 described in any one of embodiments 1 to 35 for use in a method for inducing an immune response against influenza or in protecting a subject from infection with 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 an antigenic EBV polypeptide, a ferritin particle, a composition, or a method according to 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 an 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 certain embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0050] [Figure 1-1] Figures 1A-1B show single chain gL and gH monomers (Figure 1A) (SEQ ID NO: 6) and trimers (Figure 1B) (SEQ ID NO: 11) with and without removal of the His-tag 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] 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 (FIG. 2A) as well as Coomassie (FIG. 2B) and Western blot (FIG. 2C) analyses of selected fractions from the purification (L indicates molecular weight ladder, positions of 150 and 250 kDa bands are shown on the right of FIG. 2B). Also presented are dynamic light scattering (FIG. 2D) and electron microscopy (FIG. 2E) analyses of single-chain nanoparticles. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3]Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Diagram 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 (NP) 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. [Diagram 5] 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 to single-chain gL / gH nanoparticles and negative control naked ferritin (i.e., ferritin not bound to any non-ferritin polypeptide or immunostimulatory moiety). The results show that the use of the bivalent composition does not result in interference with the anti-gL / gH antibody response compared to the single-chain gL / gH results with negative control naked ferritin. Both compositions contained AF03 adjuvant. ELISA results at individual dilutions (FIG. 5A) and binding titers (FIG. 5B) are shown. [Figure 6] 6A-6B show anti-gp220 antibody responses to a bivalent composition containing both gp220 nanoparticles and single-chain gL / gH nanoparticles as described for FIG. 5A-5B. The results show that the use of the bivalent composition does not result in interference with the anti-gp220 antibody response compared to the results of gp220 nanoparticles with the negative control naked ferritin. Both compositions contained AF03 adjuvant. ELISA results at individual dilutions (FIG. 6A) and binding titers (FIG. 6B) are shown. [Figure 7]FIG. 7A shows a design of a nanoparticle comprising an EBV polypeptide and ferritin, containing mutations replacing 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 linked to ferritin by a flexible 46 amino acid linker. FIG. 7B shows a representative Toll-like receptor agonist (SM7 / 8a with a PEG4-maleimide linker) suitable for conjugation to the construct according to FIG. 7A. FIG. 7C shows an electron micrograph (EM) image of a gL / gH nanoparticle with cysteines on the ferritin surface and SM7 / 8a conjugated via a PEG4-maleimide linker. [Figure 8A] FIG. 8A is a diagram showing the structure of a portion of ferritin containing mutations that replace surface-exposed amino acids with cysteines, with the positions of the cysteines indicated. [Figure 8B] FIG. 8B shows the conjugation of CpG adjuvant (SEQ ID NO: 247) to ferritin by adjacently juxtaposing the ferritin, linker, and CpG adjuvant, which are oriented to show a portion of each moiety that becomes bound in close proximity to one another. [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 corresponds roughly 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 corresponds roughly 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 corresponds roughly to the difference expected from conjugation of SM7 / 8a to the 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 corresponds roughly to the difference expected from conjugation of SM7 / 8a to the linker. [Figure 11] Figures 11A-11D show electron microscopy (EM) images of unconjugated (Figures 11A,C) and conjugated (Figures 11B,D) single-chain gL / gH (Figures 11A,B) and gp220 (Figures 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 separate molecules, 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 following 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 following treatment with gp220 nanoparticles conjugated to SM7 / 8a and single-chain gL / gH ferritin nanoparticles conjugated to SM7 / 8a compared to treatment with single-chain gL / gH ferritin nanoparticles conjugated to SM7 / 8a 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 following treatment with gp220 nanoparticles conjugated to SM7 / 8a and single-chain gL / gH nanoparticles conjugated to SM7 / 8a compared to treatment with gp220 nanoparticles conjugated to SM7 / 8a 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 following 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 following treatment with gp220 nanoparticles and naked ferritin, with or without mixed AF03 adjuvant and / or SM7 / 8a conjugated to gp220 nanoparticles. [Figure 18] Figure 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 key top-to-bottom symbol order corresponds to the left-to-right symbol order on the graph. [Figure 19] 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 in the graph. [Figure 20-1] 20A-20D show antibody responses in mice following 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] Continued from Figure 20-1. [Figure 21] FIG. 1 shows antibody responses in mice following treatment with gL_gH_C5 nanoparticles (SEQ ID NO: 19), with or without mixed AF03 adjuvant and / or conjugation to SM7 / 8a, as endpoint titers measured by ELISA from prime, boost, and final bleeds. [Figure 22] FIG. 1 shows antibody responses in mice following treatment with gp220 nanoparticles with or without mixed AF03 adjuvant and / or conjugation to 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] FIG. 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 of a construct. [Figure 24-2] Continued from 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 stains 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 stains 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] Continued from 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 with gp220 fused to both the light and heavy chains (Figures 26B). Figures 26E-26H show data with gp220 fused to the heavy chain and gL / gH fused to the light chain (Figures 26F). [Figure 26-2] Continued from 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] Continued from Figure 27-1. [Figure 28A] Figure 28A shows an SDS denaturing Coomassie stained gel (left) with 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] FIG. 28B shows gH / gL / gp42 NP purified from the 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 end point 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] Continued from Figure 30-1. [Figure 30-3] Continued from Figure 30-2. [Figure 30-4] Continued from 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. [Diagram 34] On the left is an SDS-reduced Coomassie gel showing 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 Angstrom crystal structure of the single-chain gH / gL / gp42-His product (SEQ ID NO: 226). Gp42 (dark grey and indicated by an arrow) interacts with the gH / gL heterodimer. [Diagram 35]Figure 35A-E are drawings 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 a rigid amino acid linker as 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 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 presented 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. 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, showing 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 PREFERRED EMBODIMENTS

[0051] Provided are EBV polypeptides that can be antigenic when administered alone, with an adjuvant as a separate molecule, and / or as part of a nanoparticle (e.g., ferritin or lumazine synthase particles) that can be self-adjuvanting. Such polypeptides and compositions that include such polypeptides can be used to induce an antibody response 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 include mutations that replace surface-exposed amino acids with cysteines, which can facilitate conjugating immunostimulatory moieties to ferritin via cysteines. Such conjugation can eliminate or reduce the need for separately administered adjuvants, and can also potentially reduce the amount of adjuvant / immunostimulatory moiety required to induce an immune response against EBV polypeptides. In some embodiments, an antigenic EBV polypeptide is provided that comprises (i) an EBV polypeptide and (ii-a) a ferritin that comprises a surface-exposed cysteine ​​or (ii-b) a ferritin and an N-terminal or C-terminal linker that comprises 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 that includes all or a portion of the amino acid sequence encoded by EBV. Similarly, gL, gH, gp42, and gp220 polypeptides refer to polypeptides that include all or a portion of the gL, gH, gp42, or gp220 amino acid sequence encoded by EBV, respectively. For example, a polypeptide that has at least 80% identity with an EBV-encoded polypeptide necessarily includes 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 an antigenic polypeptide can confer protection against infection with EBV. Unless the context states otherwise, any polypeptide disclosed herein, including an EBV polypeptide, can 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 in a single chain, or EBV gL, gH, and gp42 expressed in a single chain.

[0054] As used herein, "trimer" or "trimer construct" refers to a construct that includes EBV gL and / or gH with a trimerization domain, such as the Foldon trimerization domain from T4 phage fibritin. Other trimerization domains, such as human collagen XVIII trimerization domain (see, for example, Alvarez-Cienfuegos et al., Scientific Reports 2016;6:28643) and L1ORF1p trimerization domain (see, for example, 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 trimer construct.

[0055] "Ferritin" or "ferritin protein" as used herein refers to a protein that has detectable sequence identity with 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 serves to store iron, for example, within cells or tissues, or to transport iron to the bloodstream. Such exemplary 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 a fragment of ferritin that has one or more differences in its 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, as 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 self-assembled into a spherical form. Ferritin particles are sometimes referred to as "ferritin nanoparticles" or simply "nanoparticles." In some embodiments, the ferritin particle comprises 24 ferritin monomers (or 24 total heavy and light chains, if applicable).

[0059] "Hybrid ferritin" as used herein refers to a ferritin comprising H. pyroliferritin with an amino-terminal extension of bullfrog ferritin. An exemplary sequence used as the amino-terminal extension of bullfrog ferritin is shown as SEQ ID NO: 217. In the hybrid ferritin, the amino-terminal extension of bullfrog ferritin can be fused to H. pyroliferritin so that the binding sites of the immunostimulatory moiety are evenly distributed on the ferritin particle surface. "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 comprising bullfrog sequences can be provided without bullfrog sequences, e.g., without linkers or alternative linkers. Exemplary bullfrog linker sequences are provided in Table 2. Although Table 2 shows bullfrog linkers, the same constructs can be made without linkers or alternative linkers.

[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] "Immune response" as used herein 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 an 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, and the like. An "antibody response" is an immune response in which antibodies are produced.

[0063] As used herein, "antigen" refers to an agent that elicits an immune response when exposed to or administered to an organism, 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 additionally, 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., mice, rabbits, primates, humans), 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%, 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., including 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 (either 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 non-specifically enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed, water-in-oil or oil-in-water emulsions in which antigen solutions are emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Sometimes, killed mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) 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] "Antigenic EBV polypeptide" as used herein refers to a polypeptide that contains all or part of the EBV amino acid sequence of sufficient length that the molecule is antigenic with respect to EBV. Antigenicity can be a feature of the EBV sequence as part of a construct that further contains a heterologous sequence, 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 contains a heterologous sequence, it is sufficient that the construct can serve as an antigen to generate anti-EBV antibodies, regardless of whether the EBV sequence without the heterologous sequence can do so.

[0067] "Antigenic ferritin polypeptide" and "antigenic ferritin protein" are used interchangeably herein and refer to a polypeptide that includes 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 include an immunostimulatory moiety. Antigenicity may be a feature of the non-ferritin sequence as part of a larger construct. That is, the construct is sufficient to be able to serve as an antigen to the non-ferritin polypeptide, regardless of whether the non-ferritin polypeptide (and immunostimulatory moiety, if applicable) without ferritin can 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 that includes 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 generate 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, a "subject" refers to any member of the animal kingdom. In some embodiments, a "subject" refers to a human. In some embodiments, a "subject" refers to a non-human animal. In some embodiments, a subject includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, an insect, and / or a worm. In certain embodiments, a non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments of the invention, a subject is an adult, an adolescent, or a juvenile. In some embodiments, the terms "individual" or "patient" are used interchangeably and are intended to be interchangeable with "subject."

[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 a pathogen and / or the development of one or more symptoms, and in some embodiments, before, during, and / or immediately after exposure to a pathogen. In some embodiments, vaccination includes multiple administrations of a vaccination 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 are intended to refer to the percentage of nucleotides or amino acids that are identical, particularly in optimal alignment between the sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may be distributed over the entire length of the sequences being compared, but do not necessarily have to be distributed randomly. Comparison of two sequences is usually carried out by comparing the sequences over segments or "comparison windows" 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 according to Smith and Waterman, 1981, Ads App. Math. 2, p. 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, p. 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, p. 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0075] The percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, 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 full length of the reference sequence.For example, if the reference nucleic acid sequence is 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 full length of the reference sequence.

[0077] A nucleic acid sequence or amino acid sequence that has a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, can have at least one functional property of the given sequence, for example, in some cases, is functionally equivalent to the given 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 that has a certain degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0078] As used herein, the term "kit" refers to a packaged set of one or more compounds or related components, such as 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 to allow 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 believed to be necessary for efficient membrane fusion and binding to epithelial cell receptors required for viral entry.

[0080] Disclosed herein are antigenic polypeptides comprising EBV gL and EBV gH. In some embodiments, the polypeptides are present 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), such as by multimerization of ferritin or lumazine synthase. In some embodiments, the antigenic EBV polypeptides according to the present disclosure include 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 such improvements in 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 with 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 ​​to avoid 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 ​​to avoid 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 the 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 of 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 of 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 the sequence shown in the Sequence Listing and annotated with the description as "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, the 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 "trimer."

[0088] The gL / gH polypeptide can 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 that optionally includes 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 the gp220 Polypeptide 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. The nanoparticles did not contain any mutations providing surface exposed cysteines or linkers containing cysteines, among other differences from certain ferritins described herein.

[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 a gp220 polypeptide, hi some embodiments, the mammalian leader sequence provides for secretion of the protein when expressed in mammalian cells.

[0093] The gp220 polypeptide can be combined with any of the ferritins or lumazine synthases 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 that optionally contains 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, for example, a gL and gH polypeptide is provided as SEQ ID NO: 239. Another exemplary gp42 sequence suitable for inclusion in a fusion with, for example, a gL and gH polypeptide is provided as SEQ ID NO: 240.

[0096] In some embodiments, 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: 34. In some embodiments, 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. In some embodiments, 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: 240.

[0097] In some embodiments, a mammalian leader sequence (also known as a signal sequence) is added to the N-terminus of a gp42 polypeptide. In some embodiments, the mammalian leader sequence provides for 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 a gL polypeptide further comprises a gp42 polypeptide. Any of the EBV polypeptides comprising a gH and / or a gL polypeptide described above 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, also exemplified in SEQ ID NOs: 21 and 227-231. Thus, for example, an antigenic EBV polypeptide may comprise, in an N-terminal to C-terminal orientation, a gL polypeptide, a gH polypeptide, a gp42 polypeptide, and optionally ferritin. A linker as described herein may separate the gp42 polypeptide from the EBV polypeptide and / or ferritin located at its N-terminus 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 may also be present between the ferritin and an adjacent EBV polypeptide (e.g., a gp42 polypeptide), if present.

[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 the polypeptide, a linker separates the EBV gp42 polypeptide and the ferritin. Such a linker may have a length of at least 15 amino acids, or 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, 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 that optionally includes a surface-exposed cysteine ​​as described herein.

[0102] In some embodiments, an 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, an 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, an 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. Although the following features are described with respect to any such linker, the invention provides that a relatively long linker between the gL and gH sequences may 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 no more than 60 amino acids in length. In some embodiments, the linker is no more than 50 amino acids 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) and the cysteine ​​is 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 site 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 that contain cysteines as conjugation sites for immunostimulatory moieties, such as adjuvants, are used in constructs that contain ferritin molecules that lack an unpaired surface-exposed cysteine ​​or that contain a unpaired surface-exposed cysteine.

[0108] In some embodiments, the linker is a cysteine-thrombin-histidine linker. In some embodiments, this linker is used to directly conjugate EBV polypeptide to ferritin via click chemistry. An exemplary sequence that includes the cysteine-thrombin-histidine linker is SEQ ID NO: 39. Click chemistry suitable for the conjugation reaction involving the cysteine-thrombin-histidine linker 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 comprises a linker between gH and gL, but not between the polypeptide and ferritin, or vice versa. In some embodiments, the construct comprises a linker only between the polypeptide and ferritin.

[0111] F. Antigenic EBV Polypeptides, Including EBV Polypeptides and Ferritin or Lumazine Synthase In some embodiments, an antigenic EBV polypeptide is provided, including an EBV polypeptide and a ferritin. The EBV polypeptide can be any of the EBV polypeptides described herein, such as a gL, gH, gL / gH, gp220, gp42 polypeptide, or a combination thereof. The ferritin component of the polypeptide can be ferritin from any species, with or without a mutation, such as a mutation replacing a surface-exposed amino acid with a cysteine, as described herein. In some embodiments, the polypeptide comprises any one of the amino acids 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 from bacteria, insects, fungi, birds, or mammals. In some embodiments, the ferritin is from humans. In some embodiments, the ferritin is 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 ni heavy chain ferritin (SEQ ID NO: 211) or Trichoplusia ni 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 include all 24 subunits of heavy chain ferritin and light chain ferritin, such as human or Trichoplusia nii ferritin nanoparticles. T. nii ferritin nanoparticles may include 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, the antigenic polypeptide comprising heavy chain ferritin and polypeptide is assembled with the antigenic polypeptide comprising light chain ferritin and EBV polypeptide to allow two presentations of the same 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 polypeptides comprise a light chain ferritin and a gp220 and / or a gp42 polypeptide. In some embodiments, the antigenic polypeptides comprise a heavy chain ferritin and a gp220 and / or a 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, an antigenic polypeptide comprising a light chain ferritin and a gp220 and / or a gp42 polypeptide is assembled with an antigenic polypeptide comprising a heavy chain ferritin 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 the case of 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 that comprise an antigenic EBV polypeptide disclosed herein, including an EBV polypeptide and ferritin.

[0124] 1. Ferritin Mutations In some embodiments, the present specification discloses ferritin that comprises one or more mutations. In some embodiments, the one or more mutations include, for example, changes in the amino acid sequence of wild-type ferritin and / or insertions at N- or C-terminus. In some embodiments, 1, 2, 3, 4, 5, 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, mutation simply refers to sequence differences (such as substituted, added, or deleted amino acid residue or residues) compared to corresponding wild-type ferritin.

[0125] 2. Cysteine ​​for conjugation In some embodiments, ferritin is mutated to provide a chemical handle for conjugation of immunostimulatory moieties and / or EBV polypeptides. This can be achieved by mutations that replace surface-exposed non-cysteine ​​amino acids with cysteines. For the avoidance of doubt, phrases such as "replace surface-exposed amino acids with cysteines" necessarily imply that the surface-exposed amino acids in the wild-type or pre-mutated sequence are not cysteines. Another approach to provide a chemical handle for conjugation of immunostimulatory moieties or EBV polypeptides is to include a linker-like amino acid segment at the N- or C-terminus of ferritin, the amino acid segment comprising 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 change the secondary structure of ferritin. In some embodiments, this cysteine ​​does not change 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 assembled ferritin particles. In some embodiments, this cysteine ​​can interact with molecules and cells of interest during the assembly of ferritin particles 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 an immunostimulatory moiety does not occur in the absence of this cysteine.

[0128] In some embodiments, the non-cysteine ​​amino acid that is 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 that is replaced with cysteine ​​is the amino acid residue that corresponds 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 that is replaced with cysteine ​​can be selected from the amino acids that correspond 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 the ferritin. In some embodiments, the E12C residue provides a reactive free thiol group. In some embodiments, the agent conjugated to the E12C residue on the ferritin monomer is expressed on the surface of the 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, 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, the agent conjugated to the S26C residue on the ferritin monomer is expressed on the surface of the 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, 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 a ferritin multimer or particle.

[0132] In some embodiments, 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, the agent conjugated to the A75C residue on the ferritin monomer is expressed on the surface of the 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, 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, the agent conjugated to the K79C residue on the ferritin monomer is expressed on the surface of the 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, 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, the agent conjugated to the S100C residue on the ferritin monomer is expressed on the surface of the 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, 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, the agent conjugated to the S111C residue on the ferritin monomer is expressed on the surface of the 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 comprises a mutation that replaces an internal cysteine ​​with a non-cysteine ​​amino acid. By removing the original internal cysteine ​​residue, it is possible to ensure that there is only one unpaired cysteine ​​per ferritin monomer, avoiding undesirable reactions such as disulfide formation, which may result in a more stable and efficient outcome (e.g., adjuvant presentation). In some embodiments, C31 of H. pyrolori ferritin is replaced with a non-cysteine ​​amino acid. In some embodiments, C31 of H. pyrolori ferritin is replaced with serine (C31S), although any non-cysteine ​​residue, such as alanine, glycine, threonine, or asparagine, may be used. Similar amino acids can be found in non-H. pyrolori ferritin by pairwise or structural alignment. Thus, in some embodiments, the internal cysteine ​​that is replaced for a non-cysteine ​​is an amino acid residue that aligns with C31 of H. pyrolori 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 may play a role in safety and efficacy in recombinant drug products. Regulatory approval may be contingent on demonstrating proper glycosylation as a critical quality attribute (see Zhang et al., Drug Discovery Today 21(5):740-765 (2016)). N-glycans may result from glycosylation of asparagine side chains and may vary in structure between humans and other organisms such as bacteria and yeast. Thus, it would 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, especially when used for human vaccination.

[0138] In some embodiments, ferritin is mutated to inhibit the formation of N-glycans. 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. pyroloriferritin, or a position corresponding to position 31 of H. pyroloriferritin, as determined by pairwise or structural alignment. In some embodiments, mutating such an asparagine, for example N19 of H. pyroloriferritin, reduces glycosylation of the ferritin. In some embodiments, the mutation replaces the asparagine with a glutamine. In some embodiments, the ferritin is H. pyroloriferritin 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 against 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, the reduced glycosylation of ferritin by amino acid mutation promotes the production of the protein in bacteria or yeast. In some embodiments, the reduced glycosylation of ferritin reduces the possibility of side effects in mammals when the mutant ferritin expressed in bacteria or yeast is administered. In some embodiments, the reactogenicity of the mutant ferritin produced in bacteria or yeast in human subjects is low due to the reduced glycosylation. In some embodiments, the incidence of hypersensitivity reactions in human subjects is low after treatment with mutant ferritin with reduced glycosylation compared to wild type ferritin.

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

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

[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 contains mutations corresponding to any of the aforementioned sets of mutations, which change N to Q, C to S, and non-cysteine ​​surface-exposed amino acids to cysteines 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 the 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 the proteins share similar structures despite significant sequence variation and many members of the family have been structurally characterized, and can also be used to identify the 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-Cyprinus tepidum. 4cmy-FR+Fe-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-cryo EM. 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) modeling the structure of the first sequence using the known structure of the second sequence, or (ii) comparing the structures of the first and second sequences, both of which are known, and identifying the residue in the first sequence that is most similarly located to the residue of interest in the second sequence.Corresponding residues are identified in some algorithms based on minimizing the distance of alpha carbons in the superimposed structures (e.g., which set of alpha carbon pairs provides the smallest mean square deviation for alignment).When identifying positions in non-H. pylori ferritin that correspond to positions described for H. pylori ferritin, H. pylori ferritin can be the "second" sequence. If 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 the model with the H. pyrolori ferritin structure to identify the desired corresponding residues in the ferritin of interest.There is an extensive 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 a discussion of modeling structures 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 60 subunit lumazine synthase particles. 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 can be 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 contains one or more mutations to remove potential succinimide / isoaspartate or deamidation sites. For example, the gL sequence may contain 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 comprises one or more mutations to remove potential deamidation sites. For example, the linker sequence may comprise 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 corresponding to 1150 of SEQ ID NO:227, an M to I mutation at position corresponding to 1168 of SEQ ID NO:227, an M to L mutation at position corresponding to 1177 of SEQ ID NO:227, a G to A mutation at position corresponding to 1188 of SEQ ID NO:227, or an N to Q mutation at position corresponding to 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, an aspartate, or a 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 an amino acid exposed on the surface of ferritin. In some embodiments, more than one immunostimulatory moiety, such as an adjuvant, is attached to an amino acid exposed on the surface of 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 a ferritin nanoparticle, the immunostimulatory moieties are all identical. In some embodiments where multiple immunostimulatory moieties are attached to a ferritin nanoparticle, 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 response.

[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 replacing the appropriate terminal atom (e.g., hydrogen) of the moiety, for example, with a bond to ferritin as described herein at the sulfur of a surface-exposed cysteine, 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] It is.

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

[0170] c) TLR9 agonists 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, TLR9 agonist is CpG oligodeoxynucleotide (ODN).In some embodiments, TLR9 agonist is unmethylated CpG ODN.In some embodiments, CpG ODN contains partial or complete phosphorothioate (PS) backbone instead of 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 including 5' purine (Pu)-pyrimidine (Py)-CG-Py-Pu3'; has a fully phosphorothioated (i.e., PS-modified) backbone; and has a length of 18-28 nucleotides. In some embodiments, the CpG ODN comprises the sequence of SEQ ID NO: 210, and optionally includes 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 protein, also called 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, the STING agonist is a cyclic dinucleotide (CDN).See, for example, 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 agonists also include synthetic agonists such as DMXAA. [ka]

[0176] 2. Conjugated EBV Polypeptides In some embodiments, the EBV polypeptide is conjugated to the 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 by itself, 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, 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) is used to conjugate an immunostimulatory moiety, such as an adjuvant, or an EBV polypeptide to 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, the linker, or the 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 a cysteine. In some embodiments, the cysteine ​​is a surface-exposed unpaired cysteine, i.e., a cysteine ​​that lacks a partner cysteine ​​in the appropriate position to form a disulfide bond. In some embodiments, the cysteine ​​is an unpaired cysteine ​​that includes 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, the 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 mutations 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, for example, 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 performed 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 the adjuvant to ferritin. In some embodiments, copper-free conjugation is used in the reaction of binding the 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 via 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 Ferritin as used herein can include cysteines that contain thiols, also known as sulfhydryls, that 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, vinylsulfone, 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., formation of a thioether bond). In some embodiments, the sulfhydryl-reactive chemical group is conjugated to the sulfhydryl of a cysteine ​​by disulfide exchange (i.e., formation of 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 a maleimide. In some embodiments, the reaction of maleimide with cysteine ​​results in the formation of a stable thioester bond that is, for example, not reversible. In some embodiments, the maleimide does not react with tyrosine, histidine, or methionine in ferritin. In some embodiments, the 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 of conjugating an immunostimulatory moiety, such as an adjuvant, to ferritin is thiol-disulfide exchange, also known as disulfide interchange. In some embodiments, the reaction involves the formation of a mixed disulfide that includes a portion of the original disulfide. In some embodiments, the original disulfide is a cysteine ​​that is introduced into ferritin by mutation of a surface-exposed amino acid or by adding 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., a 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. The PEG linker is 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 ​​of 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 comprises a sulfhydryl-reactive chemical group at one end and an amine-reactive group at the other end.In such a 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 that is bound to ISM.In this way, 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 ​​that has been introduced into ferritin by mutation of a surface-exposed amino acid or by 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, maleimide-linker-DBCO is conjugated to ferritin after reduction of ferritin. In some embodiments, maleimide-linker-reagent is conjugated to ferritin by reaction of maleimide with cysteine ​​of ferritin in the first step. In some embodiments, DBCO is used to link to ISM that is linked to azide. In some embodiments, ISM that is linked to azide is ISS-1018. In some embodiments, adjuvant that is coupled to 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] It 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] It is.

[0204] In some embodiments, the PEG4-maleimide linker is conjugated to SM7 / 8a. An exemplary structure of SM7 / 8a conjugated to a PEG4-maleimide linker is: [ka] It 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] It 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 against EBV in a subject. In some embodiments, the method of the present invention comprises administering to a subject an effective amount of a pharmaceutical composition described herein. In some embodiments, the method of the present invention comprises administering to a 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 pharma- ceutically acceptable vehicle, adjuvant, or excipient.

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

[0209] In some embodiments, the protective immune response reduces the incidence of hospitalization. In 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 cancer, 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 age less than or equal to 60 years).

[0215] In some embodiments, the composition is formulated appropriately for the 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 nanoparticles 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 comprises 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 US Patent No. 9703095). 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, hi some embodiments, the adjuvant comprises incomplete Freund's adjuvant (IFA).

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

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

[0227] 3. Pharmaceutical Compositions In various embodiments, pharmaceutical compositions are provided that include 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 comprising (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 polypeptide sequences of gL and gH.

[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 compositions comprise antibodies that bind to and / or compete with the antigenic polypeptides described herein. Alternatively, the antibodies may recognize virus particles or bacteria that contain the non-ferritin polypeptide components 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 response, 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 pharma- ceutically 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 sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. 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 participate in 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 minor 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, they can contain auxiliary agents, stabilizers, thickeners, lubricants, and colorants.

[0233] In various embodiments, pharmaceutical compositions can be formulated to suit 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, powders, sustained release preparations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dry powders, or any other form suitable for use.General discussions of pharmaceutical formulation and manufacture can be found, for example, in Remington's Pharmaceutical Sciences, 1999, incorporated herein by reference. th ed., Mack Publishing Co., Easton, PA, 1995.

[0234] The pharmaceutical composition can be administered via any route of administration. The routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, mucosal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, intrarectal, intratracheal instillation, bronchial instillation, inhalation, or topical routes of administration. Administration can be local or systemic. In some embodiments, administration is performed orally. In another embodiment, administration is by parenteral injection. In some cases, administration results in the release of the antigenic ferritin polypeptide 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, and the like. 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 injections are prepared in the usual form, i.e., liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, injection 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, through the inclusion of 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 a 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 to be prevented or treated, the particular composition used, and its mode of administration.

[0239] In some embodiments, the pharmaceutical composition according to the present invention is administered in a single or multiple doses. In some embodiments, the pharmaceutical composition is administered in multiple doses administered on different days (e.g., a prime-boost vaccination strategy). In some embodiments, the pharmaceutical composition is 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 that the therapeutic agents be administered at the same time, provided that the pharmacological activity of the additional therapeutic agent and the active ingredient in the pharmaceutical composition overlaps in time, thereby exerting a combined therapeutic effect. In general, each agent is administered at a dose and time schedule determined for that agent.

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

[0242] 5. Kit Also provided herein are kits that include one or more of the antigenic EBV polypeptides, nucleic acids, antigenic ferritin particles, antigenic lumazine synthase particles, compositions, or pharmaceutical compositions described herein. In some embodiments, the kits further include 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 amounts, percentages, or proportions, as well as other numerical values ​​used in this specification and the appended 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, within 10%, 5%, 2%, or 1% variation. Thus, 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 scope of the claims and the application of the doctrine of equivalents, each numerical parameter should be construed at least by considering the 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 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 grammatical variations thereof are intended to be open-ended, and the recitation of items in a list is not the exclusion of 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 text 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

[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 inducing antibodies against EBV We developed antigenic polypeptides that induce antibodies against EBV, 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 fractionation of gL and gH trimers (SEQ ID NO: 11) as absorbance traces on a Superose® SEC column and by Coomassie, along with a Western blot to confirm cleavage of the His-tag by thrombin protease. For the construct of SEQ ID NO: 6, 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 purification and characterization by Superose® 6 SEC fractionation (2A), Coomassie 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 as 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 with a 3 week dosing interval, together 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, the 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 to gL / gH and gp220 Bivalent immunization was performed with a composition comprising single-chain gL / gH nanoparticles and gp220 nanoparticles. The inclusion of gp220 nanoparticles (SEQ ID NO: 1) had no significant interference 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 assay using sera from mice vaccinated as described above (Figures 5A-5B, showing 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, showing 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, the conjugation of adjuvants to ferritin nanoparticles was evaluated. Figure 7A illustrates a construct in which ferritin contains a mutation that replaces a surface-exposed amino acid with an available cysteine ​​for conjugation. Figure 7B shows an exemplary immunostimulatory moiety (SM7 / 8a, a TLR-7 / 8 agonist) linked to a PEG4 linker and a maleimide. The maleimide can be used to covalently conjugate the linker (which itself is bound to SM7 / 8a) to the surface-exposed cysteine ​​of ferritin. A polypeptide containing a single-chain gL / gH polypeptide fused to ferritin conjugated to SM7 / 8a is shown in the electron micrograph of Figure 7C.

[0257] The cysteines resulting from mutation of surface-exposed amino acids are illustrated in the structure of the ferritin molecule in Figure 8 A. Conjugation of the 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 that bind in close proximity to one another.

[0258] gL / gH nanoparticles (SEQ ID NO: 19) were reduced with 2 mM TCEP and then oxidized by adding 1X PBS and removing 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 by a 100 kD microspin column. Mass spectrometry (MS) data showed that approximately 100% of the polypeptides, including single-chain gL / gH and ferritin (SEQ ID NO: 19), were conjugated to SM7 / 8a (FIG. 9B) based on the shift of the major MS peak relative to the spectrum of the unconjugated polypeptide (FIG. 9A). The difference in mass 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 1X PBS and removing 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 by a 100 kD microspin column. MS data showed 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 of 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 unconjugated sample in Figure 11A) or to polypeptides containing gp220 and ferritin (Figure 11D compared to 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 NP, Figures 12A and 12B) or nanoparticles containing gp220 (Figures 13A and 13B). Nanoparticles were combined with 1 μg of naked ferritin and were either conjugated or not conjugated to SM7 / 8a. Unconjugated nanoparticles were administered with or without mixed AF03 adjuvant. Each mouse received 100 μL of the nanoparticle composition described above. For mice receiving AF03 adjuvant, AF03 and nanoparticles were mixed at a volume ratio of 1:1. BALB / c mice (n=5 / group) were immunized twice with a 3-week dosing interval. Blood was collected for ELISA analysis at week 5. The strongest ELISA responses were seen with nanoparticles administered in AF03 adjuvant. Conjugation to SM7 / 8a produced a stronger ELISA response compared to unconjugated nanoparticles without adjuvant.

[0262] The effect of co-administration of 1 μg each of gL_gH_C5 nanoparticles conjugated to SM7 / 8a and gp220 nanoparticles conjugated to SM7 / 8a was also evaluated in Figures 14A-B and 15A-B, 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-B, without and with AF03, respectively) or gp220 (Figures 15A-B, without and with AF03, respectively).

[0263] 4.Long-term immunogenicity A study was conducted to evaluate the immunogenicity 3 months after administration of nanoparticles containing single-chain gL / gH (gL / gH_C5, SEQ ID NO: 19). BALB / c mice (n=5 / group) were immunized twice with a 3-week dosing interval. 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, which were formulated with or without mixed AF03 adjuvant. Blood was collected for ELISA analysis at week 13. For mice receiving AF03 adjuvant, AF03 and the nanoparticle composition were mixed 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 the 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 seen with these nanoparticles 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 seen with these nanoparticles, with the mixed AF03-containing formulation producing the strongest response, and with these nanoparticles also showing a strong immune response 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 vaccine were mixed at a volume ratio of 1:1. At the final 13th week, blood was collected for ELISA analysis. No interference was observed with the immune response against single-chain gL / gH (FIG. 18) and gp220 (FIG. 19) due to the administration of the combined nanoparticles compared to either nanoparticle administered in combination with naked ferritin.

[0267] Further experiments with 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 nanoparticles were administered. For mice receiving AF03 adjuvant, AF03 and vaccine were mixed at a volume ratio of 1:1. Blood was collected for ELISA analysis at 2 weeks (prime), 5 weeks (boost), and 13 weeks (final). Parallel experiments were performed with gp220 nanoparticles (SEQ ID NO: 1) and similar long-lasting responses were observed for 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 that contains a cysteine ​​as a conjugation site for the immunostimulatory moiety. The linker may 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 1X 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 by a 100 kD microspin column.

[0269] Mice were administered 1 μg of these gL / gH nanoparticles conjugated or unconjugated to 7 / 8a and 1 μg of naked ferritin. 100 μL of nanoparticle composition containing 1 μg nanoparticles were administered. BALB / c mice (n=5 / group) were immunized at 3-week dosing intervals. For mice receiving AF03 adjuvant, AF03 and nanoparticle composition were mixed at a volume ratio of 1:1. Blood was collected for ELISA analysis at 2 weeks (prime), 5 weeks (booster), and 13 weeks (final). These nanoparticles elicited an immune response when formulated in AF03 or conjugated to SM7 / 8a, as measured by ELISA endpoint titers in the prime bleed (Figure 20A). Similar results were seen in samples from the booster bleed (Figure 20C) or terminal bleed (Figure 20D). These nanoparticles were also conjugated to CpG oligodeoxynucleotides and administered in the same manner. Results for the CpG conjugates were similar to unconjugated nanoparticles at 5 weeks (FIG. 20B).

[0270] 5. Characterization of Nanoparticles Containing Trichoplusia ni Ferritin Nanoparticles containing Trichoplusia nii ferritin and gp220 and / or gL / gH polypeptides were also developed. Trichoplusia nii ferritin nanoparticles contain self-assembled heavy and light chains in a 1:1 ratio. It was found that by combining one non-ferritin polypeptide with a light chain and another non-ferritin polypeptide on a heavy chain, 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 with heavy chain fused to gp220 (SEQ ID NO: 24) or single chain gL / gH (SEQ ID NO: 25) and light chain fused to gp220 (SEQ ID NO: 26) or single chain gL / gH (SEQ ID NO: 27) (constructs are illustrated in FIG. 23B and visualized by Coomassie gel staining in FIG. 23A, showing the expected increase in molecular weight compared to light and heavy chains alone). The combination of 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, containing only gp220 in both the heavy and light chains (shown in Figure 24E) or gp220 in the heavy chain and gH_gL in the light chain (shown in Figure 25E). The purification followed two steps. The first purification step was an ion exchange chromatography step (Q column, see Figure 24A and Coomassie results Figure 24C, and Figure 24A and Coomassie results Figure 25C). This step was followed by size exclusion chromatography (see Figure 24B and Coomassie results Figure 24D, and Figure 25B and Coomassie results Figure 25D).

[0273] Nanoparticles containing Trichoplusia nii light and heavy chains fused to gp220 (SEQ ID NOs: 24 and 26, shown in FIG. 26B) showed profiles consistent with the formation of nanoparticles containing heterologous gp220 polypeptides based on Coomassie staining (FIG. 26A), increased DLS radius compared to naked T. nii ferritin (FIG. 27C) (FIG. 26D), and EM analysis revealing more peripheral density around the nanoparticle core compared to naked nanoparticles (FIG. 26C) (FIG. 27B). Similar results were seen for SEQ ID NOs: 24 and 27 indicating the presence of heterologous gL / gH and gp220 polypeptides in the nanoparticles (light chain containing Trichoplusia nii gL / gH polypeptides and heavy chain containing gp220 polypeptides, see FIG. 26E-H for visualization by Coomassie staining, construct illustration, electron micrographs, 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 individual nanoparticles.

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

[0276] The crystal structure of 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 (Figure 34 and Figure 35B). In Figure 34 and Figure 35B, Gp42 (shown as a 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 presented on a nanoparticle. There are 24 copies of single-chain gH / gL / gp42 presented 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 response elicited by monovalent (gH / gL / gp42 NP+naked ferritin nanoparticles) or bivalent (gH / gL / gp42 NP+gp220 NP) compositions was evaluated. gH / gL / gp42 NP had the sequence of SEQ ID NO:227, and gp220 NP had the sequence of SEQ ID NO:1. 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 with AF03 adjuvant (AF03 mixed with vaccine at a volume ratio of 1:1). Boost indicates serum collected 5 weeks after the second immunization. Serum collected from mice at 5 weeks was used for EBV virus neutralization assay analysis in B cells (FIG. 29A) and epithelial cells (FIG. 29B). No interference was observed with administration of the bivalent formulation of nanoparticles compared with administration of the monovalent forms (naked ferritin and gH / gL / gp42).

[0279] Bivalent immunization of ferrets was performed with compositions comprising single-stranded 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-B) or gL / gH / gp42 NPs (SEQ ID NO: 227) and gp220 nanoparticles (SEQ ID NO: 1) in the presence of adjuvant AF03 (Figures 30C-E). 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-E, 30F-G show EBV virus neutralization assays (in B cells and epithelial cells, respectively) of sera from ferrets bivalently vaccinated with 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 purification of SEQ ID NO:227 after expression in 293Expi cells. Denaturing SDS Coomassie gel shows that gH / gL / gp42 fused to ferritin exceeds 150 kD due to glycosylation. Negative staining electron microscopy analysis of the purified product shows that single chain gH / gL / gp42 fused to ferritin successfully forms nanoparticles and displays gH / gL / gp42 antigen on the surface (Figure 35E). Potential destabilizing sequences were identified from sequence analysis or mass spectrometry of single chain gH / gL / gp42 nanoparticles of SEQ ID NO:227 by temperature, oxidation, and / or deamidation stress testing 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 substitution mutations will be added to SEQ ID NO:227. Conservative amino acid substitutions at each location 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 an Epstein-Barr Virus (EBV) gL polypeptide, an EBV gH polypeptide and an EBV gp42 polypeptide, the polypeptide comprising an amino acid sequence of amino acids 31 to 1156 of SEQ ID NO:

242.

2. The polypeptide of claim 1, further comprising an amino acid sequence of a ferritin protein comprising the amino acid sequence of any one of SEQ ID NOs:201-208, further comprising one or more amino acid substitutions, wherein the amino acid sequence of the ferritin protein has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs:201-208, and wherein the amino acid sequence of the ferritin protein comprising one or more amino acid substitutions is located within the polypeptide at the C-terminus of amino acids 31-1156 of SEQ ID NO:

242.

3. The polypeptide of claim 2, wherein the amino acid sequence of the ferritin protein has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

208.

4. The polypeptide of claim 2, wherein the amino acid sequence of the ferritin protein has at least 97% sequence identity to the amino acid sequence of SEQ ID NO:

208.

5. The polypeptide of claim 1, further comprising a ferritin protein, wherein the amino acid sequence of the ferritin protein consists of the amino acid sequence of any one of SEQ ID NOs:201-208, and wherein the amino acid sequence of the ferritin protein is located within the polypeptide at the C-terminus of amino acids 31-1156 of SEQ ID NO:

242.

6. A composition comprising a first polypeptide according to any one of claims 1 to 5, and a second polypeptide comprising an EBV gp220 polypeptide and ferritin.

7. The composition of claim 6, wherein the amino acid sequence of the EBV gp220 polypeptide has at least 99% sequence identity to the amino acid sequence of SEQ ID NO:

38.

8. The EBV gp220 polypeptide consists of the amino acid sequence of SEQ ID NO:

38. Item 7. The composition according to item 6.

9. The composition according to any one of claims 6 to 8, wherein the amino acid sequence of the ferritin protein of the second polypeptide consists of any one of the amino acid sequences of SEQ ID NOs: 201 to 208.

10. The composition of claim 6, wherein the amino acid sequence of the second polypeptide has at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1 lacking the leader sequence.

11. The composition of claim 6, wherein the amino acid sequence of the second polypeptide comprises the amino acid sequence of SEQ ID NO:1 lacking a leader sequence.

12. (a) a first polypeptide consisting of an amino acid sequence of amino acids 31 to 1156 of SEQ ID NO:242 and an amino acid sequence of a ferritin protein that is at least 97% identical to the amino acid sequence of SEQ ID NO:208, wherein the amino acid sequence of the ferritin protein is located C-terminally to the amino acid sequence of amino acids 31 to 1156 of SEQ ID NO:242 within the first polypeptide; and (b) a second polypeptide consisting of the amino acid sequence of SEQ ID NO:1 lacking a leader sequence. A composition comprising:

13. The composition of claim 12 further comprising an adjuvant.

14. (a) the amino acid sequence of amino acids 31-1156 of SEQ ID NO: 242, and (b) the amino acid sequence of ferritin protein of SEQ ID NO: 208 having five amino acid substitutions including N25Q and C37S according to the numbering of SEQ ID NO: 208; It consists of: The polypeptide of claim 1, wherein the amino acid sequence of the ferritin protein is located within the polypeptide at the C-terminus of amino acids 31 to 1156 of SEQ ID NO:

242.

15. The polypeptide of claim 14, wherein two of the five amino acid substitutions other than N25Q and C37S are N to Q substitutions in SEQ ID NO:

208.

16. 16. The polypeptide of claim 15, wherein the N to Q substitution is a surface-exposed amino acid residue in SEQ ID NO:

208.

17. The polypeptide of claim 14, wherein the five amino acid substitutions include N25Q, C37S, and one or more N to Q substitutions in SEQ ID NO:

208.

18. The polypeptide of claim 14, wherein the five amino acid substitutions include one or more amino acid substitutions selected from N25Q, C37S, and an N to Q substitution and a Q to S substitution in SEQ ID NO:

208.

19. 16. The polypeptide of claim 15, wherein the amino acid substitution other than N25Q, C37S and N to Q substitution is a Q to S substitution in SEQ ID NO:

208.

20. 17. The polypeptide of claim 16, wherein the amino acid substitution other than N25Q, C37S and N to Q substitution is a Q to S substitution in SEQ ID NO:

208.

21. A composition comprising a first polypeptide according to any one of claims 14 to 20 and a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 lacking a leader sequence.

22. A polypeptide consisting of the amino acid sequence of a ferritin protein consisting of amino acids 31-1156 of SEQ ID NO:242 and amino acids 1157-1329 of SEQ ID NO:242 having one amino acid substitution, wherein the amino acid sequence of the ferritin protein is located within the polypeptide at the C-terminus of amino acids 31-1156 of SEQ ID NO:

242.

23. 2. The polypeptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:242 lacking the leader sequence.

24. The polypeptide of claim 22, wherein the amino acid substitutions are to surface-exposed amino acid residues in amino acids 1157-1329 of SEQ ID NO:

242.

25. A composition comprising a first polypeptide according to any one of claims 22 to 24 and a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 lacking a leader sequence.

26. A ferritin particle comprising a polypeptide according to any one of claims 14 to 20 and 22 to 24.

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